Molecular Genetics of Kaposi's Sarcoma-Associated Herpesvirus (Human Herpesvirus 8) Epidemiology and Pathogenesis

Abstract

Kaposi's sarcoma had been recognized as unique human cancer for a century before it manifested as an AIDS-defining illness with a suspected infectious etiology. The discovery of Kaposi's sarcoma-associated herpesvirus (KSHV), also known as human herpesvirus-8, in 1994 by using representational difference analysis, a subtractive method previously employed for cloning differences in human genomic DNA, was a fitting harbinger for the powerful bioinformatic approaches since employed to understand its pathogenesis in KS. Indeed, the discovery of KSHV was rapidly followed by publication of its complete sequence, which revealed that the virus had coopted a wide armamentarium of human genes; in the short time since then, the functions of many of these viral gene variants in cell growth control, signaling apoptosis, angiogenesis, and immunomodulation have been characterized. This critical literature review explores the pathogenic potential of these genes within the framework of current knowledge of the basic herpesvirology of KSHV, including the relationships between viral genotypic variation and the four clinicoepidemiologic forms of Kaposi's sarcoma, current viral detection methods and their utility, primary infection by KSHV, tissue culture and animal models of latent- and lytic-cycle gene expression and pathogenesis, and viral reactivation from latency. Recent advances in models of de novo endothelial infection, microarray analyses of the host response to infection, receptor identification, and cloning of full-length, infectious KSHV genomic DNA promise to reveal key molecular mechanisms of the candidate pathogeneic genes when expressed in the context of viral infection.

Full text

MICROBIOLOGY AND MOLECULAR BIOLOGY REVIEWS, June 2003, p. 175–212 Vol. 67, No. 2
1092-2172/03/$08.00⫹0 DOI: 10.1128/MMBR.67.2.175–212.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.
Molecular Genetics of Kaposi’s Sarcoma-Associated Herpesvirus
(Human Herpesvirus 8) Epidemiology and Pathogenesis
Lyubomir A. Dourmishev,
1
Assen L. Dourmishev,
1
Diana Palmeri,
2
Robert A. Schwartz,
3
and David M. Lukac
2
*
Department of Dermatology, Medical University of Sofia, Sofia Bulgaria,
1
and Department of Microbiology and
Molecular Genetics, International Center for Public Health,
2
and Dermatology,
3
University of
Medicine and Dentistry of New Jersey/New Jersey Medical School, Newark, New Jersey
INTRODUCTION .......................................................................................................................................................175
CLINICOEPIDEMIOLOGY OF KSHV ..................................................................................................................176
Classic KS................................................................................................................................................................176
AIDS-Associated KS ...............................................................................................................................................177
Endemic (African) KS............................................................................................................................................179
Iatrogenic KS...........................................................................................................................................................180
OTHER KSHV-ASSOCIATED DISEASES.............................................................................................................181
CLINICAL DETECTION OF KSHV, AND ESTIMATES OF INFECTION IN
THE GENERAL POPULATION ......................................................................................................................182
PRIMARY INFECTION BY KSHV..........................................................................................................................183
HISTOPATHOGENESIS OF KS AND ITS RELATIONSHIP TO KSHV INFECTION .................................184
HIV-1 in KS Histopathogenesis............................................................................................................................185
TISSUE CULTURE MODELS OF ENDOTHELIAL KSHV INFECTION........................................................185
KSHV INFECTION IN ANIMAL MODELS ..........................................................................................................186
PHYSICAL AND GENETIC STRUCTURE OF KSHV.........................................................................................187
KSHV Subtypes and Geographic Variability ......................................................................................................187
LATENT VERSUS LYTIC GENE EXPRESSION IN KSHV ...............................................................................192
Latent Genes and KSHV Pathogenesis ...............................................................................................................193
The major latent locus: ORF73, ORF72, and K13 ........................................................................................193
K12/kaposin A .....................................................................................................................................................194
K11.5/vIRF2 .........................................................................................................................................................194
K10.5/LANA-2......................................................................................................................................................194
K15/LAMP ...........................................................................................................................................................194
Lytic Genes and KSHV Pathogenesis ..................................................................................................................194
K2/vIL-6 ...............................................................................................................................................................195
ORF74/vGPCR ....................................................................................................................................................195
K6/vMIP, K4/vMIP-II, and K4.1/vMIP-III......................................................................................................195
K9/vIRF-1 and K10.5/K10.6/vIRF-3..................................................................................................................195
K1..........................................................................................................................................................................195
ORF16/vBCl-2......................................................................................................................................................196
K7/vIAP ................................................................................................................................................................196
K3/MIR1, K5/MIR2 ............................................................................................................................................196
K14/viral OX2......................................................................................................................................................196
LYTIC REACTIVATION OF KSHV ........................................................................................................................196
PERSPECTIVES AND FUTURE DIRECTIONS ...................................................................................................198
ACKNOWLEDGMENTS ...........................................................................................................................................199
REFERENCES ............................................................................................................................................................199
INTRODUCTION
In 1872, Moritz Kaposi described an angiomatous neoplasm
that affected elderly men of Italian, Jewish, or Mediterranean
origin and had a relatively aggressive evolution, a disorder later
known as classical Kaposi’s sarcoma (KS) (reviewed in refer-
ence 216). Years later, other epidemiologic forms of KS were
also reported, including endemic KS in equatorial Africa and
iatrogenic KS among immunosuppressed organ transplant re-
cipients. In 1981, disseminated and highly aggressive KS in
young homosexual men with a poor prognosis was identified as
a part of a new AIDS epidemic and was thus termed “epidemic
KS” (49, 192, 226). Analyses of numerous American and
European human immunodeficiency virus type 1 (HIV-1)-
infected populations revealed that up to 30% of HIV-1-posi-
tive homosexual men develop AIDS-associated KS (37). Fur-
ther epidemiologic studies suggested an infectious etiology of
this previously rare cancer (37, 528), confirming that the major
risk factor is homosexual sex among young men (37, 528).
* Corresponding author. Mailing address: Department of Microbi-
ology and Molecular Genetics, UMDNJ/NJ Medical School, Interna-
tional Center for Public Health, Room E350T, 225 Warren St., P.O.
Box 1709, Newark, NJ 07101-1709. Phone: (973) 972-4483, ext. 20907.
Fax: (973) 972-8981. E-mail: Lukacdm@umdnj.edu.
175

Indeed, herpesvirus-like particles in tissue culture of KS
specimens from different geographic regions was observed
more than 30 years ago (182), and an association with human
cytomegalovirus (HCMV) infection had originally been pro-
posed (170, 183). However, the major breakthrough in the
hunt for the KS agent came in 1994, when Chang et al. used
representational difference analysis to discover two DNA frag-
ments associated uniquely with diseased dermal tissue from a
KS lesion of a patient with AIDS (90). Confirming the sus-
pected infectious nature of KS, the sequences did not repre-
sent heritable human polymorphic loci but had 39 and 51%
amino acid identity to the capsid and tegument proteins, re-
spectively, of two transforming primate gammaherpesviruses,
Epstein-Barr virus (EBV) and herpesvirus saimiri (HVS) (90).
Concordant with its homology to lymphotropic viruses, the
discovery of KS-associated herpesvirus (KSHV) DNA in KS
specimens was soon followed by detection of the virus in cul-
tured cells from patients with primary effusion lymphoma
(PEL), a rare AIDS-associated B-cell non-Hodgkin’s lym-
phoma (NHL) (82, 83, 366, 424). The ability of PEL cells to
support continuous KSHV infection and conditional, produc-
tive replication (342, 355, 424) was crucial for the subsequent
cloning and nucleotide sequencing of the entire viral genome
(436) and for direct visualization of mature herpesvirus-like
KSHV virions by electron microscopy (384, 424, 439). The
complete nucleotide sequence of KSHV (also called human
herpesvirus-8 [HHV-8]) confirmed its classification as a rhadi-
novirus, or gamma-2-herpesvirus, joining the gamma-1-herpes-
virus EBV as the only human gammaherpesviruses (355, 436).
Initial seroepidemiologic studies using PEL cells as a source of
KSHV-specific antigen established that all pathogenic forms of
KS are associated with infection by KSHV (176, 250, 290, 343,
466). The fascinating epidemiology of KS and the creative
molecular virologic studies of KSHV have ignited vigorous
interest in this DNA tumor virus. Although the resulting ex-
plosion of the literature has brought us closer to understanding
the relationship between KSHV replication and the unique
malignancies associated with its infection of humans, many
questions remain unanswered.
CLINICOEPIDEMIOLOGY OF KSHV
The individual clinicoepidemiologic forms of KS have been
classified as classic or sporadic (in the Mediterranean region),
epidemic (or AIDS associated), endemic (in Africa), and iat-
rogenic (in organ and tissue transplant recipients receiving
immunosuppressive therapy). Historically, each form has been
distinguished by numerous clinical parameters, including the
extent of anatomic involvement, the aggressiveness of lesion
formation, and associated morbidity and mortality, as well as
by divergent patient risk factors, notably age, sex, and ethno-
geographic origin (each of these is discussed individually below
[452, 549]). Despite these clinical differences, it is now well
established that all four epidemiologic forms of KS have in
common KSHV infection of the host (12, 13, 161, 176, 250,
290, 343, 390, 412, 419, 449, 466), with the four forms of KS
being histologically indistinguishable (1). Seroconversion for
KSHV occurs before progression to KS and is a strong predic-
tor of clinical disease (175, 250, 325, 356, 425). The risk of KS
in most populations (AIDS and non-AIDS) is directly propor-
tional to the prevalence of KSHV; however, in HIV-uninfected
populations, the prevalence varies widely in geographically dis-
tinct patterns (35, 176, 290, 382, 412, 466, 531).
Although KSHV infection is a prerequisite for KS develop-
ment, the clinical disparities between the individual epidemi-
ologic forms of KS suggest a significant role for cofactors in the
outcome of infection. For example, coinfection by HIV-1 is
clearly the major risk factor unique to AIDS-associated KS and
distinguishes its aggressive course from the other forms. How-
ever, the pathogenic consequences of genetic variation within
the KSHV type species and within different human popula-
tions, together with the interactions between them, is not yet
well understood. Integration of further epidemiologic studies
(e.g., route of transmission) with analyses of molecular genetic
host-virus interactions in tissue culture and nonhuman models
of infection promise to reveal key mechanisms in the patho-
genesis of KSHV.
Classic KS
Classic KS occurs as a rare and indolent form in elderly
Mediterranean men, with particularly high incidence in Italy,
Greece, Turkey, and Israel (230, 452). Historically, KS inci-
dence in the Mediterranean is up to 10-fold higher than in the
rest of Europe and the United States (40, 133, 204). Classic KS
lesions tend to remain confined to the lower extremities and
preferentially afflict men rather than women, at approximately
a 15:1 ratio. Those affected often live with the disease for 10
years or more and are usually not killed by it (216).
In Italy, seroprevalence and incidence increase with age,
particularly in males who have reached their 50th birthdays
(443). Unlike AIDS-associated KS, HIV-1 coinfection is not
typical, but the observation that KS develops annually in only
0.03% of KSHV-infected men older than 50 years in the Medi-
terranean strongly suggests the existence of a cofactor(s) (516).
Extensive epidemiologic studies in Italy demonstrate a
strong influence of ethnogeography on KSHV seropositivity
and KS incidence, with a marked gradient increasing from the
north to the south. Among a population of 910 blood donors
and lymphoma patients, the lowest incidences were in northern
Italy (7.3% KSHV positive, 0.605 case of KS/100,000 popula-
tion/year), the highest incidences were in the south (24.6% and
1.495/100,000), and a moderate number were in central Italy
(9.5% and 0.5/100,000) (531). Two particular southern Italian
hot spots for KS are on the islands of Sardinia and Sicily: for
example, a high rate of KS in northern Sardinia is associated
with a general seroprevalence of 35%, with a range of 15.3 to
46.3% in five areas (443). However, hot spots also occur in
low-incidence regions: in the Po Valley in northern (mainland)
Italy, the incidence is 2.5/100,000 men and 0.7/100,000 women,
with one rural zone having double the male rate and four times
the female rate (24). Elsewhere in Mediterranean Europe, a
group of blood donors in Spain had a low KSHV seropositivity
similar to that of northern Italians (171). The high prevalence
of KSHV in the Mediterranean contrasts with non-Mediter-
ranean European countries such as Latvia, where a recent
screening of 150 healthy blood donors by PCR detected human
cytomegalovirus, HHV-6, and HHV-7 but failed to detect
KSHV (269).
The indolent nature of classic KS has made the determina-
176 DOURMISHEV ET AL. MICROBIOL.MOL.BIOL.REV.

tion of its prognostic factors challenging, but the geographic
gradient of seropositivity would appear to make Italy an ideal
setting to identify environmental risk factors. In northern Sar-
dinia, the occupation of cereal farming enhances KS risk (116),
lending support to Ziegler’s hypothesis that chronic exposure
to the aluminosilicate-rich volcanic soils specific to southern
Italy may contribute to localized immune suppression in the
extremities and increased KS risk (546). However, other stud-
ies have failed to confirm the connection between type of soil
and KS incidence in northeast Sardinia (350). In the Po Valley,
residence in areas where malaria was formerly endemic corre-
sponded to regions of high KSHV seroprevalence (24).
Coluzzi et al. make a provocative argument that many of the
risk factors identified for classic KS might be attributable to
high exposure of those populations to blood-sucking arthro-
pods (108). The authors hypothesize that local immunosup-
pression engendered by injection of insect saliva following a
bite would prime the tissue microenvironment to enhance (i)
transmission of KSHV (from an infected adult’s saliva applied
as a “salve” to a bitten child) or (ii) reactivation of KSHV in an
elderly person, whose immune system is already declining.
Although this evidence is largely anecdotal, the authors pro-
pose prospective studies to test their hypotheses.
Two other studies explored the risk associated with age and
immune function. Brenner et al. (58) retrospectively analyzed
all of the classic KS patients (a total of 248) treated in two
hospitals in northern Israel and Tel Aviv between 1960 and
1995. Using progression-free survival as the end point of dis-
ease, univariate and multivariate analyses showed that increas-
ing age at KS diagnosis was prognostic of increasing disease
progression and that immunosuppression (from steroid treat-
ment or renal failure associated with organ transplantation)
predicted dissemination. A second study found that KS risk
was associated with both mild immunosuppression (lower lym-
phocyte and CD4
⫹
cell counts) as well as immune activation
(increased serum neopterin and ␤
2
-microglobulin levels) (503).
Behaviors that influence KS risk have been examined by
Goedert et al., who demonstrated that increased cigarette
smoking (measured both in packs per day and in “pack-years”)
reduces the risk of classic KS fourfold (189). This study used
the powerful comparison of patients with both histologically
confirmed KS and serologically confirmed KSHV infection to
age- and sex-matched controls who were KSHV positive with-
out KS. Topical corticosteroid use, infrequent bathing, and
asthma independently increased risk for KS in this study.
Transmission of KSHV in classic KS probably occurs by both
sexual and nonsexual routes. Both the close family members
and heterosexual partners of KS patients show increased
KSHV seroprevalence (19, 56), while a group of 51 Catholic
nuns in Italy had a seroprevalence indistinguishable from that
of matched female controls (517). Similar to AIDS-associated
KS patients, the high frequency of detection of KSHV DNA in
tonsillar swabs from HIV-negative KS patients in Italy suggests
that transmission in saliva is a predominant route (75). Con-
versely, only 2 of 27 seropositive Italian women had KSHV
DNA in cervicovaginal smears (428). Chemotherapy and/or
radiotherapy has been used successfully to treat classic KS in
France, but KSHV DNA remains detectable in postlesional
skin and peripheral blood mononuclear cells (PBMCs), sug-
gesting a potential explanation for the frequently observed
relapse of classic KS (206).
AIDS-Associated KS
KS is the most common neoplasm in homosexual and bisex-
ual men with AIDS (186); the disease is extremely aggressive in
this population and displays a more frequent mucosal progres-
sion than in the other epidemiologic forms (452). It commonly
presents multifocally and frequently on the upper body, head,
and neck (216), and it evolves quickly, both in local progression
of lesions to tumors and in visceral dissemination leading to
organ dysfunction and high mortality.
In the United States, the frequency of dual KSHV- and
HIV-positive subjects during the height of the AIDS epidemic
corresponded to AIDS epicenters: from 3% in Kansas and 6%
in Iowa to 30% in California and 31% in New York (38). The
incidence of AIDS-associated KS in homosexual men in San
Francisco in the early 1980s was nearly 40% (325), but the
decline in new infections reduced its frequency in AIDS pa-
tients in the mid-1990s to about 15 to 25% (137, 336, 425).
Recent data suggest that KSHV was highly prevalent in the
homosexual population prior to the AIDS epidemic: in San
Francisco in the first 6 months of 1978, the prevalence of
KSHV was 24.6%, with a concurrent HIV-1 prevalence of only
1.8% (386).
Similar to classic KS, the relative seroprevalence of KSHV
matches the KS incidence in HIV-infected populations. Sero-
logical surveys show that KSHV prevalence is increased in
patients with a high risk of developing KS (176, 325, 343, 466)
and that infection with KSHV anticipates the development of
KS (175, 250, 325, 356, 425, 530). For example, in Thailand,
where there is a high prevalence of HIV but a low prevalence
of AIDS-associated KS, only 2 to 12% of HIV-1-positive ho-
mosexual men were KSHV seropositive (a value only slightly
higher than that for the general population in that country)
(27). In regions where classic KS is common, such as Rome,
patients at a sexually transmitted disease (STD) clinic with the
highest relative KS risk were homosexual HIV-positive males
(429), also suggesting a coevolving AIDS-related KS epidemic.
A direct correlation between HIV-1 infection, AIDS patho-
genesis, and KS progression has been demonstrated in numer-
ous studies. In a cohort of homosexual men from New York
and Washington, D.C., 12.5% of men who enrolled with a
CD4
⫹
count of ⬍330 cells/␮l developed KS while none with a
count of ⬎550 cells/␮l presented with KS (187). Similarly,
other reports have shown that (i) KSHV seropositivity in-
creased as CD4
⫹
counts decreased (325), (ii) detection of
KSHV DNA in peripheral blood corresponded to a lower
mean CD4
⫹
cell count in HIV-1-positive patients without KS
(344), and (iii) KS progression was proportional to an in-
creased rate of CD4
⫹
cell loss and increased HIV-1 RNA
levels (233).
HIV-1 probably exacerbates KSHV pathogenesis at multiple
levels, including through immunosuppression, by priming of
target cells and the tissue microenvironment for KSHV infec-
tion and replication, and by exerting direct effects on KSHV
gene expression and viral replication. Direct and reciprocal
interactions between HIV-1 and KSHV at the molecular level
have received a lot of recent attention in the literature; they
VOL. 67, 2003 KSHV MOLECULAR GENETICS 177

suggest mutual positive-feedback loops during replication of
both viruses. This is discussed further below.
Many conflicting studies have demonstrated, however, that
the relative contributions of HIV-1 burden and immunosup-
pression to KS development are not always directly propor-
tional. There was no correspondence between CD4
⫹
count
and the risk of KSHV seroconversion in a prospective study of
259 Danish homosexual men (336). Likewise, specific T-cell
responses to purified KSHV virions were reduced in HIV-1-
positive men but were proportional to KS risk in HIV-1-neg-
ative men; however, both were independent of CD4
⫹
counts
(486). Similarly, there was no correlation between CD4
⫹
count, HIV-1 RNA levels, and KS progression in an AIDS-
associated KS cohort in Zimbabwe; instead, KS progression
was proportional to the KSHV burden in the peripheral blood
(68). Finally, Veugelers, et al. found that age at the time of
KSHV seroconversion may carry more risk than absolute
CD4
⫹
numbers: older HIV-1-positive men have higher CD4
⫹
numbers at the time of seroconversion than do younger men
(512).
Furthermore, in the Amsterdam Cohort, decreasing CD4
⫹
numbers had no influence on the rate of KSHV seroconversion
but, instead, correlated with increased KSHV replication; this
was suggested by an increase of antibody titers to a viral struc-
tural protein but no change in antibody titers to a viral latent
protein as CD4
⫹
levels decreased (193). This study also dem-
onstrated that KSHV seroconversion following HIV-1 sero-
conversion carries a higher risk for KS development than does
KSHV seroconversion prior to HIV-1 seroconversion (193). In
AIDS-associated KS patients in Italy, a 5.4-fold increase in
detectable KSHV DNA in plasma was associated with patients
having more than 50 HIV-1 RNA copies per ml of plasma;
however, a 7.24-fold increase in detectable KSHV DNA in
plasma was correlated with a higher, not a lower, CD4
⫹
cell
count (498). Similarly, Quinlivan et al. established real-time
PCR as a sensitive, specific method for KSHV detection in
PBMCs and demonstrated that the KSHV burden in PBMCs,
but not CD4
⫹
counts or HIV-1 RNA, was predictive of KS
outcome (410).
Sexual transmission of KSHV in the HIV-infected popula-
tion is supported by numerous data, and sexual behavior is the
most significant epidemiologic variable in AIDS-associated KS
(325). The risk of developing KS is ⬎10,000 times higher in
HIV-infected homosexuals than in the general population
(186). Conversely, hemophiliacs and injection drug users with
AIDS have historically had a markedly reduced KSHV preva-
lence and KS risk (38). Furthermore, the disease is 300 times
more frequent in homosexual and bisexual men than in other
immunosuppressed individuals (38). Sexual exposure is the
strongest risk factor for KSHV infection, and a history of STDs
prior to KS diagnosis is strongly associated with KSHV sero-
positivity (325). Women with AIDS have a fourfold-increased
risk of KS if their partners are bisexual rather than addicted
intravenous drug users (38).
Recent studies have suggested that orogenital rather than
anogenital sex may be the most significant behavioral risk fac-
tor for KSHV infection. Although receptive anal intercourse
was a KSHV risk factor among homosexual men in the Am-
sterdam Cohort Study (336), participation in orogenital sex
was a predictor for KSHV seroconversion (140). In the period
from 1985 to 1996, the HIV-1 seroconversion rate in that
population was declining while the KSHV seroconversion rate
remained relatively steady. These trends were accompanied by
a decreasing percentage of cases of unprotected anal sex with
a decreasing number of partners. However, virtually 100% of
the population reported at least one occurrence of unprotected
oral sex in every year of the study, with an increasing number
of partners over that period. Studies of three different popu-
lations with high STD and AIDS prevalences in San Francisco
agree with this behavioral risk factor, suggesting that insertive
oral sex may be the highest risk behavior, as inferred from the
easy detection of KSHV in saliva but a low viral load in semen
(386). In agreement, many studies have found KSHV in the
saliva of seropositive patients (45, 266, 392, 481, 513), and
KSHV has been detected in prostate tissue and the male uro-
genital tract (132, 348, 481, 483); detection of KSHV in the
ejaculate has been reported but remains controversial (255,
300, 348, 396, 481; P. Gupta, M. K. Singh, C. Rinaldo, M. Ding,
H. Farzadegan, A. Saah, D. Hoover, P. Moore, and L. Kings-
ley, Letter, AIDS 10:1596–1598, 1996). Taken together, these
studies affirm a route of sexual transmission for KSHV that
differs significantly from that for HIV-1. Indeed, 474 HIV-
negative men who have sex with men were monitored for 1
year in Seattle and showed a higher seroconversion for KSHV
(3.8/100 person-years; similar to that for herpes simplex virus
type 1) than for HIV-1 (74).
The increasing and successful use of highly active antiretro-
viral therapy (HAART) has afforded important clinicoepide-
miological insights into the reversal of AIDS and the conse-
quent fate of KSHV pathogenesis. Widespread HAART has
resulted in a yearly declining trend in AIDS-related deaths in
the United States since 1996 (77a). In agreement, the Centers
for Disease Control and Prevention has reported an 8.8%
annual decline in KS incidence in the United States between
1990 and 1998 (239). In raw numbers, a cancer surveillance
program of the general population in San Francisco has re-
corded a dynamic incidence of KS over the past 30 years: in
1973 (prior to AIDS), 0.5 KS case per 100,000 people per year
was reported; during the AIDS peak from 1987 to 1991, this
value increased to 31.1 to 33.3, but then sharply declined to 2.8
in 1998 (post-HAART) (146). Others predict further reduc-
tions in KS incidence (501). Reduced KS risk has been asso-
ciated with triple, but not single or double, therapy (239), and
one study showed complete KS remission within 6 months of
HAART initiation in 9 of 10 early-stage AIDS-associated KS
patients (387). Tam et al. calculated an 81% reduction in risk
of death for AIDS-KS patients treated with HAART; the con-
trol group consisted of AIDS-KS patients who received no
therapy or single or double therapy (495). HAART was also
shown to be efficacious if initiated following KS diagnosis
(495).
There is clear evidence that immune restoration due to
HAART is one key to improving the prognosis of AIDS-KS
patients. Over 52 weeks of HAART, 19 AIDS patients dem-
onstrated increased CD4
⫹
counts, increased CD8
⫹
anti-
KSHV cytotoxic T-lymphocyte (CTL) activity, and increased
anti-KSHV antibody titers (534). This immune restoration was
associated with decreased HIV-1 and KSHV viral loads in the
blood, and patients without KS at baseline experienced a
greater decrease in KSHV load than did patients with KS.
178 DOURMISHEV ET AL. MICROBIOL.MOL.BIOL.REV.

However, patients with KS had a greater number of CTLs
specific for lytic viral antigens, suggesting that replication of
KSHV is faster in patients with KS than in infected patients
without KS. HAART also restores anti-PEL natural killer
(NK) cell lytic activity in AIDS-associated KS patients (469).
This immune restoration is associated with loss of detection of
KSHV DNA in PBMCs in these patients. At baseline, high
CD4
⫹
counts and undetectable KSHV DNA in PBMCs are
predictive of HAART success in KS regression (395).
HAART may reverse KS progression not only by inhibiting
HIV-1 replication but also by exerting direct effects on KS
tumors. The HIV-1 specific protease inhibitors (PIs) indinavir
and saquinavir have recently been demonstrated to inhibit an-
giogenic lesion formation by primary AIDS-associated KS cells
and injected basic fibroblast growth factor (bFGF) in nude
mice (459). The PIs were also antiangiogenic in chorioallantoic
membrane (CAM) assays and blocked the invasion of modified
human umbilical vein endothelial cells (HUVECs) in in vitro
assays. Mechanistic studies demonstrated that the PIs inhibited
cleavage and activation of matrix metalloprotease-2 (459), a
protein that is highly expressed in human KS lesions (502) and
is required for basement membrane degradation prior to en-
dothelial cell invasion; interestingly, the PIs did not directly
inhibit the cleavage of matrix metalloprotease-2 but instead
acted upstream.
As expected, lack of response to HAART is associated with
AIDS-associated KS progression: 2 of 14 non-HAART re-
sponders among Italian AIDS-associated KS patients failed to
show a reduction in HIV-1 RNA load or an increase in CD4
⫹
numbers; consequently, their KS progressed (77). Nonre-
sponders also failed to recover NK activity with respect to cells
latently infected with KSHV (PEL cells) and failed to clear
KSHV from PBMCs (469). A case report clearly demonstrated
the inverse relationship between HAART and KS: after 6 years
of antiretroviral therapy, including 3 years of HAART, KSHV
DNA became undetectable in PBMCs (388). However, a 2-
month interruption of HAART in this patient resulted in a
rapid rebound of KSHV seropositivity and viral DNA load.
HAART was subsequently reintroduced, and KSHV periph-
eral DNA became undetectable after 4 months. Considering
only current therapies, the fate of AIDS-associated KS in du-
ally infected patients will arguably depend on the continued
success of HAART treatment.
Endemic (African) KS
Prior to the emergence of HIV, African KS was an endemic
disease that affected mainly two age groups: young men with an
average age of 35 years and children with an average age of
3 years (519). In Cameroon from 1986 to 1993, the latter form
accounted for about 4% of childhood cancers (247). The high-
est prevalence of African KS prior to AIDS was found in a
broad band crossing equatorial Africa, with particularly high
rates in northeastern Zaire and western Uganda and Tanzania.
This geographic pattern conformed to areas of frequent podo-
coniosis, a lymphatic disease of the legs presenting as lymph-
edema etiologically associated with chronic, barefoot exposure
to volcanic soils (548). In podoconiosis, microparticles of silica
dust in these soils penetrate the skin of the foot during bare-
foot walking and are then taken up by the lymphatics and cause
localized inflammation and lymphedema. Aluminosilicates are
cytotoxic to macrophages in animal models (168) and may
contribute to localized immune suppression; this had been
proposed as an environmental contributor to African KS (548).
However, KS on that continent has evolved to an epidemic
magnitude as the spread of the AIDS pandemic has exacer-
bated the already elevated prevalence of endemic KS. Current
clinicoepidemiologic studies of African KS as an independent
disease entity are thus virtually impossible in most contempo-
rary African populations.
Clinically, KS in Africa is more frequent in children (247,
526, 547) and females than anywhere else worldwide and oc-
curs in four forms (216). One form is similar to classic KS in its
course but strikes young adults. The other three forms are
more aggressive and are similar to AIDS-associated KS in their
progression: one of these, however, remains cutaneous with
local tissue invasion, while another occurs most often in young
children with a mean age of 3 years, is aggressive with visceral
progression, but often lacks the cutaneous involvement (216).
In Tanzania, the highest incidence of pediatric KS was in
children younger than 5 years, possibly reflecting a low resis-
tance to KSHV infection (14). Among children in Africa, there
are age-specific discrepancies in clinical course: comparing
Zambian children over the age range of 3 to 15 years, the
younger patients presented primarily with lymphadenopathy
while the older patients had cutaneous involvement (247).
A comparison of the prevalence of KS prior to AIDS with its
prevalence during the AIDS pandemic in Africa clearly dem-
onstrates the brutal potentiation of KS by HIV-1 coinfection.
In the pre-AIDS periods from 1954 to 1960 and 1968 to 1970
in Uganda, KS was diagnosed in 6.4 to 6.6% of male cancer
patients, respectively, with no female cases. However, in 1989
to 1991, KS prevalence in male cancer patients rose to 48.6%
(incidence of 30.1/100,000), becoming the most frequently re-
ported cancer in men, while prevalence in female cancer pa-
tients climbed to 17.9% (incidence of 11.0/100000) (519). KS is
also the leading cancer in Zimbabwe, comprising almost 33%
of cancers there (102), and accounts for 15% of male cancers
in Zaire (376). Since the emergence of AIDS, the incidence of
KS in children has increased 40-fold in Uganda (547), while in
South African women it has increased at a higher rate than in
men: the male-to-female ratio has decreased from 7:1 in 1988
to 2:1 in 1996 (471). Paradoxically, calculations of the relative
risks of KS in HIV-1-positive patients in Rwanda, Uganda, and
South Africa were 35, 62, and 54, respectively, much lower
than that in the United States and Europe (approximately
300); Sitas and colleagues hypothesized, however, that the cu-
mulative risk relative to the West was similar due to the higher
pre-AIDS prevalence of KS in Africa (472). In the rare coun-
tries where the KS incidence has not dramatically changed
since AIDS, the progression of the disease remains dramati-
cally altered; for example, in Tanzanian children, the overall
incidence and the ratio of KS in boys to girls (about 5:1) have
remained consistent pre- and post-AIDS, but the anatomical
distribution now typically resembles that of more aggressive
KS (14). Overall, while the prevalence of KS within the HIV-
positive community has skyrocketed, even the prevalence of
KS in the HIV-negative, “contemporary”-endemic population
has risen.
Despite clinical heterogeneity, all forms of KS in Africa are
VOL. 67, 2003 KSHV MOLECULAR GENETICS 179

associated with KSHV infection (62, 92, 104, 176, 290). HIV-
1-positive South African patients have shown higher titers of
antibodies to KSHV latent proteins than have HIV-1-negative
patients and have had a higher risk of developing KS (470).
KSHV seropositivity in Africa is quite heterogeneous geo-
graphically, ranging from 36 to 100% in different countries and
populations in sub-Saharan Africa (reviewed in reference 93).
Similar to the situation for U.S. populations, numerous studies
support the inference that KSHV was highly prevalent preced-
ing the emergence of HIV-1; nonetheless, these studies affirm
the critical role of HIV-1 as a cofactor in African KS progres-
sion. For instance, the age-specific prevalence of KSHV in
Uganda has been virtually unchanged over the period of enor-
mous increases in KS incidence during the emergence of AIDS
(129). Interestingly, studies of Gambians suggest that those
coinfected by HIV-1 have a 12.4-fold higher risk of KS devel-
opment than those coinfected by HIV-2, even with equal se-
roprevalence for KSHV in the two groups (22).
Unlike the strict correlation between KSHV prevalence and
disease incidence in Western countries, however, different Af-
rican populations that have similar KSHV prevalences may
have significant variations in KS incidences. For example, a
cohort of 249 Ethiopian immigrants to Israel had KSHV sero-
prevalence typical of many African countries: 39.1% in HIV-
negative people and 57% in HIV-positive people, yet none
developed KS (201).
The endemic pattern of KS in Africa, especially the high
prevalence in childhood, reflects the occurrence of primary
KSHV infection before puberty (including during infancy). In
fact, in many African populations, seroprevalence reaches
adult levels during adolescence. During the AIDS epidemic in
Uganda, 37% of children younger than 5 years were seroreac-
tive to at least one KSHV antigen, and this proportion in-
creased to 58% in children 5 to 9 years old and leveled off at an
adult-like level of 49% after the age of 20 years (331). These
authors noted that this pattern of prepubescent acquisition of
KSHV infection is similar to that seen for EBV and hepatitis B
virus in many African countries. A pre-AIDS population in
Uganda showed a similar trend, but the overall KSHV sero-
prevalence was higher, reaching 57% at 11 to 13 years old, 89%
at 14 to 17 years old, and 84% at 23 to more than 50 years old
(129). In Cameroon, seroprevalence increases gradually during
childhood to 48% by the age of 15 years, nearly equal to the
seroprevalence among pregnant women (54.5%) living in sim-
ilar areas (180). Similarly, the seroprevalence of anti-lytic an-
tibodies to KSHV in Egyptian children increased from 16.6%
at younger than 1 year to 58% at older than 12 years; however,
there was no consistent increase in the levels of antibodies to
latent KSHV proteins (16).
This early appearance of KSHV in children in Africa sug-
gests that transmission is primarily nonsexual, possibly occur-
ring from mother to fetus (142), with clear evidence of intra-
familial and horizontal spread. Indeed, a significant risk factor
for seroconversion of a child is having a KSHV-infected moth-
er: 42% of children born to seropositive mothers were also
seropositive when tested between 0 and 14 years of age, while
only 1% of those born to seronegative mothers were seropos-
itive (53). In this study, the potential for vertical transmission
of KSHV was suggested, since one of four infants younger than
18 months born to an infected mother was also seropositive.
However, many studies argue that a majority of seropositive
infants acquire maternal immunoglobulins passively from their
mothers, with vertical transmission of virus remaining possible
but rare. For example, 83% of Zambian infants were KSHV
seropositive if born to a mother who was also seropositive;
however, only 3% of the infants had detectable KSHV DNA in
their PBMCs (322). Frequent studies have demonstrated a loss
of KSHV seropositivity in children after infancy. A total of
46% of infants in Cameroon were seropositive prior to 6
months of age, but only 13% were seropositive between 7 and
12 months of age (180). A total of 25 and 58% of Ugandan
infants were seropositive in two studies, but at 1 year old, none
were seropositive (129, 331), and 14 infants born to HIV- and
KSHV-positive mothers were also seropositive before 1 month
of age, but all had lost seropositivity by 6 months old (316).
Outside of Africa, similar results have been reported for 32
HIV-1-infected mother-infant pairs of Ghanian and Italian
descent (67).
Childhood transmission in Africans therefore seems to fol-
low mostly horizontal patterns, with a few vertical instances.
An increasing risk of KSHV infection in Egyptian children
(detected by serologic or DNA testing) is proportional to hav-
ing contact with more than two other children or with having
siblings (18). A unique perspective on intrafamilial transmis-
sion of KSHV has come from a study of the Noir-Marron of
French Guiana, who are descendants of African slaves (403).
The seroprevalence rate there reached a plateau of about 15%
by 15 years old and then rose sharply to about 30% after the
age of 40 years. Intrafamilial correlations of seropositivity were
calculated and showed a high correlation between (i) children
and their mothers, but not their fathers, and (ii) children and
their siblings. The mother-child correlation was even greater
for children younger than 10 years old.
Other evidence suggests that transmission in Africa can also
occur by population-delimited routes. Blood-borne transmis-
sion has been suggested for an Eritrean tribe that has detect-
able KSHV DNA in serum and practices ritual skin piercing
(148). In Djibouti, although street prostitutes have a vastly
higher rate of HIV-1 infection than do women who are not
prostitutes, the two populations have a very similar KSHV
seroprevalence, suggesting different risk factors for transmis-
sion of KSHV and HIV-1 there (323). In South Africa and
Kenya, seroprevalence does not plateau during puberty, but
continues to rise throughout life (470), similar to that of hep-
atitis B virus (352).
Iatrogenic KS
Iatrogenic KS represents an additional clinicoepidemiologic
peculiarity of KSHV infection that presents either chronically
or with rapid progression (216). The induction of iatrogenic KS
by immunosuppressive therapy and its subsequent regression
on removal of immunosuppression provided some of the ear-
liest clinical recognition of the reversibility of KS (216). Iatro-
genic KS shows extreme ethnogeographic associations, occur-
ring in only about 0.4% of transplant patients in the United
States and Western Europe (157, 398) but in about 4.0 to 5.3%
of renal transplant patients in Saudi Arabia (399, 411). Strik-
ingly, KS represents 87.5% of posttransplantation neoplasia in
Saudi Arabia (412), and a recent study found KS in 80% of
180 DOURMISHEV ET AL. MICROBIOL.MOL.BIOL.REV.

posttransplantation cancers in Turkey (141). In the latter
study, KS developed within 1 year in 46% of those cases. The
high frequency of iatrogenic KS in Saudi Arabia reflects the
7% endemic seroprevalence of KSHV in healthy Saudi donors
or patients with non-KS malignancies (412). Interestingly,
transplant-associated KS is seen predominantly in kidney allo-
graft recipients and not other solid-organ or bone marrow
transplant recipients (230); kidney recipients have a greater
than two-fold-higher seroprevalence than those at low risk of
KS in France (85). Although Andreoni et al. found that 21.4%
of liver recipients, but only 8.6% of kidney recipients, serocon-
verted for KSHV, the risk of KS was higher in the kidney
transplant recipients. In fact, 75% of those who progressed to
KS were KSHV seropositive prior to transplantation (17).
Differences in immunosuppressive therapy might favor KSHV
reactivation in the kidney recipients. KS has historically been
seen more often in patients whose treatment includes cyclo-
sporine (397), a drug that can reactivate KSHV from latency to
lytic replication in tissue culture (225). Likewise, remission of
iatrogenic KS on cessation of immunosuppression is the norm.
A recent study in Turkey demonstrated that 8 of 11 iatrogenic
KS patients showed complete regression of visceral and cuta-
neous KS within 6 months of cessation of cyclosporine (141). A
recent case study showed regression of cutaneous and mucosal
KS after cessation of immunosuppressive therapy in a kidney
allograft recipient; however, KSHV DNA persisted in the re-
gressed lesions (367).
A more comprehensive association between KSHV infection
and kidney disease was suggested by a study that demonstrated
93% KSHV seroprevalence in Saudi patients with posttrans-
plantation KS, 28% in transplant patients without KS, and
29% in patients with end-stage renal disease (412). In fact,
end-stage renal disease and kidney dialysis were found to be
risk factors for KSHV seropositivity (11). In a case report,
Sarid et al. proposed that the kidney may be a site of latent
KSHV, describing two KSHV-seronegative patients who re-
ceived infected kidney tissue, seroconverted, and subsequently
developed KS; a third patient was weakly seropositive prior to
transplantation and seemed to reactivate a latent KSHV infec-
tion within 1 year (445).
Many data support the inference that most iatrogenic KS
patients are KSHV positive prior to transplantation, suggesting
that reactivation of latent viral infection leads to disease. One
study demonstrated that 10 (91%) of 11 kidney or heart trans-
plant patients who developed iatrogenic KS were KSHV pos-
itive prior to their procedures (390). Cattani et al. calculated a
relative risk of 34.4 for kidney recipients who were KSHV
seropositive versus seronegative prior to transplant (76). In a
transplant cohort in Paris, 8% of 400 kidney transplant recip-
ients were KSHV positive prior to transplantation: 28% of
these developed KS within 3 years of their procedures, but
none of those who were seronegative prior to receiving the
kidney developed KS (165). Females, Africans, or those with
severe bacterial and/or Pneumocystis carinii infection had the
highest risks for KS development in that population. Female
sex as a risk factor was also found in an Italian cohort and
reflected a higher seroprevalence in women than in men prior
to transplantation (17). Four of five transplant recipients in
Pittsburgh, Pa., who were seropositive prior to transplantation
showed a greater than fourfold increase in antibody titer to
lytic KSHV antigens (235), supporting the inference of viral
reactivation in seropositive organ recipients. Although less fre-
quent, seroconversion following transplantation suggests that
seronegative recipients can be infected by KSHV from the
donated organ (390).
OTHER KSHV-ASSOCIATED DISEASES
As discussed above, epidemiologic proof that KSHV is the
etiologic agent in all clinical forms of KS has come largely from
universal detection of viral nucleic acids in diseased but not
healthy tissue and from direct correlations between detection
of KSHV, risk of developing KS, and progression to disease.
Epidemiologic proof that KSHV infection is etiologically as-
sociated with diseases other than KS in the HIV-negative pop-
ulation, however, has been more elusive. For example, in the
absence of AIDS, a second KSHV-associated cancer, PEL, is
extremely rare, thus frustrating attempts to establish statisti-
cally significant correlations with KSHV infection. The etio-
logic connection between PEL and KSHV infection has thus
relied on the HIV-positive population. Although PEL is rare
even in AIDS patients, constituting only 0.13% of all AIDS-
associated lymphomas in the United States, previous KS diag-
nosis confers an increased risk of PEL relative to all other
AIDS-associated NHLs (332). Nonetheless, the term “classic
PEL” has been coined by Ascoli et al. to describe 20 cases of
PEL in HIV-negative elderly patients of Eastern European/
Mediterranean and Jewish descent (25), two populations with
high seropositivity for KSHV and elevated incidence of classic
KS.
KSHV infection may also be associated with heart disease.
Classic PEL patients show a high incidence of congestive heart
failure, a condition shared with classic KS patients (25). In-
versely, Italian patients with cardiovascular disease have a
higher KSHV seroprevalence than does the general population
(72). Furthermore, HIV-1-positive patients with KS had an
increased odds ratio of 3.35 for developing atheroma relative
to those without KS (195). In animal models of heart disease,
two non-human gammaherpesviruses, murine herpesvirus 68
and bovine herpesvirus 4, both accelerate atherogenesis (7,
305), with the former requiring hyperlipidemia. Furthermore,
macaques coinfected with rhesus rhadinovirus and simian im-
munodeficiency virus frequently develop large-vessel arteritis
(321). Interestingly, a KSHV-encoded chemokine, viral mac-
rophage inflammatory protein 1 (vMIP-1), is chemotactic for
endothelial cells expressing its receptor, CC chemokine recep-
tor 8 (CCR8) (214). CCR8 is itself expressed on KS spindle
cells, as well as on endothelial cells found in atherotic plaques;
thus, vMIP-1 may be mimicking the action of its cellular coun-
terpart, the chemokine I-309, which is released by endothelial
cells in response to the atherogenic apolipoprotein A (214).
Apart from KS and PEL, KSHV is also variably found in
patients with multicentric Castleman disease (MCD) (111,
479), a rare angiolymphoproliferative disorder. More than
90% of patients with AIDS and MCD are infected with KSHV,
while no more than 40% of HIV-seronegative MCD patients
are infected (196). In MCD lesions, LANA-1 is found in im-
munoblasts around the hair follicle (142). The ratio of lytically
infected to latently infected cells in MCD is much greater than
in PEL or KS, suggesting that the pathogenic role of KSHV in
VOL. 67, 2003 KSHV MOLECULAR GENETICS 181

these different diseases might be attributable to different gene
expression programs of the virus (196, 249, 380, 389, 431, 482,
499). Furthermore, the occurrence of MCD increased the risk
of subsequent NHL development in an HIV-positive popula-
tion, hypothetically by (i) clonal expansion of a lesional MCD
cell or (ii) oncogenic stimulation of a pre-NHL cell in the
MCD microenvironment (379). In this study, in fact, 9 of 14
subjects had concurrent KS, MCD, and PEL.
Other clinical conditions have also been connected with
KSHV infections, including angiosarcoma (333), angiolym-
phoid hyperplasia with eosinophilia (211), skin carcinomas
(squamous cell carcinoma and Bowen disease) in immunosup-
pressed individuals (416), sarcoidosis (131), multiple myeloma
(MM) (427), pemphigus vulgaris and pemphigus foliaceus
(337, 338), AIDS-associated immunoblastic lymphoma (149),
primary central nervous system lymphoma (190), posttrans-
plantation lymphoproliferative disorders (244, 329), and
pulmonary inflammatory myofibroblastic tumor (191). A con-
dition of immunocompetent patients named germinotropic
lymphoproliferative disorder, in which KSHV- and EBV-coin-
fected B-cell plasmablasts invade lymphoid follicle germinal
centers, has also been described (139).
The etiologic connection between KSHV infection and most
of these disorders, however, has not been confirmed: these
disorders include skin carcinomas in noncompromised patients
(2; N. Dupin, I. Gorin, J. P. Escarde, V. Calvez, M. Grand-
adam, J. M. Huraux, and H. Agut, Letter, Arch. Dermatol.
133:537, 1997), angiosarcomas (299), MM (117, 328, 497), my-
cosis fungoides (217), paraneoplastic pemphigus, pemphigus
vulgaris, and pemphigus foliaceus (107).
The connection between KSHV infection and multiple mye-
loma (MM) has been extremely controversial. KSHV DNA has
been detected more frequently in bone marrow stromal cells
and fractionated PBMCs enriched for dendritic markers in
MM patients than in healthy controls (33, 39, 427). However,
only 1 of 15 serologic studies (173) has demonstrated increased
anti-KSHV reactivity associated with MM patients (reviewed
in reference 496), and MM patients with strong anti-EBV CTL
responses unanimously failed, nonetheless, to demonstrate
CTL responses to KSHV open reading frame 65 (ORF65) or
ORF73 proteins (57). Furthermore, the specific detection of
KSHV DNA in MM but not healthy (control) donors has not
been confirmed for all populations studied (496).
CLINICAL DETECTION OF KSHV, AND ESTIMATES OF
INFECTION IN THE GENERAL POPULATION
Serologic methods have remained the most sensitive ap-
proach for detecting exposure to and infection by KSHV since
their first use in cementing the etiology of KS. The genesis of
KSHV seroepidemiology came from studies employing easily
cultured, uniformly KSHV-infected B-lymphoma cell lines
(PEL cell lines) as the source of viral latent antigen (176, 250,
355). PELs have since served as the primary target for indirect
immunofluorescent-antibody (IFA) approaches to measure
levels of antibody to both latent and lytic KSHV proteins in
clinical serum samples and have been successfully used as a
source of protein extract in confirmatory immunoblots (93,
175, 176, 250, 290, 343, 355, 474, 545). When clinical sera are
incubated with latently infected PEL cells or nuclei, they are
scored positive for previous viral exposure if a characteristic
punctate nuclear stain is observed (176, 250, 355). Very elegant
experiments identified this putative latency-associated nuclear
antigen (LANA-1 or LNA-1) as the product of the KSHV
ORF73 gene, which is sufficient to generate the punctate stain
when overexpressed in uninfected 293HEK (human embryonic
kidney) cells (252, 253, 355, 417). Among other functions (dis-
cussed below), LANA-1 maintains the latent viral genome as a
stable episome (30, 115). KSHV virions purified from induced
PEL cells have been used as targets in enzyme-linked immu-
nosorbent assays (ELISAs) (94), and publication of the KSHV
sequence has allowed rational design of whole-protein and
peptide-based ELISAs (87, 304, 413, 450, 466).
The plethora of diagnostic KSHV methods has revealed that
the choice of viral antigen can profoundly influence the quan-
titation of seroprevalence (93, 414). While the prevalence of
KSHV in populations with KS (and PEL) is consistently mea-
sured at 90 to 100%, the frequency of the virus in the general
population has been reported to vary over a wide range. The
relative seroprevalences calculated for geographically distinct
populations remains consistent if similar antigens are com-
pared between groups (414). However, intrageographic mea-
surements are typically higher when the prevalence of anti-lytic
antibodies, as opposed to anti-latent antibodies, is measured
(93). For example, 20% of one U.S. cohort of blood donors
were seropositive for anti-lytic antibodies but none of the do-
nors were seropositive for anti-latent antibodies (290). Like-
wise, a Swedish cohort showed 33% anti-lytic but only 6% anti-
latent seroreactivity (147). In the general population of the
countries of Western Europe, seroprevalence ranges from 2.0
to 4.0% when measured by latent IFA assay, which corre-
sponds to the frequency of KS in those countries (142).
Such discrepancies in measures of seroprevalence have fo-
mented controversy concerning the need to screen donated
blood for KSHV. A study of blood samples from blood donors
in Texas, using both anti-latent and anti-lytic tests, showed an
overall KSHV seroprevalence of 15%, much higher than an-
ticipated from samples in the general American population
and reflective of a possible need for screening blood donors in
America (28). Blood-borne transmission was also supported by
a prospective study of HIV-infected women in the United
States that identified intravenous drug use as the most signif-
icant risk factor for KSHV seropositivity, with the frequency of
intravenous drug use being proportional to the risk (70). How-
ever, two studies using identical anti-latent IFA assays to test
both donor and recipient blood demonstrated no transmission
of KSHV from seropositive donors to seronegative recipients
(150, 383). Determinations of the age association with KSHV
transmission also suffer from the same discrepancies: healthy
children outside Africa have shown reactivity to lytic antigens
but are often seronegative for latent antigens (290, 458).
Some experts have proposed that the seemingly increased
sensitivity of anti-lytic viral antigen methods may come at the
expense of decreased specificity (93). However, recent studies
using recombinant KSHV proteins suggest that the lytic glyco-
protein K8.1 is more sensitive and specific as a diagnostic
antigen than is the latent LANA-1 protein (229). The proteins
were expressed by infection of BHK-21 cells with recombinant
Semliki Forest virus vectors, and the study also revealed that
recombinant K8.1 was 100% specific compared to PEL-based
182 DOURMISHEV ET AL. MICROBIOL.MOL.BIOL.REV.

lytic assays. An ELISA based on a four-branch, multiple anti-
genic peptide derived from K8.1 likewise demonstrated high
sensitivity and specificity (282).
A study that comprehensively compared multiple diagnostic
methods suggested that a combination of anti-latent and anti-
lytic assays will provide the best diagnostic procedure for de-
tecting KSHV serologically. In a search for the “gold standard”
for KSHV detection and KS prediction, Schatz et al. compared
six PEL-based IFA assays and eight ELISAs (with whole-pro-
tein and optimized peptide antigens) to detect both latent and
lytic antigens and found that a dual, anti-lytic/anti-latent IFA
assay showed a good balance of specificity (89.1%) and sensi-
tivity (94.9%) (450). A whole virus ELISA followed by a PEL-
based IFA assay also showed specific and sensitive results (73).
Of course, PCR-based methods have also been employed
successfully to detect KSHV. In clinical samples, viral copy
number is highest in affected skin, followed by saliva and
PBMC (277), and PCR of KS lesions (among other tissues)
gives the highest specificity for diagnosis (124, 277). The de-
tection of KSHV DNA in both PBMCs (277, 395) and plasma
suggests that both cell-associated and plasma viremia may con-
tribute to viral dissemination. However, one study detected
KSHV DNA unanimously in the plasma of all 14 participants
but in the PBMCs of only 1 donor, suggesting that plasma
viremia is a better indicator of active replication (498). Overall,
the relatively high titers of KSHV in peripheral blood but the
virtual lack of evidence for blood-borne transmission suggests
that blood is an inefficient tissue for transmission.
In general, many studies agree that serologic testing for
KSHV is far more sensitive for detecting infection than is PCR,
especially during latency. Early studies estimating the time
from detection of infection to appearance of KS strikingly
demonstrate this difference. Kaplan-Meier estimates showed
that HIV-1-positive men who were seroreactive to LANA-1 at
baseline had a 49.6% probability of developing KS 10 years
later (i.e., a median time of 10 years) (325). However, quanti-
tation of KSHV DNA in peripheral blood estimated the me-
dian time to KS as 3.5 years (530), suggesting that the detection
of viral DNA required a higher viral burden and a more ad-
vanced infection than detection of seroreactivity. Comparison
of the sensitivity of the two detection methods has thus pro-
vided insight into relationships between the stage of infection
(primary, latent, and lytic) and KSHV pathogenesis. In gen-
eral, a patient who is seropositive without detectable DNA in
the peripheral blood is considered to be carrying a latent
KSHV infection (175, 466), whereas seronegative patients who
are DNA positive are hosting an early primary infection (18,
523); by inference, those who are positive by both tests have a
reactivated infection or a late primary infection.
PRIMARY INFECTION BY KSHV
Unlike reactivation of KSHV from latency, virtually all cur-
rent studies have demonstrated that primary infection by the
virus is not associated with significant concurrent morbidity in
immunocompetent populations. The vast majority of prospec-
tive studies of primary KSHV infection have evaluated popu-
lations of children. Of 53 Zambian children presenting with
their first febrile illness and accompanying respiratory symp-
toms, 4 had KSHV DNA detectable by semiquantitative PCR
in whole blood at levels suggesting active viremia (247). Two of
the KSHV-positive children were negative for HHV-6 (a com-
mon cause of childhood fever), suggesting that their symptoms
may have been attributable to a primary KSHV infection. Of
56 Japanese children who presented with acute febrile illness,
including many younger than 2 years, 36 (64%) had PCR-de-
tectable KSHV DNA in PBMCs (although these authors im-
plicated bacterial infections as the pathogenic culprits) (257).
Of 81 Egyptian children with acute febrile illness, 6 were
KSHV seronegative but had KSHV DNA in plasma or saliva,
suggesting primary infection; 5 of the 6 also showed a cutane-
ous craniocaudal maculopapular rash (18). Primary infection
can (rarely) occur vertically, and viral DNA has been detected
in Zambian infants within their first 24 h of life (322).
Measurements of seroconversion in infants to assess primary
KSHV infection are problematic due to the observation that
infected mothers passively transfer anti-KSHV antibodies to
their offspring (see discussion, above) (67, 129, 180, 316, 331).
However, seroconversion can be temporally compared to other
clinical parameters and symptoms in order to monitor the
natural history of KSHV infection in adults. The first prospec-
tive study of primary KSHV infection in immunocompetent
adults found that symptoms were similar to those of primary
infection during childhood. In a cohort of HIV-negative ho-
mosexual men in Pittsburgh, 5 of 108 seroconverted for KSHV
(as determined by measuring anti-lytic antibodies) over a 15-
year period (523). Their symptoms were fever, diarrhea, fa-
tigue, localized rash, and lymphadenopathy; the first detection
of KSHV DNA preceded seroconversion and coincided with
the appearance of a broad CTL response to at least three viral
lytic proteins in each patient. The CTL response then declined
over several years, while viral DNA was persistent and sporad-
ically detectable. Unlike mononucleosis following primary
EBV infection, there was no detectable expansion of CD8
⫹
cell numbers during these episodes of KSHV infection. The
low KSHV seropositivity in the general population of the
United States and western countries suggests that primary in-
fection is not acquired during childhood but, instead, is ac-
quired in sexually mature adults in those regions.
Symptoms of primary KSHV infection in immunosup-
pressed hosts obviously are more difficult to directly attribute
to infection by KSHV. However, a case report of an HIV-1-
positive male noted seroconversion to KSHV 5 weeks prior to
the sudden onset of symptoms including fever, arthralgia, cer-
vical lymphadenopathy, and splenomegaly (381). PCR of the
cervical mass specimen revealed KSHV DNA, vascular hy-
perplasia, and “intense” activation and proliferation of B
cells and was negative for EBV gene expression. The febrile
episode was prolonged but resolved spontaneously within 2
months.
Primary infection by KSHV in different immunosuppressive
settings may lead to drastically different outcomes. In the Am-
sterdam cohort, patients who seroconverted for KSHV follow-
ing HIV-1 seroconversion experienced a greater relative risk
for progression to KS than did those who had seroconverted
for KSHV prior to HIV-1 infection (193). This suggests that
primary KSHV infection has a higher morbidity in those who
are HIV-1 infected than in those who reactivate a previous,
latent KSHV infection. However, primary KSHV infection
during iatrogenic immunosuppression following organ trans-
VOL. 67, 2003 KSHV MOLECULAR GENETICS 183

plantation carries a lower risk for KS than does reactivation of
a previous infection (235). It is plausible that these differences
in risk of KS following primary infection in the two populations
reflect drastically different titers of KSHV in the respective
inocula. For example, transmission in the homosexual popula-
tion is likely to be through saliva, a site of viral shedding that
contains high-titer KSHV, potentially from an infected person
who is immunosuppressed himself. Conversely, transmission
from immunocompetent organ donors is likely to occur via
blood, a tissue shown not to be a significant source of virus
(150, 383).
HISTOPATHOGENESIS OF KS AND
ITS RELATIONSHIP TO KSHV INFECTION
KS lesions contain a variety of cell types, including endothe-
lium, extravasated erythrocytes, infiltrating inflammatory cells,
and characteristic “spindle” cells (154). The spindle cells ex-
press markers of both endothelium and macrophages (364,
420, 505, 506). The spindle cell population in vivo may be
heterogeneous, especially early in KS disease: although a
marker specifictofibroblasts and macrophages is expressed on
100% of cultured KS spindle cells, it is expressed on only a
subset of cells in situ in KS specimens (465).
Whether spindle cells are derived from vascular or lymphatic
precursors has been controversial (143, 240, 270, 318, 529,
539). A new monoclonal antibody that recognizes an O-linked
sialoglycoprotein reinforces the argument for a lymphatic ori-
gin of these cells. This antibody stains (i) lymphatic, but not
vascular, endothelium in normal tissues and (ii) transformed
tissues of lymphatic but not vascular origin (241). In cutaneous
KS specimens, this antibody stains 100% of the spindle cells
from all pathologic stages of KS.
Extensive and aberrant neoangiogenesis in KS lesions is
accompanied by elevated levels of many cytokines, including
bFGF, interleukin-1 (IL-1), IL-6, IL-8, platelet-derived growth
factor, tumor necrosis factor (TNF), gamma interferon (IFN-
␥), and vascular endothelial growth factor (VEGF) (reviewed
in reference 154). Many of these cytokines are secreted by
spindle cells, are essential for spindle cell viability in culture,
and are themselves proangiogenic (152, 153, 370), suggesting
that maintenance of KS lesions in vivo is dependent on the
dysregulation of these growth factors in the tumor milieu.
Although elevation of the levels of these cytokines is detect-
able in the microenvironment of the tumor specimens and is
critical in KS pathogenesis, their concentrations in serum do
not necessarily correlate with KSHV status or KS incidence
(326, 426).
Early PCR-based studies confirmed that KSHV DNA is
specifically found in lysates only from affected tissue from KS
patients (12, 13, 90, 354, 449). The lymphotropism of KSHV
was demonstrated by PCR amplification of viral DNA from the
CD19
⫹
B-cell subset of PBMCs from AIDS-associated KS
patients (13, 340), as well as in PEL specimens (80, 161, 245,
267, 366), suggesting a mechanism by which the virus may
disseminate in the host. Active KSHV replication and in-
creased detection by PCR of viral DNA in the peripheral blood
are strongly correlated with (i) increased risk of progression to
KS (13, 530) and (ii) increased severity of pathogenic stage of
KS (47, 68); the peripheral viral load predicts the pathogenic
outcome of the infection (410). KSHV has also been found in
the neutrophil subset of PBMCs (285) and in circulating KS-
like spindle cells that express markers of both macrophages
and endothelium (60; M. C. Sirianni, S. Uccini, A. Angeloni, A.
Faggioni, F. Cottoni, and B. Ensoli, Letter, Lancet 349: 255,
1997).
Localization of KSHV DNA was demonstrated in clinical
specimens by PCR in situ hybridization (PCR-ISH); when us-
ing this technique in combination with immunohistochemistry
of tissue-specific protein expression, Boshoff et al. demonstrat-
ed specific KSHV signals in vessel endothelial cells and the
majority of spindle cells in cutaneous KS nodule specimens
(52). However, KSHV may also infect nonendothelial cells in
KS lesions: by using limited tryptic digestion, Foreman et al.
used in situ PCR to detect KSHV DNA in epidermal keratin-
ocytes in four of five KS lesions and eccrine (salivary) epithelia
in two of four KS lesions (161). Reverse transcription-in situ
PCR for KSHV nucleic acid has recently been used to suc-
cessfully differentiate KS from dermatofibromas and to di-
agnose KS in two of eight atypical vascular lesions (375).
Stamey et al. have demonstrated the successful use of real-time
PCR in detection and quantitation of KSHV in KS tissue
(481).
Expression of KSHV genes in clinical specimens from all
stages of histologically recognizable KS was revealed by ISH
with RNA probes (483). More than 85% of the spindle cells in
these tumors expressed transcripts from the latent kaposin
gene (540), encoding a transforming protein (362) that stimu-
lates the guanine-nucleotide exchange activity of cytohesin-1
upstream of mitogen-activated protein kinase pathways (263;
also see discussion below). About 10% of the spindle cells ex-
pressed both delayed early (nut-1/PAN) and late (major capsid
protein [MCP]) lytic transcripts suggesting that a minor pop-
ulation of tumor cells supports productive viral replication
(483). A later study used ISH with a more extensive array of
probes to confirm this relationship between latent and lytic
gene expression in KS tissue (489). Interestingly, two viral
genes encoding cytokine homologs (viral IL-6 [vIL-6] and
vMIP-1) and one encoding an anti-apoptotic protein (vBcl-2)
were expressed exclusively in cells supporting lytic viral rep-
lication, suggesting that the KSHV lytic cycle may directly
contribute to the pathogenesis of KS tumors (489). A similar
approach was used to detect viral lytic transcripts in lesional
monocytes, suggesting a second mechanism, exclusive of B
lymphocytes, for viral dissemination (46).
Immunohistochemistry also demonstrated that detection of
LANA-1-positive cells in KS lesions increases during progres-
sion to nodular KS (143). LANA-1 was detected in cells sur-
rounding slit-like vascular spaces and colocalized with cells
expressing a marker of lymphatic and precursor endothelium.
Using a catalyzed signal amplification method without PCR,
Reed et al. demonstrated that viral cyclin D was expressed in
the latently infected portion of lesional spindle cells and that
the number of staining cells increased with KS progression
(418). Viral cyclin D expression was also detected in keratin-
ocytes, eccrine epithelium, and the endothelium of the perile-
sional dermis (418).
184 DOURMISHEV ET AL. MICROBIOL.MOL.BIOL.REV.

HIV-1 in KS Histopathogenesis
Whether KS is a true malignancy, a reactive proliferation, or
both remains unclear. Thus, early KS seems to be a reactive
process of polyclonal nature, whereas it may progress in time
into a true sarcoma (154). The aggressiveness of AIDS-asso-
ciated KS implicates infection with HIV-1 as a necessary co-
factor in rapid KS progression; indeed, the time of KSHV
seroconversion until the onset of KS may be decades in classic
KS (78, 268), while it is estimated at 2 to 10 years in AIDS-
associated KS (175, 325, 425). In dually infected individuals, a
local and systemic increase in the level of Th1-type cytokines
seems to activate KSHV infection, resulting in increased viral
load, antibody titers, expanded cell tropism, and KS (488).
The HIV-1 Tat protein has been directly implicated in the
development of AIDS-associated KS. Tat induces KS-like le-
sions when overexpressed alone in transgenic mice (518) and
synergizes when coinjected with bFGF in nude mice (152).
Ex vivo studies demonstrate that extracellular Tat stimulates
monocyte-derived macrophages to secrete IL-8 and Gro␣, two
chemokines that are potently proangiogenic for endothelial
cells (284). Extracellular Tat also can sustain AIDS-associated
KS cell growth and survival (8, 32, 151) and exerts a biphasic
antiapoptotic effect on KS cells by (i) global induction of anti
apoptotic gene expression and (ii) activation of Akt kinase
activity (128). These functions of Tat are mediated by direct
interactions with VEGF receptor 2 (VEGFR-2) and insulin-
like growth factor (IGF) receptor I (128). Similarly, integrin
binding by extracellular Tat is responsible for many of its pro-
liferative and proangiogenic functions (reviewed in reference
511). KSHV virions also utilize integrins as receptors and ac-
tivate downstream signaling after binding target cells (4; also
see below), similar to other herpesviruses (464, 544). There-
fore, the interaction of both extracellular Tat and KSHV viri-
ons with integrins might represent a common mechanism for
priming a target cell physiologically for subsequent viral infec-
tion and pathogenic exacerbation.
HIV-1 also has direct effects on KSHV. Conditioned media
from T cells infected by HIV-1 induces lytic reactivation of
KSHV in PEL cells (339), as does replication of HIV-1 in PEL
cells (510). HIV-1 Tat and Vpr proteins can induce KSHV
gene expression, and, reciprocally, the lytic switch protein of
KSHV can synergize with Tat in activating the HIV-1 long
terminal repeat (LTR) (224).
TISSUE CULTURE MODELS OF ENDOTHELIAL
KSHV INFECTION
Critical proof of the transforming potential of KSHV came
from de novo infection of cultured bone marrow microvascular
endothelial cells and HUVECs. KSHV infection conferred
long-term survival of both cell types, and anchorage-indepen-
dent growth of the HUVECs (159). However, after long-term
passage, only 5% of HUVECs maintained KSHV DNA; unlike
primary cultures, the uninfected cells retained the ability to
respond to VEGF, suggesting that their survival was depen-
dent on paracrine VEGF provided by the infected cells of the
population (159). However, serial passage of telomerase-im-
mortalized microvascular endothelial cells infected de novo by
KSHV leads to complete loss of virus, similar to spindle cells
explanted from KS lesions, without growth transformation of
the cells (278). KSHV infection of cultured primary human
dermal microvascular endothelial cells (DMVECs) induces
colonies of latently infected spindle-shaped cells and can be
maintained only by diluting infected cultures into uninfected
cells (106). The inability to passage latently infected cells sug-
gested that the cultures were not transformed by KSHV (106);
however, KSHV infection could enhance the transformed phe-
notype of DMVECs stably expressing the human papilloma-
virus E6 and E7 proteins prior to KSHV infection (358).
Sakurada et al. demonstrated that induced PEL cells can very
efficiently transmit KSHV directly to HUVECs in culture, high-
lighting the potential for direct cell-to-cell transmission of
KSHV (441).
Primary endothelial cells isolated from (i) the dermis of
human neonatal foreskin (fMVDEC) or adult breast and (ii)
human uterine myometrium are all efficiently infected by pu-
rified KSHV (130). Cerimele et al. recently provided ex vivo
confirmation of the detection of KSHV DNA in nonendothe-
lial cells of KS lesions by demonstrating de novo infection of
primary human keratinocytes (79). Like infection of primary
HUVECs, the keratinocytes were transformed by KSHV but
did not maintain the viral genome (79).
Purified KSHV virions bind readily to the surface of B cells
and monocytes, but not T or NK cells, when mixed with pe-
ripheral blood specimens (130). Although the two cell types in
which KSHV DNA is most consistently observed in vivo, en-
dothelial cells and B lymphocytes, correspond to the sites as-
sociated most strongly with KSHV pathogenesis, the virus ex-
hibits a very broad host range in tissue culture (44, 79, 106, 159,
163, 358, 422, 514). By analogy to many other herpesviruses,
recent studies have demonstrated that heparan sulfate, a ubiq-
uitous cell surface molecule, can serve as a receptor for KSHV,
specifically interacting with the KSHV virion proteins K8.1 and
glycoprotein B (gB) (3, 5, 42, 521). A more restricted cell
surface protein, ␣
3
␤
1
-integrin, also functions as a KSHV re-
ceptor on human foreskin fibroblasts and human DMVECs
through interactions with gB (4). Independent cell surface
expression of gB, gH, and gL can mediate cell fusion between
heterologous, uninfected cells, suggesting that this model re-
creates both binding and postbinding functions in entry of
KSHV (400). However, similar to some endothelial models of
infection, virtually all cultured cells tested do not support “one-
step,” productive viral infection, nor are they permissive for
latent KSHV infection. Providing one potential explanation for
this shortfall, Friborg et al. have shown that 293 cells support
efficient replication of KSHV derived from KS lesions but not
from BCBL-1 (PEL) cells (166). Thus, the tropism of KSHV
depends only partly on successful entry of host cells; consider-
ing that clinical infection by herpesviruses leads to lifelong viral
persistence, postentry events that support the establishment
and maintenance of latency are critical for KSHV pathogene-
sis.
De novo models of KSHV infection suggest a complex pat-
tern of viral gene expression and replication following entry. In
fMVDEC, steady-state levels of two viral lytic RNAs (nut-1/
pan and K8.1) increased linearly after infection until the
cells were reductively divided, but that of the viral G-protein-
coupled receptor (vGPCR) cycled with a 48- to 72-h periodicity,
suggesting a dynamic, synchronous infection (130). Analyses of
VOL. 67, 2003 KSHV MOLECULAR GENETICS 185

viral gene expression and detection of both closed circular (i.e.,
latent episomes) and linear (lytic) genomic viral DNA sug-
gested that both lytic and latent replication were occurring
simultaneously in the infected-cell population (130), but the
relationship of this provocative observation to the multiplicity
of infection it is not known.
De novo infection of cultured endothelial cells has also pro-
vided a critical starting point to evaluate postentry effects of
KSHV on host cell gene expression and physiology. Global
microarray studies of latently infected DMVECs have demon-
strated a significant upregulation of transcripts from IFN-in-
duced genes (especially interferon response factor 7 [IRF-7]),
proinflammatory genes (such as MCP-1, GPCR kinases, CD36,
and RDC1), genes involved in cell division (including CDC25B),
and tumorigenic genes (c-jun, junD, c-kit, c-mer, and others)
(359, 406). Many inhibitors of the above cellular processes are
also down-regulated by infection (359, 406). The c-kit inhibitor
STI 571 (Gleevec) inhibits the proliferation of KSHV-infected
DMVECs in response to the c-kit ligand, scatter factor, and a
dominant negative mutant of c-kit reverses the transformation
of DMVECs by KSHV infection (359). Independent overex-
pression of c-kit in uninfected DMVECs induces their mor-
phologic conversion to spindle cells, suggesting that induction
of c-kit expression by KSHV infection contributes to the ability
of the virus to engender endothelial growth deregulation (359).
In particular, KSHV infection of cultured endothelial cells
induces a complex response of the expression and function of
angiogenic genes that seems to be influenced not only by the
pretransformed status of the cells but also by the uniformity of
infection. In HUVECs that are immortalized by KSHV infec-
tion but maintain the virus in only 1 to 5% of cells, VEGF,
VEGF-C, VEGF-D, placental growth factor, and VEGFR-1
through VEGFR-3 were all induced relative to the situation in
uninfected cells (327). Furthermore, treatment of the infected
cells with VEGFR-2-neutralizing antibody or VEGF antisense
oligonucleotides impaired their growth. A uniform, latent
KSHV infection of HPV E6/E7-immortalized DMVECs
also induced VEGFR-2 (359); however, infection of primary
DMVECs by KSHV had no effect on any VEGF cytokines or
receptors (406). Similarly, neutralizing antibodies to VEGF
failed to inhibit angiogenesis stimulated by KSHV-infected
DMVECs implanted in SCID mice or chemotaxis of endothe-
lial cells induced by the infected cells in vitro (284). Instead,
this study showed that IL-8 and GRO␣ were the KSHV-in-
duced mediators of these effects. Prior transformation of
DMVECs also may be responsible for blunting of the induc-
tion of the IFN pathway in response to KSHV infection (359).
Treatment of KSHV-infected primary DMVECs with TPA
(12-O-tetradecanoylphorbol-13-acetate) allowed an analysis of
effects of the KSHV lytic cycle on endothelial gene expression
(406). Many, but not all, of the induced genes were those that
were up-regulated by infection in the absence of TPA; they
included 10 IFN pathway genes, IRF-7, STAT1, and IGF bind-
ing protein 1 (IGFP-1). A set of genes with the potential for
both complementary and antagonistic function to the induced
genes were down-regulated after TPA treatment. Finally, an
interesting observation was that many genes induced by infec-
tion of DMVECs with KSHV were regulated similarly to treat-
ment of uninfected DMVECS with TPA.
KS spindle cells that have lost the virus after ex vivo culture
have provided an interesting paradigm for understanding the
high ratio of male to female KS cases in humans. Implanted in
nude mice, these cells grew more rapidly and formed larger
tumors in males than in females (9). In female mice, tumor
development was inhibited during early pregnancy when a cho-
rionic gonadotropin (CG)-like activity is high, and crude prep-
arations of human CG (hCG) caused regression of tumors and
inhibition of focus formation of these cells (314) by inducing
apoptosis (442). Further purification of hCG preparations
demonstrated that the anti-KS activity could be separated from
the hCG molecule and resided in an RNase called eosinophil-
derived neurotoxin (EDN) (199, 200, 313). Recombinant EDN
had potent, cancer-specific, cytotoxic effects on KS cells if it
contained the four proximal amino acids of its cognate signal
peptide but not if it was without them (373). Treatment of
humans with crude hCG preparations confirmed its clinical
efficacy, leading to regression of cutaneous and visceral KS
lesions (181, 218).
KSHV INFECTION IN ANIMAL MODELS
The transplantion of human tissue to mice with severe com-
bined immunodeficiency disease (SCID/hu mice) not only has
provided valuable models for KSHV pathogenesis but also has
demonstrated the strict species barrier for infection by the
virus. Injection of infected PEL (BCBL-1) cells into SCID
mice evoked MCD-like immunoblastic lymphomas that were
prominently neovascularized by murine-derived cells; the mu-
rine cells, nonetheless, remained uninfected (401). Although
peritoneal tumors and ascites were produced, only subcutane-
ous and not intraperitoneal (i.p.), injection of the PELs led to
dissemination of the lymphomas. The majority of tumor cells
in this model remained latently infected by KSHV, and the
virus could not infect coinjected human PBMCs even if the
PELs were pretreated with TPA to induce reactivation.
A similar approach using nonobese diabetic (NOD)/SCID
mice revealed a significant pathogenic difference resulting from
injection of PEL cell lines that were either singly (KSHV
⫹
)
or dually (KSHV
⫹
/EBV
⫹
) infected. PEL-like effusions were
produced following i.p. injection of both types of PEL cells,
whereas only the dually infected, but not monoinfected, PEL
produced an effusion following intravenous injection (51).
These authors demonstrated that singly infected PELs express
an array of cell surface homing receptors very different from
most lymphomas, with the potential for both positive and neg-
ative effects on effusion formation. The dually infected PELs
expressed adhesion molecules that were very similar to EBV-
positive Burkitt’s lymphoma cells, suggesting an explanation
for the differential metastasis to solid tumors (as in Burkitt’s
lymphoma) between the PEL types.
A third study suggested that PELs secrete VEGF to accel-
erate the vascular permeability of peritoneal vessels, leading to
effusion rather than to neovascularization of tumors (21). The
ability of various lymphoma lines (including both PELs and
BLs) to form effusions following i.p. inoculation correlated di-
rectly with their respective magnitudes of VEGF release. Coin-
jection of antibodies specific for VEGF, but not control anti-
bodies, blocked effusion formation by the PELs.
De novo infection of SCID/hu mice by KSHV has been doc-
umented for transplanted fetal thymus and liver (thy/liv) (135),
186 DOURMISHEV ET AL. MICROBIOL.MOL.BIOL.REV.

and human skin grafted in keratome strips (162). The thy/liv
model demonstrated a transient lytic and persistent latent in-
fection of CD19
⫹
B cells that did not spread to mouse tissue,
suggesting an appropriate model for initial infection and per-
sistence of the virus in its authentic reservoir. In the skin
model, six of eight mice developed KS-like lesions with angio-
genesis. Similar to human infection, keratinocytes in the epi-
dermis and spindle cells in the dermis supported a largely
latent infection, with rare cells expressing lytic genes. The lack
of universal infection suggests that the skin model may allow
an analysis of viral and host determinants of permissiveness.
PHYSICAL AND GENETIC STRUCTURE OF KSHV
The coding capacity of the KSHV genome was determined
by sequencing of viral DNA isolated from both a PEL cell line
and biopsy specimens of KS (372, 436). Complementing this
approach, Gardella gel analyses were used to demonstrate the
size and conformation of the viral nucleic acid (423). Similar to
the HVS and EBV genomes, the KSHV genome is maintained
during latency in PEL cell lines as a circular, multicopy epi-
some and contains multiple GC-rich, 801-bp terminal repeats
(279) flanking approximately 145 kb of “unique” sequence
(351) (Fig. 1). During productive replication, viral progeny
DNA is ultimately synthesized as linear, single-unit genomes
destined for incorporation into independent virions (423).
Purified virions released from PEL cells treated with TPA
and sodium butyrate contain three types of capsid, named A,
B, and C (371). Fully mature C capsids have a total mass of 300
MDa and, in declining order of abundance, contain the poly-
peptides ORF25/MCP (major capsid protein), ORF65/SCIP
(small capsomer-interacting protein), ORF26/TRI-2 (triplex-
2), ORF62/TRI-1, and the 160- to 170-kb (unique plus repeats)
viral genome (371). A and B capsids contain the four proteins
listed above but lack viral genomic DNA; B capsids contain, in
addition, the scaffolding protein encoded by ORF17.5 (371).
Cryoelectron microscopy at 24 Å resolution and digital recon-
struction demonstrate that the capsid is icosahedral with a
diameter of approximately 1,140 to 1,300 Å (504, 535). The
characteristic herpesvirus tegument and envelope presumably
are added to the capsids during egress.
KSHV contains at least 87 open reading frames (ORFs)
(372, 436) (Fig. 1). Comparison of its genome with that of HVS
(the prototype gammaherpesvirus) reveals a remarkably simi-
lar genetic organization (436). The two family members share
68 conserved genes that are arranged colinearly, with short,
interspersed regions of genes unique to each virus. Each con-
served gene has been given the prefix “ORF” and numbered
consecutively from left to right along the genome, while the
unique genes have been designated K1 to K15 (436). The more
recent publication of the complete genomic sequences of the
rhesus rhadinovirus (10, 455) and murine gammaherpesvirus
68 (515) has confirmed and extended the conservation of this
genetic organization to additional nonhuman gammaherpesvi-
ruses. The genes with highest conservation among these viruses
are those predicted to perform metabolic and catalytic roles in
viral DNA replication (or virion structure [463]) and are in-
cluded in a set of “ancient” genes conserved in all mammalian
herpesviruses (334). In KSHV, these include the DNA poly-
merase and processivity factor (ORF9 and ORF59, respec-
tively), DNA helicase-primase (ORF40, ORF41, and ORF44),
thymidylate synthase (ORF70), and thymidine kinase (ORF21).
Conversely, many of the K genes are homologs of cellular
growth control and signaling molecules (372, 436) and are
postulated to have been “captured” from the human host ge-
nome over a wide coevolutionary window (Fig. 1) (334). The
KSHV genome also contains two origins that support lytic rep-
lication of heterologous plasmids and are inverted duplications
of each other: oriLyt-L lies between K4.2 and K5, and oriLyt-R
lies between ORF69 and vFLIP (26) (Fig. 1).
These analyses have also revealed that the viral DNA poly-
merase gene has the highest intervirus identity, facilitating the
construction of a densely branched rhadinoviral phylogenetic
tree which includes KSHV, HVS, and nine additional pri-
mate rhadinoviruses that have been identified over the last half
decade (197) (the rhadinovirus tree does not include EBV, the
other human gammaherpesvirus). The rhadinoviruses have
since been subdivided into those of New World and Old World
primates; the Old World viruses, including KSHV, have been
further subdivided into groups RV-1 and RV-2 (197). The
branching order of the RV-1 group, which includes KSHV,
follows that of the host species (197). Although not completely
sequenced at the time of publication, Pan troglodytes (chim-
panzee) rhadinovirus 1 is most probably the closest KSHV
homolog, encoding a DNA polymerase gene that has 93.2%
amino acid identity to the KSHV polymerase (198).
KSHV Subtypes and Geographic Variability
Although KSHV occupies a single branch within the dense
phylogenetic tree of nonhuman rhadinoviruses, its nucleic acid
displays significant intragenomic heterogeneity. In an attempt
to uncover the molecular basis for the extreme pathogenic and
epidemiologic variation in the different forms of KS, numerous
groups have initiated a comprehensive, comparative sequence
analysis of KSHV DNA from specimens from AIDS-associ-
ated, classic, and endemic KS patients worldwide. The earliest
studies revealed that the ORF25/26 and ORF75 loci of dif-
ferent isolates displayed approximately 1.5% sequence diver-
gence (407, 553). Although small, these differences were sig-
nificant enough to divide individual isolates into subgroups A,
B, C, and N with intertypic recombination (6, 407, 553). How-
ever, as predicted by the original full-length sequence compar-
isons within the gammaherpesviruses, it was later discovered
that the genomic variability is not evenly distributed within the
virus; instead, individual genes are evolving at different rates
(215, 334). In fact, the greatest sequence divergence is not in
ORF25/26 and ORF75 but is concentrated within the K1 locus
at the extreme left side of the genome (551, 552) (Fig. 1).
The amino acid sequence of K1 differs by 0.4 to 44% be-
tween KSHV isolates and has permitted further differentiation
of the virus into six subtypes in multiple studies (A, B, C, D, E,
and Z, with more than 24 clades [41, 215, 242, 246, 551, 552]).
These data support an extremely strong linkage of the KSHV
subtype to the geographic origin of the infected host. Subtypes
A and C are most similar and prevail in Europe, the United
States, and northern Asia (551). Subtypes B and A5 are char-
acteristic for Africa (242, 551), and subtype D is found in the
countries from the Pacific Islands region (551). Subtype E has
more recently been identified in Brazilian Amerindians (41),
VOL. 67, 2003 KSHV MOLECULAR GENETICS 187

FIG. 1
188 DOURMISHEV ET AL. M
ICROBIOL.MOL.BIOL.REV.

and subtype Z has been found in a small cohort of Zambian
children (246). Conclusive evidence of the geographic associ-
ation of genotype comes from studies of infected emigrants,
who harbor virus subtypes characteristic of their countries of
origin but not their adopted homes (551). Strain analysis of the
viruses responsible for 15 KS cases in ethnically diverse eastern
Taiwan showed the rarely described strain D in four aborigines
and strain C more common among Han Chinese KS patients
(222); furthermore, two isolates obtained from African immi-
grants to the United States were B subtype virus, not the
prevailing U.S. forms (551). Among classic KS patients of
Ashkenazi origin in Israel, strain A1 predominates, whereas
among North African Sephardic Jews, C2 and C6 variants
prevail instead (551).
The mutation rates in K1 approximate those in genes in
other human pathogens (for instance, HIV-1 env), but there is
no evidence for a herpesvirus error-prone replication mecha-
nism that would permit rapid positive selection of the K1 gene
(215, 433). Furthermore, isolation of multiple K1 subtype vi-
ruses from a single individual is not universal. A comparison of
PEL specimens and anatomically distinct KS lesions taken over
time from nine individuals harbored identical viruses (551,
552), suggesting that K1 variation does not occur over the
lifetime of a single infected host. However, K1 genes isolated
from biopsy specimens obtained 4 months apart in Russian pa-
tients had acquired up to 5 nucleotide and amino acid changes
in four of the seven patients (276). Although these current
PCR-based genotyping methods have suggested that infection
by a single KSHV variant predominates, and may be exclusive,
in many individuals, isolation of recombinant genomes argues
strongly that coincident infection of a single host by different
KSHV subtypes must occur. A population that might be inves-
tigated to address the issue of viral evolution is KSHV-positive
Zambian children presenting with either febrile illness (i.e., no
KS) or childhood endemic KS (246). In one study, all 13 chil-
dren harbored the rare Z variant of K1, regardless of concom-
itant KS or HIV seropositivity; due to their young age, these
variants probably represented primary infections. A prospec-
tive analysis of strain status and progress of infection would be
highly informative; of course, a potential complication would
be multiple exposures to variant viruses. Nonetheless, this may
confirm the superinfection/coinfection hypothesis.
FIG. 1. ORF map of the KSHV genome. A linear representation of the KSHV genome, showing the position of the ORFs as published in
references 63, 309, 351, 431, 436 is shown. Each ORF is represented by an arrow pointing in the direction in which it is expressed; ORFs are
displayed linearly in two groups based on leftward or rightward direction. Approximate nucleotide positions are indicated in kilobases in the scale
at top, as in reference 436. The expression kinetics of each ORF in PEL models of latency and reactivation are indicated by the fill of each arrow,
as shown in the figure: latent (65, 122, 134, 156, 184, 431, 444, 483), IE (430, 448, 487, 489, 542), and lytic ⬍24 h postinduction (hpi) and ⬎24 h
postinduction (236, 393) are all shown. An asterisk above an ORF indicates that at least one transcript encoding the ORF is spliced (either
removing part of the ORF or introducing additional exons from outside the ORF). ORFs with published functions are indicated by bold lettering,
and their respective functions are shown (see the text for relevant references). The nomenclature of each ORF follows that of reference 436 and
other references in the text. CBP, complement binding protein; DHFR, dihydrofolate reductase; TK, thymidine kinase; PF, processivity factor;
UDG, uracil-DNA glycosylase; RR, ribonucleotide reductase; other abbreviations are defined in the text.
V
OL. 67, 2003 KSHV MOLECULAR GENETICS 189

Adding to the divergence at the K1 locus is the observation
that the K15 gene, from the extreme right side of the viral
genome (Fig. 1), exists as two distinct alleles. The M and P (for
“minor” and “predominant”) alleles of K15 are 67% divergent
(407). Although the overall divergence within each allele ap-
proximates the natural variation found in most of the KSHV
genome, four isolates with K15 alleles that have diverged fur-
ther have been described (242) or postulated (551). Like K1,
the K15 alleles are geographically delimited and are partially
linked to particular K1 subtypes. The M K15 allele was iden-
tified in 14 of 49 West and Central, but not East, African
samples by Lacoste et al. (275); however, it was not detected
among 21 African KSHV specimens by Zong et al (with the
exception of three U.S. AIDS patients with West African her-
itage) (551). In a small Russian cohort, four of seven samples
harbored virus with M alleles (276). It has been postulated that
the M K15 alleles are derived from a novel, possibly nonhu-
man, rhadinovirus by ancient recombination events and that
the M allele may be evolving toward a higher prevalence in the
population (551). Linkage analyses have shown that up to 20
kb, including “M alleles” of eight neighboring genes, is fre-
quently linked to the M K15 allele, suggesting a generous
transfer of genetic information from the M virus (551).
The proteins encoded by the K1 and K15 genes are attrac-
tive candidates for playing a direct role in KSHV pathogenesis.
K1 and K15 are positional homologs of transforming genes in
EBV and HVS (215), and K1 can functionally replace its ho-
molog in transformation of lymphoid cells by HVS (289). Mice
transgenic for K1 develop KS-like tumors and plasmablastic
lymphomas in which there is constitutive activation of NF-␬B,
Oct-2, and Lyn (408). K1 may also assist immune evasion of
infected cells by its ability to inhibit transport of B-cell receptor
complexes to the surface of B cells (286). The K1 product is a
transmembrane protein localized to the cell surface that sig-
nals constitutively through its cytoplasmic immunoreceptor ty-
rosine activation motif (ITAM), activating the well-character-
ized cellular nuclear factor of activated transcription (NFAT)
growth control pathway (281, 288). This function of K1 is
critical for KSHV replication, since truncation of the ITAM
creates a mutant K1 that represses viral replication following
reactivation from latency, in a dominant negative fashion
(280). Furthermore, the ITAM is conserved in all diverged
variants of K1 (41, 246, 551, 552). Additional studies demon-
strated that overexpression of wild-type K1 can also inhibit
TPA induction of viral reactivation in PELs without altering
the expression of the viral lytic switch protein; this occurs by
inhibition of cellular AP-1, NF-␬B, and Oct-1 activity, suggest-
ing that K1 may help fine-tune viral gene expression to mod-
ulate KSHV reactivation (287).
The K15 gene encodes a latently expressed protein with a
cytoplasmic domain that contains SH2 motifs, interacts with
TRAF (tumor necrosis factor receptor-associated factor) and
antiapoptotic proteins, and represses B-cell receptor signaling
(101, 184, 460). The M and P forms of K15 conserve a similar
splicing structure and two C-terminal SH2 domains (407).
However, whether a particular viral genotype can be con-
nected to (i) development or progression of KS (or any of the
other KSHV-associated diseases) or (ii) a particular clinico-
epidemiologic form of KS remains an open question. Geno-
typing of numerous, geographically distinct AIDS populations
has shown no clear correlation between a particular K1 or K15
variant and pathogenesis of KS, PEL, or MCD (215, 222, 242,
275, 551). A recent case report demonstrated that KSHV iso-
lated from individual KS, PEL, and MCD specimens from the
same patient all had the identical K1 genotype (484). Likewise,
classic KS in Italy correlates geographically with KSHV sero-
prevalence rather than KSHV subtype (109). In France, a
genotyping study of only ORF26 demonstrated that subtype A
was isolated from the more aggressive forms of KS, but the
viral genotype was incomplete, since linkage of ORF26 to
other loci was not evaluated (48).
Nevertheless, studies of populations in which seroprevalence
is high but KS incidence is low have affirmed a role for both
virus and host genotype in disease phenotype. Most of sub-
Saharan Africa experiences a high KSHV seroprevalence, with
the B subtype predominating throughout, yet endemic KS is
confined primarily to East and Central Africa and variably in
West Africa (275). Conversely, a population of 781 Brazilian
Amerindians who have a 53% KSHV seroprevalence but a 0%
KS incidence are infected with a rare K1 E subtype virus, while
other South American isolates are A or C subtypes (the au-
thors concede, however, that reporting of KS in Amerindians
may be incomplete). The molecular basis of host susceptibility
to KS has been virtually unexplored (319); however, sex-spe-
cific discrepancies in KS incidence (188, 251, 532) might be
partially explained by hormonal factors (54, 313) (see discus-
sion, above).
Heterogeneity within the ORF73 gene, which encodes the
pathognomic, seroepidemiologic target antigen LANA-1 (dis-
cussed above), has independently been utilized to genotype
individual viral isolates. In the prototype BC-1 virus, the
ORF73 gene contains a 3,489-nucleotide (nt) ORF that in-
cludes an internal repeat domain (IRD) within nt 929 to 2826
(538) (Fig. 2). The IRD itself can be further subdivided into
three repeat regions, with the outer two encoding mostly acidic
amino acids and the inner one encoding a preponderance of
glutamine residues. The ORF73 IRD is highly variable among
individual KSHV isolates, with variations due to insertions,
deletions, and point mutations but never frameshifts or non-
sense codons (538). As a result, LANA-1 from different iso-
lates contains variable numbers of acidic repeats, with the
glutamine-rich region (region II), in particular, being a hot
spot for amino acid variation (177, 537).
Comparison of the respective electrophoretic mobilities of
products of PCR amplification across the IRD provides a char-
acteristic KVNAtype, or genotype, of individual viruses from
distinct PEL cells and clinical specimens (177). Multiple spec-
imens from single patients had the same KVNAtype, although
two KS specimens showed evidence of a minor, second KVNA
type. Furthermore, various PEL cell lines carry latent virus
with unique KVNAtypes (177). Combining this PCR approach
with restriction digestion at sites within the IRD (i.e., PCR plus
restriction fragment length polymorphism), allowed even
higher resolution of individual viral genotypes (538). This ap-
proach confirmed that there is little or no variation in viral
genotype in multiple KS lesions from the same patients and
demonstrated that variability in the IRD is independent of
both the ORF26 (538) and K1 (110) genotypes. PCR plus
restriction fragment length polymorphism of ORF73 has more
recently been used to track inter- and intrafamilial transmis-
190 DOURMISHEV ET AL. MICROBIOL.MOL.BIOL.REV.

sion of virus isolated from both mouth rinses and peripheral
blood of Malawian KS patients (110).
The IRD may be a crucial domain for mediating protein-
protein interactions of the cognate LANA-1 protein in tran-
scriptional regulation of both viral and cellular genes. Al-
though mutational studies have suggested that the role of
LANA-1 in maintenance of the latent KSHV episome is inde-
pendent of the intact IRD (178, 179, 223) (discussed below),
most truncated derivatives of LANA-1 that retain both positive
and negative transcriptional effects include all or part of the
IRD (Fig. 1). Transcriptional activation by LANA-1 is proba-
bly independent of direct DNA binding, since LANA activates
transcription directed by a wide array of simple, synthetic pro-
moters containing binding sites for the cellular proteins ATF,
AP-1, CAAT, or Sp1, linked to a TATA box (421). Although
the TATA box alone was also activated by LANA-1 in these
studies (421), activation of the cellular IL-6 promoter by
LANA requires both a TATA box and an upstream AP-1
element (15), and activation of the HIV-1 LTR in BJAB cells
requires both the TATA box and the core enhancer elements
(NF–IL-6, Ets, NF-␬B, and Sp1 sites) (227). However, LANA-
1 can activate the HIV-1 LTR independently of the core en-
hancer if the Tat protein is coexpressed; this effect is mediated
by the carboxy-terminal 400 amino acids (aa) of LANA-1,
which includes the third repeat region (227). Truncated LANA-
1 containing the IRD plus the C terminus activates the tran-
scription of an EBV latency promoter containing PU.1, ATF,
and Sp1 elements (202). Mechanistically, LANA-1 can modify
the DNA binding activity of Sp1 to activate the transcription of
the telomerase reverse transcriptase promoter (265). Finally,
similar to the oncogenes of small DNA tumor viruses, a
LANA-1 truncation containing aa 803 to 990 (including the
third repeat region [Fig. 2]) binds to the pocket domain of RB1
to facilitate E2F-dependent activation of the cyclin E pro-
moter; this disruption of RB1 function suggests one mecha-
nism by which LANA-1 cooperates in cellular transformation
with H-ras (415). Taken together, these studies suggest that
LANA-1 activates transcription by undergoing promiscuous
interactions with multiple cellular transcription factors; further
structure-function studies of LANA-1 are necessary to deter-
mine the influence of the IRD in these interactions.
LANA-1, however, can also contribute to broad repressive
effects on transcription. In Cos cells, LANA-1 represses the
transcriptional activity of NF-␬B, and repression of the HIV-1
LTR in this study (421) suggests that the interactions of
LANA-1 with the cellular transcription apparatus is cell spe-
cific. LANA-1 also represses transcription controlled by the
ATF4/CREB2 protein, independently of ATF4 DNA binding;
FIG. 2. Primary structure-function map of KSHV ORF73/LANA-1. This schematic shows a linear representation of the amino acid content and
predicted structural motifs of the LANA-1 protein. The amino acid numbering is as published in reference 436 and corresponds to the virus from
BC-1 cells. The internal repeat domain (IRD) is shown by the bracket, as in reference 538, and is described in the text. The approximate position
of each functional domain is shown by a black bar, corresponding to the function shown in the column on the right (see the text for references).
No amino acid boundaries are indicated for the functional domains, since functional mapping has used different isolates of LANA-1 that are
variable in size. Abbreviations: DE, aspartic acid/glutamic acid rich; Q, glutamine, LZ, putative leucine zipper; QE, glutamine/glutamic acid rich.
V
OL. 67, 2003 KSHV MOLECULAR GENETICS 191

this effect is mediated by LANA-1 aa 751 to 1162, which in-
cludes the third repeat region (297) (Fig. 2). Similarly, the first
acidic repeat (aa 340 to 431) of LANA-1 is one of two domains
required to competitively bind the cellular cyclic AMP-respon-
sive element binding protein (CREB) binding protein (CBP)
and block its histone acetyltransferase activity, thus inhibiting
the ability of CBP to coactivate transcription with c-fos (296).
This mechanism might reflect the general ability of LANA-1 to
repress transcription driven by other cellular and viral CBP-
dependent transactivators, including the KSHV lytic switch
protein ORF50/Rta (208). The entire IRD and C terminus of
LANA-1 also directly binds to the p53 tumor suppressor to
inhibit transactivation as well as apoptosis (167) (since p53 can
repress the promoter of LANA-1, the interaction between the
two proteins may also result in derepression of transcription, to
contribute to autoactivation by LANA-1 and maintenance of
its expression during infection [238, 421]). Thus, it is provoc-
ative to speculate that IRD polymorphisms in ORF73 (177,
538) might influence heterooligomerization of LANA-1 in a
KVNAtype-specific manner, to tune the cell-specific, positive
and negative interactions of LANA-1 with the cellular tran-
scriptional machinery, resulting in variable consequences for
viral replication and pathogenesis.
A final divergent locus that has not yet been evaluated for
viral classification surrounds the K12 ORF (Fig. 1), which
encodes the transforming protein kaposin (263, 291, 362, 363,
437). Different KSHV isolates derived from PELs or KS tissue
demonstrate variable numbers of direct repeats of genomic
sequence upstream of K12 (437). The repeats preserve all
three respective ORFs and are transcribed in infection, encod-
ing proteins containing variable numbers of peptide repeats
with unknown function (discussed in more detail below) (437).
LATENT VERSUS LYTIC GENE EXPRESSION IN KSHV
Analagous to the other herpesviruses, KSHV exhibits both
latent (nonproductive) and lytic (productive) replication, both
of which are characterized by virtually distinct gene expression
programs. This biphasic life cycle of KSHV was recognized
early in both KS lesions (483) and cultured PEL specimens
(342, 343, 424, 540). Productive infection by herpesviruses
leads to cell lysis and death and obviously is not consistent with
the ability of an infecting virus to transform its host cell. There-
fore, classifying the latent or lytic cycle expression of individual
KSHV ORFs is critical for predicting their potential roles in
pathogenesis of the viral infection.
Assigning the expression of KSHV genes to the latent or
lytic phase has benefited immensely from the ease of culturing
PEL cells latently infected with KSHV and inducing lytic re-
activation with common laboratory chemicals (such as phorbol
esters or sodium butyrate). Individual PEL cell lines carry 40 to
150 copies of KSHV DNA per cell genome, respectively, with
every cell in the culture infected (reviewed in reference 138).
In normal passage of the cells, the virus is maintained as a
latent episome, with highly restricted viral gene expression and
lack of virus production. On chemical induction, viral gene
expression switches from the latent program to an ordered
cascade of lytic gene expression, leading to viral replication,
virion production, cell lysis, and viral release (423, 424, 444,
540).
However, the differentiation of a viral gene as latent or lytic
strictly by analysis of RNA expressed in bulk PEL cultures has
been complicated by the characteristic small percentage of
every cultured PEL population that experiences spontaneous
lytic reactivation (424, 540). One powerful approach to over-
come this problem is by analyzing KSHV gene expression at
the single-cell level by ISH. When ISH was performed with KS
specimens, the kaposin gene (K12) was expressed in at least
85% of spindle cells while the ORF25/MCP, a lytic structural
protein in PELs (371) (and conserved across the Herpesviri-
dae), was expressed in no more than 10% of the spindle cells
(483). Kaposin was thus classified as a latent gene (483), and
this approach provided a seminal paradigm for classifying ex-
pression of other KSHV genes.
Further genome-wide analyses of KSHV gene expression
have also utilized PEL models of infection. The first such study
compared the respective expression patterns of each viral ORF
during normal culture of PELs (i.e., latency) to the response of
each to TPA treatment and lytic viral induction (444). Each
viral gene was thus distinguished as class I (constitutively ex-
pressed regardless of TPA treatment), class II (expressed with-
out TPA and further induced by TPA addition), or class III
(undetectable without TPA but induced by the chemical), re-
spectively (444). This study revealed a cluster of three class I
genes that included LANA-1, ORF72 (viral cyclin D [vCyc]),
and K13 (fas-ligand IL-1 ␤-converting enzyme inhibitory pro-
tein [vFLIP]) (Fig. 1); their wide expression in KS specimens
confirmed their latent classification (122, 134). The class III
genes, in contrast, encoded mostly the viral structural and
replication genes (typically late [L] genes in herpesviruses); the
identification of kaposin as a class III gene in these cells (444),
however, demonstrates that not all latent genes (437, 483) are
class I. The class II genes consisted of typical herpesvirus reg-
ulatory and viral DNA replicative genes, as well as the majority
of the viral homologs of cellular genes (discussed below) (444).
More recent studies using DNA microarrays have permitted
simultaneous comparisons of the transcription kinetics of vir-
tually all the KSHV genes (236, 393). While confirming the
original PEL-based classifications of the viral genes based on
the addition of TPA, microarrays have demonstrated a pow-
erful means of determining the kinetics of first appearance and
peak expression of the lytic genes. For example, the peak ex-
pression of some candidate structural genes, such as ORF8/gB
and ORF47/gL, occurs at early (12 h), rather than late (24 h)
times, postinduction (393) (Fig. 1).
These genome-wide analyses of KSHV gene expression ki-
netics in PEL cells have complemented many individual studies
of single viral genes or loci; two additional genes identified as
latent, K10.5/LANA-2 and K11.5, encode homologs of IRFs
(65, 431), and a third (expressed from K15) encodes the laten-
cy- associated membrane protein (LAMP) (184) (Fig. 1). Ad-
ditional studies of gene expression following reactivation of
latent virus have demonstrated that, typical of regulatory genes
of herpesviruses, immediate early (IE, or ␣) transcripts could
be identified based on their resistance to treatment of the
PELs with cycloheximide (CHX); six IE loci with multiple
transcripts (discussed below) have thus been identified, con-
taining (Fig. 1) (i) ORF50 (replication and transcriptional ac-
tivator [Rta]), K8, and K8.2 (K-basic-leucine zipper (bZIP)/
replication-associated protein [RAP]), (ii) ORF45, (iii) K4.2,
192 DOURMISHEV ET AL. MICROBIOL.MOL.BIOL.REV.

K4.1, and K4 (vMIPs), (iv) ORF48 and ORF29b, (v) K3 and
ORF70, and (vi) a transcript with no apparent coding potential
(489, 490, 542). Similarly, typical of structural genes of herpes-
viruses, late (L, or ␥) genes have been identified by their
sensitivity to inhibitors of viral DNA replication, for example
the ORF17 viral assembly protein (AP)/protease (Pr) and
K8.1, a glycoprotein (87, 508). Studies of transcript architec-
ture from individual loci have also demonstrated that numer-
ous KSHV transcripts are spliced (Fig. 1), a gene expression
strategy that is not axiomatic in the Herpesviridae, and many
are polycistronic.
Remarkably, the low level of spontaneous lytic gene expres-
sion detected against the backdrop of latent expression in most
PEL cultures (156, 236, 393, 444) is extremely similar to what
is detected in KS clinical samples. Indeed, most infected cells
in KS specimens display latent KSHV gene expression, with
occasional cells expressing lytic transcripts (86, 143, 249, 301,
384, 389, 483, 489), suggesting that the low level of spontane-
ous lytic gene expression is not an artifact of tissue culture
models. More recent models of de novo infection of cultured
endothelial cells have also demonstrated a similar mixed pat-
tern of gene expression (106, 278, 358).
Latent Genes and KSHV Pathogenesis
Similar to the latent expression of genes required for EBV
transformation of primary B lymphocytes (reviewed in refer-
ence 256), KSHV expresses seven latent genes with demon-
strated modulatory effects on host cell growth (Fig. 1); all are
thus candidate effectors of KS pathogenesis.
The major latent locus: ORF73, ORF72, and K13. ORF73
(encoding LANA-1), ORF72 (v-cyclinD) and K13 (vFLICE)
are all expressed from the same locus in polycistronic, differ-
entially spliced mRNAs whose transcription is coordinately
regulated by a common promoter (84, 134, 205, 447, 494). This
promoter is bidirectional, controlling the constitutive expres-
sion of the latent genes to the left but lytic, TPA-inducible
expression of the K14 and ORF74/vGPCR to the right (134,
238, 447, 494) (Fig. 1). LANA-1 can autoactivate transcription
of the promoter in the latent direction (238, 421), and, similar
to the human cyclin D1 promoter, the latent promoter is in-
duced at the G
1
-to-S transition of synchronized cells (447). In
transgenic mice, the LANA-1 promoter is highly expressed in
CD19
⫹
B cells, similar to its constitutive activity in PEL cells,
but is not expressed in CD3
⫹
T cells (237). This tissue-specific
activity of the LANA-1 promoter may thus help specify posten-
try persistence of KSHV in CD19
⫹
B cells of infected humans
(13, 340).
As discussed above, LANA-1 (Fig. 2) both activates and
represses transcription (15, 202, 227, 238, 265, 296, 421); it also
subverts the tumor suppressors p53 and RB1, blocks apoptosis,
and stimulates cellular transformation (167, 415). The obser-
vation that LANA-1 also associates with cellular chromatin
(30, 115, 272, 273, 330, 402, 451, 492, 493) reflects the crucial
role for this large protein in maintaining latent persistence of
the viral episome. LANA-1 interacts directly with both com-
ponents of host chromatin (Fig. 2), the protein (histone H1,
DEK1, and RING3) (115, 272, 330, 404) and the DNA (31,
178, 179, 223, 298, 402, 493), to tether the viral episome to the
host genome. In tissue culture cells expressing LANA-1, a sin-
gle copy of one viral terminal repeat (TR) introduced into a
heterologous plasmid is sufficient for its episomal maintenance
(30, 31). The C-terminal 233 aa of LANA-1 binds as a se-
quence-specific dimer to two tandem 17-bp direct repeats
found within each TR of the virus, with cooperative kinetics
(31, 178, 179, 298, 451). The LANA-1 DNA binding site and an
adjacent GC-rich region contained within a single TR together
function as the putative origin of latent episomal replication
when present on a plasmid; expression of the C-terminal DNA
binding fragment of LANA-1 is sufficient to mediate this effect
(178, 223), although the N-terminal 90 aa may also contribute
(298). Reflecting its ability to bind the TR at the origin of
replication, LANA-1 also interacts with the origin recognition
complex 1 and 2 (Orc-1 and Orc-2) proteins (298).
LANA-1 also targets the protein component of chromatin,
tying its role in viral replication to a role in transcriptional
repression (Fig. 2). When placed upstream of a heterologous
promoter, the TRs can function as transcriptional enhancers
but are potently suppressed by the DNA binding domain of
LANA-1 (178, 179, 298, 451). In fact, independent fusions
of either the N or C terminus of LANA-1 to the Gal4 DNA
binding domain can reproduce the transcriptional repression
function of LANA-1 on heterologous promoters containing
Gal4 binding sites (273, 451). The N-terminal repression do-
main binds directly to the transcriptional corepressor proteins
SAP30, Sin3A, and CIR (273), while the C terminus binds to
CBP (208). Cognate LANA-1 protein preferentially associates
with heterochromatin and dominantly redistributes the cellular
RING3 protein, a putative chromatin remodeling factor, from
euchromatin to heterochromatin (330, 404, 493). Expression of
the C-terminal domain in uninfected cells is sufficient to gen-
erate the characteristic punctate subnuclear localization of
LANA-1 (402, 451) and also mediates binding to RING3 and
DEK1, a second putative chromatin-remodeling factor (272,
404). However, the C-terminal domain is not sufficient to
target LANA-1 to heterochromatin; instead, aa 1 to 15 of
LANA-1 are tethered to heterochromatin through interactions
with methyl-CpG binding protein 2 (MeCP-2) (272). Deletion
of the N-terminal chromatin binding site abolishes the ability
of LANA-1 to mediate episomal persistence, and can be res-
cued by fusion of the mutant protein to histone H1 but not
histone H2B (462).
ORF72 encodes viral cyclin D (vCyc or kCyc) (Fig. 1), a
protein that has 32% identity and 54% similarity to the cellular
cyclin D2 (294). Like its cellular counterpart, vCyc forms func-
tional complexes with cellular cyclin cdk6, phosphorylates RB1
and histone H1, and stimulates the G
1
-to-S transition of the
cell cycle (91, 185, 294, 491). However, the CDK inhibitors
p16
Ink4a
, p21
Cip1
, and p27
Kip1
, which limit the activity of the
cellular cyclin-cdk complexes, are unable to block vCyc/cdk6
(491). For p27
Kip1
this difference is directly attributable to
divergence of critical amino acids in the CDK inhibitor inter-
face of vCyc that alter its conformation and prohibit a direct
interaction between the proteins (71, 491). Furthermore, stim-
ulation of cdk6 activity by vCyc does not require the cdk-
activating kinase cycH/cdk7; however, complete resistance to
p16, and efficient RB1 phosphorylation, do depend on cdk-
activating kinase activation of vCyc/cdk6 (99, 243).
Viral cyclin also diverges from its cellular counterpart in
having evolved a wider range of substrates as targets when
VOL. 67, 2003 KSHV MOLECULAR GENETICS 193

associated with cdk6. The phosphorylation of p27
Kip1
by vCyc/
cdk6 leads to degradation of the CDK inhibitor by the cellular
proteasome and is required for efficient stimulation of cell
cycling by the vCyc (145, 320). The vCyc/cdk6 complex can also
phosphorylate the cellular Bcl-2 protein, which leads to induc-
tion of apoptosis in cells expressing high levels of cdk6; inter-
estingly, overexpression of the viral lytic cycle protein bcl-2
(ORF16), but not cellular bcl-2, can inhibit this effect (377,
378). Origin recognition complex 1 (ORC-1) protein is also a
target of vCyc/cdk6 phosphorylation, which contributes to the
ability of vCyc/cdk6 to stimulate cellular DNA replication even
in the presence of the cdk inhibitor roscovotine (283). This
raises the possibility that the latently expressed KSHV cyclin D
not only stimulates cell cycling and replication of the host
genome but also ensures replication of the latent viral genome.
K13 encodes the vFLIP protein (Fig. 1), which inhibits
FADD-mediated apoptosis downstream of the Fas receptor
(34, 136, 500). vFLIP blocks the protease activities of caspase-
3, caspase-8, and caspase-9; it directly interacts with procas-
pase-8 to inhibit its activation (34, 136). Overexpression of
vFLIP in Fas-sensitive, A20 B lymphoma cells allows their
clonal outgrowth in the presence of Fas receptor activation and
promotes tumor establishment and progression of A20 cells in
immunocompetent mice, presumably by allowing evasion of
CTL-mediated killing (136). vFLIP also constitutively activates
the NF-␬B pathway by direct interaction with the I␬B kinase
(IKK) complex in the cell cytosol (307), an activity that allows
activation of the HIV-1 LTR (34). Inhibition of constitutive
NF-␬B activity in PEL cells induces programmed cell death
(254); however, it is not clear whether vFLIP constitutively
activates NF-␬B for survival of infected endothelial cells.
K12/kaposin A. The 60-aa hydrophobic protein encoded by
K12 (called kaposin A) (Fig. 1) transforms cultured cells and
drives tumorigenesis when these cells are introduced into nude
mice (263, 362, 363). Cells transformed by kaposin A show
enhanced activation of numerous serine/threonine kinase
pathways (363), increased adhesion to intercellular cell adhe-
sion molecule 1 (ICAM-1), and reorganization of their cellular
F-actin (263). Genetic and biochemical experiments have dem-
onstrated that kaposin A activates the ERK1/2 pathway by
recruiting the guanine nucleotide exchange factor cytohesin-1
to membranes, leading to enhanced GTP binding of the
GTPase, ARF1 (263).
The K12 locus expresses the most abundant latent tran-
script(s) in KS tissue and PEL cells and is also strongly induced
following lytic reactivation of KSHV (437, 483, 487, 540). The
major transcript in this locus initiates at least 2 kb upstream of
the K12 ORF, at either of two start sites separated by a 5-kb
intron (291, 437). The KSHV genomic sequence between the
start sites and the K12 ORF is highly polymorphic in compar-
isons of viruses from different KS specimens and PEL cell
lines, containing a variable number of direct, GC-rich repeats
(291, 437). Transcription through these polymorphisms yields
RNAs that differ in length by up to 0.8 kb in different isolates;
although the only predicted translational start codon within the
locus is the AUG of ORF K12, the direct repeats contain
CUGs, which are preferentially used for translational initiation
(437). Downstream of these CUGs, the direct repeats lack stop
codons in all three reading frames, but only one reading frame
is open to the end of K12 (437). Hence, the major protein
product of this locus is approximately 48 kDa (437); the 6-kDa
protein predicted by K12 is detectable only after treatment of
BCBL-1 cell lysates with strong reducing agents (263).
K11.5/vIRF2. K11.5 expresses a latent homolog of cellular
IRFs (Fig. 1) that inhibits (i) NF-␬B and cellular IRF1- and
IRF3-mediated transactivation (65), (ii) apoptosis of T-cell
receptor (TCR)/CD3-stimulated Jurkat cells (258), and (iii)
double-stranded RNA protein kinase (63).
K10.5/LANA-2. K10.5 expresses LANA-2, which inhibits
p53-mediated transactivation and apoptosis but is expressed
latently only in B cells, not KS tissue (431).
K15/LAMP. One of the two K15 transcripts (Fig. 1) is ex-
pressed latently in PEL cells, is a positional homolog of trans-
forming genes in EBV and HVS, and interacts with growth
control proteins (discussed above) (101, 184, 407, 460).
Lytic Genes and KSHV Pathogenesis
The vast majority of infected cells in KS lesions express
latent viral proteins, whose growth-regulatory properties sug-
gest that they contribute prominently to viral pathogenesis,
especially in an autocrine fashion. However, the control of lytic
reactivation of KSHV is also a crucial event in the pathogen-
esis of KS. B-cell infection predicts future development of KS
(13), and latent infection by KSHV is established well before
the onset of KS (325, 356). In keeping with the KSHV classi-
fication, the primary target of viral infection is the CD19
⫹
B
lymphocyte (13, 340, 354, 530) (although some other mononu-
clear cells may also be susceptible to infection [46]). Therefore,
reactivation of productive (lytic) KSHV infection from the
latently infected B-cell reservoir appears to be a necessary
antecedent step in KS development. The presence of KSHV-
infected B cells in the peripheral blood (13, 138, 340) and the
ability of TPA-treated PEL cells to efficiently transmit KSHV
to primary endothelial cells in culture (441) suggest that cir-
culating B cells may help disseminate the virus to extralym-
phoid targets such as lymphatic endothelium, where the virus
then establishes a secondary latent infection (483). Supporting
this notion, active KSHV replication and increased viral load
in the peripheral blood predicts the pathogenic outcome of the
infection (410) and is strongly correlated with (i) increased risk
of progression to KS (13, 530) and (ii) increased severity of the
pathogenic stage of KS (47, 68). Furthermore, treatment of
high-risk patients with the antiviral ganciclovir blocks lytic
KSHV replication and reduces KS risk (324). Likewise, AIDS-
associated KS regression due to HAART is associated with
decreased KSHV loads in the peripheral blood (469, 534)
whereas HAART nonresponders fail to clear KSHV from
PBMCs (469). Posttransplantation KS is also usually associ-
ated with reactivation of KSHV from a latent infection rather
than with a primary infection (17, 165, 235, 390). Growth of
PEL cells under hypoxic conditions also induces lytic KSHV
gene expression, providing a provocative connection between
diseases affecting tissue oxygenation (e.g., malaria) and KS
progression (121). Conversely, transformation of B cells by
KSHV would probably be less dependent on lytic reactivation
for dissemination.
Molecular virologic studies support the hypothesis that
KSHV reactivation not only enhances dissemination but also
potentially contributes directly to KS through expression of
194 DOURMISHEV ET AL. MICROBIOL.MOL.BIOL.REV.

viral lytic genes (81, 155), many of which encode growth-de-
regulatory and immunomodulatory proteins (specifically dis-
cussed below) (156, 233, 236, 393, 444, 489). Numerous lytic
proteins also have the potential to enable infected cells to
avoid or inhibit the host immune system. Collectively, these
viral proteins counteract multiple levels of the immunological
response to viral infection and may play dual roles in growth
modulation and immune evasion; for example, antiapoptotic
proteins could enable infected-cell proliferation while inhibit-
ing destruction by host immunocytolytic activities.
K2/vIL-6. vIL-6 (Fig. 1) retains sequence and functional
homology to cellular IL-6 but stimulates multiple cellular path-
ways to induce cell proliferation and extrahepatic acute-phase
responses through engagement of the gp130 coreceptor inde-
pendently of the IL-6 (gp80) receptor (103, 220, 264, 345, 360,
374, 385, 520). vIL-6, but not human IL-6, protects PELs and
heterologous cells from the antiviral, cytostatic effects of
IFN-␣, which down-regulates the surface expression of gp80
but not gp130 (95). In fact, vIL-6 transcription is induced
indirectly by IFN-␣ in an IE-like fashion through two IFN-
stimulated response elements in its promoter (95). vIL-6 in-
duces human IL-6 secretion, supports the growth of IL-6 de-
pendent cell lines, and is an autocrine growth factor for PEL
cells (164, 169, 357). Cells stably expressing vIL-6 secrete in-
creased VEGF and induce hematopoiesis, tumorigenesis, and
angiogenesis when injected into nude mice (20, 306). In KS
specimens, vIL-6 is expressed in a lytic pattern in a minor
fraction of infected cells (59, 69, 482, 489).
ORF74/vGPCR. vGPCR (Fig. 1) is a 7-transmembrane, IL-8
receptor homolog that constitutively engages pathways down-
stream of multiple G protein subunits in a phospholipase C-
and phosphatidylinositol 3-kinase-dependent manner. These
pathways include protein kinase C, protein kinase B, Akt,
NF-␬B, and mitogen-activated protein kinases, leading to in-
creased transcriptional activity of their nuclear targets, stimu-
lation of cellular proliferation, promotion of cell survival, and
transformation (23, 29, 118, 349, 361, 391, 453, 473). Micro-
array experiments have shown that the global gene expression
response to vGPCR expression is very divergent in B lympho-
cytes and endothelial cell lines: two CC chemokines were most
highly induced in B cells, while IL-6 and GRO␣ were highly
induced in endothelial cells (405). In transient transfections,
vGPCR also activates the promoters of multiple latent and
lytic KSHV genes (100). Ultimately, cells expressing vGPCR
secrete increased levels of autocrine and paracrine cytokines
and growth factors (IL-1␤, TNF-␣, IL-6, IL-8, granulocyte-
macrophage colony-stimulating factor, VEGF, bFGF, and
MCP-1), and produce conditioned medium that is chemotactic
(29, 391, 453, 461, 475). In transgenic mice, vGPCR induces
multifocal, angioproliferative, KS-like lesions (536), whose
high incidence requires agonist modulation of vGPCR by het-
erologous chemokines (221, 361, 435, 473).
vGPCR is encoded by a major bicistronic transcript that also
encodes the K14/viral OX2 (Fig. 1) and by a minor monocis-
tronic transcript and is expressed in PEL cells and infected KS
tissue in a lytic pattern (100, 260, 365, 494). Its transcription is
controlled by the same promoter as the major latent tran-
scripts, but it is transcribed in the opposite direction (134, 238).
K6/vMIP, K4/vMIP-II, and K4.1/vMIP-III. The vMIP-I, v-
MIP-II, and v-MIP-III proteins are homologs of human MIP-
I␣,a␤CC chemokine. While vMIP-I is most probably a prod-
uct of DE transcription (353, 374), vMIP-II and vMIP-III are
encoded together on an IE mRNA (542)(Fig. 1).
Numerous proinflammatory roles have been attributed to
the viral chemokines. vMIP-I and vMIP-II both engage the
chemokine receptor CCR-8 (120, 214, 480) and are highly
angiogenic in chicken CAMs (50). Treatment of PELs with
vMIP-I induces the secretion of VEGF-A, and dexametha-
sone-induced apoptosis of PELs is blocked by exogeous vMIP-
I and vMIP-II (306). vMIP-III engages the CCR-4 chemokine
receptor, is a selective chemoattractant for Th2 cells, and is
also angiogenic in CAM assays (485). Paradoxical to the syn-
ergistic pathogenesis of KSHV and HIV-1, vMIP-I and vMIP-
II can inhibit CCR3- and CCR5-dependent HIV-1 infection
(50, 262, 353).
vMIP-II has been implicated in the seemingly contradictory
function of both immune evasion and proinflammation; its
biological function therefore remains controversial. It is a
broad antagonist (binding without activation) of endogenous
chemokine signaling (120, 315) and chemotaxis (262), but oth-
ers have shown that it selectively activates and chemoattracts
eosinophils, Th2 cells, monocytes, and endothelial cells (50,
214, 480). However, when human DMVEC monolayers were
used under “flow” conditions, vMIP-II blocked chemotaxis of
monocytes and Th1 lymphocytes by antagonism of CCR1 and
CCR5 and blocked chemotaxis of eosinophils and Th2 lym-
phocytes by antagonism of CCR3 (527).
K9/vIRF-1 and K10.5/K10.6/vIRF-3. In addition to the la-
tently expressed vIRFs discussed above, KSHV encodes two
other homologs of these proteins (Fig. 1). vIRF-1, encoded by
K9, transforms cells in culture, is tumorigenic in nude mice,
and inhibits apoptosis induced by Sendai virus infection,
IFN-␣, IFN-␤, TNF-␣, TCR/CD3 cross-linking, and p53 (64,
160, 174, 234, 258, 293, 302, 369, 456, 457). It also blocks
programmed cell death mediated by cooperation of the cellu-
lar protein GRIM19 with IFN-␤ and retinoic acid (456).
Although vIRF-1 does not directly bind to DNA, it activates
transcription when targeted to promoters by a heterologous
DNA binding domain (432); in fact, direct transactivation of
the c-myc promoter by vIRF-1 is required for transformation,
and CBP coactivates c-myc with vIRF-1 (234). However,
vIRF-1 has been best characterized for inhibiting the transcrip-
tional programs induced by exogenous IFNs (174, 293), spe-
cifically by blocking transcriptional activation by the cellular
proteins IRF-1 and IRF-3. This is mediated by multiple mech-
anisms, including direct binding of vIRF to its cellular ho-
mologs, competitive binding to the transcriptional coactivator
p300, and inhibition of the histone acetyltransferase activity of
p300 (64, 160, 258, 292, 302, 550). vIRF-1 also directly binds
p53 to inhibit its phosphorylation and acetylation and blocks its
ability to transactivate transcription (369, 457). Antisense in-
hibition of K9 expression in PELs demonstrated a critical role
for IRF-1 in reactivation of KSHV from latency and lytic gene
expression (293). The other lytic viral IRF, vIRF-3, is most
closely related to the cellular IRF-4 and the latent vIRF-2 and
also blocks IFN signaling by functioning as a dominant nega-
tive inhibitor of IRF-3 and IRF-7 (309).
K1. K1 encodes a transforming and immunomodulatory
protein; it is the most highly divergent ORF in the KSHV ge-
nome and is discussed in full above.
VOL. 67, 2003 KSHV MOLECULAR GENETICS 195

ORF16/vBCl-2. The product of KSHV ORF16 retains high-
est sequence similarity to human bcl-2 in its BH1 and BH2
domains but little similarity in the BH3 domain (98, 446). It is
expressed in a lytic pattern in spindle cells and monocytes in
KS lesions, and its transcript is lytically induced in PEL cells
(Fig. 1), although the protein has evaded easy detection there
(98, 446, 533). As predicted by sequence homology, vBcl-2 is
antiapoptotic; unlike its cellular counterpart, it can inhibit apo-
ptosis induced by KSHV vCyc in the presence of high cdk6
(378). Also unique to vBcl-2, it cannot be converted to a pro-
apoptotic form by caspase-mediated cleavage (36). In rabbit
reticulocyte lysates, vBcl-2 does not directly interact with cel-
lular Bcl-2 family members, but in yeast two-hybrid assays, it
does interact with cellular Bcl-2; it also partially reverses cell
death induced by Bax (98, 446). The antiapoptotic mechanism
of vBcl-2 may be attributable to its interaction with the pro-
apoptotic cellular protein Diva, which binds to the caspase-9
regulator Apaf-1 to prevent Bcl-XL from blocking cell death
(228).
K7/vIAP. The product of K7 (Fig. 1) is a homolog of the
cellular protein survivin-⌬Ex3 and is not conserved in other
close relatives of KSHV (522). Both proteins contain a BH2
domain and a partial baculovirus inhibitor of apoptosis protein
(IAPs) repeat domain. The 19- to 21-kDa glycoprotein product
of K7 localizes to mitochondrial membranes and inhibits apo-
ptosis induced by the Fas and TRAIL pathways, Bax, TNF-␣
plus CHX, staurosporine, ceramide, and other chemicals (158,
522). Mechanistically, vIAP targets two critical arms of the
early and late cellular apoptotic response. It acts as a protein
bridge to help target Bcl-2 to activated caspase-3 to inhibit its
function as an effector of cell death (522). It also binds to the
cellular calcium-modulating cyclophilin ligand to enhance the
cytosolic Ca
2⫹
flow and protect cells from mitochondrial dam-
age and apoptosis (158).
K3/MIR1, K5/MIR2. The modulators of immune recogni-
tion (MIRs) are eponymous proteins that actively eliminate
the cell surface expression of receptors recognized by the cy-
tolytic arm of the immune system. Both MIR1, encoded by K3,
and MIR2, encoded by K5, specifically increase the rapid en-
docytosis of mature major histocompatibility complex MHC
class I from the surface of infected cells and stimulate its
degradation by cellular proteases; MIR2 but not MIR1 also
stimulates the scavenging of B7.2 and ICAM-1 proteins from
the surface (112, 113, 212, 213, 231, 232, 394). The K3 and K5
products are endoplasmic reticulum proteins that both contain
plant homeodomain ring finger motifs (similar to those found
in cellular E3 ubiquitin ligase proteins), a single transmem-
brane domain, and a tyrosine-based sorting motif that is a
candidate target for cellular adaptor molecules in intracellular
sorting (112, 114, 212, 231, 232, 335, 394). Both MIR proteins
selectively target MHC class I but not class II; however, al-
though K3 targets all four HLA allotypes, K5 specifically tar-
gets HLA-A and HLA-B (232). Target specificity is deter-
mined by the transmembrane domain and cytoplasmic tail of
the respective target proteins (114, 231, 232), and dominant
negative dynamin mutants that inhibit endocytosis block the
functions of K3 and K5 (112, 113). Cells expressing K5 are
impaired in the induction of CD28-dependent and indepen-
dent T-cell stimulation (113) and in the stimulation of activity
of NK cell lines (231), but not primary NK cells (113).
Despite their homology to each other, MIR1 and MIR2 may
function by related but different mechanisms. As predicted by
its PHD domain, MIR2/K5 is an E3-ubiquitin ligase that stim-
ulates the ubiquitylation of lysines in the C termini of its
cellular targets, in a ring finger-dependent fashion (114). How-
ever, the inability to detect ubiquitylated MHC class I complex
in response to MIR I/K3 expression has led to the suggestion
that the direct target of ubiquitylation by K3 is not the MHC
class I molecule and that cysteine and aspartyl proteases, but
not the ubiquitin proteasome, mediate MHC class I degrada-
tion (308). MIR1/K3 dominantly targets MHC class I com-
plexes to dense lysosomal compartments via the trans-Golgi
network, in a fashion that requires an intact diacidic cluster
and tyrosine-based signaling motif in K3, respectively (335).
K3 and K5 are both expressed in KS tissue in a lytic pattern,
and K3 has been detected as part of a bicistronic transcript in
a screen for IE gene expression in PEL cells (213, 430). The K5
transcript expression is resistant to low, but not high, concen-
trations of CHX in PEL cells (212) but has not been detected
as an IE product in subtractive screens (430, 542).
K14/viral OX2. K14 (Fig. 1) encodes a homolog of the cel-
lular OX2 protein, a glycosylated cell surface protein that is a
member of the immunoglobulin superfamily and restricts cy-
tokine production in a paracrine fashion. Viral OX2 shares all
of these structural features but instead potently activates in-
flammatory cytokine production (IL-1␤, TNF-␣, and IL-6)
from primary, PBMC-derived monocytes/macrophages and
dendritic cells (105). It cooperates in paracrine induction with
IFN-␥ when added solubly to media or when expressed stably
on BJAB (B-lymphocyte) cells (105).
LYTIC REACTIVATION OF KSHV
Among a number of KSHV genes tested, only forced over-
expression of the product of the ORF50 gene, a protein named
Rta, can reactivate the virus from latency in PEL cells (194,
311, 312, 490). The major ORF50 transcript is expressed with
IE kinetics (489, 542) and is tricistronic, also encoding the
downstream genes K8/K-bZIP/RAP and K8.1 (203, 303, 312,
454, 490, 542). Alternative splicing events in the downstream
exons also leads to the expression of two minor tricistronic
transcripts (542). The ORF50 locus has thus been deemed the
major IE locus because two noncoding RNAs are transcribed
antisense to Rta in an IE fashion (311, 448, 542) and the K8
gene is also transcribed with IE kinetics independently of the
upstream ORF50 gene (448). As expected, the Rta transcript is
expressed in a lytic pattern in KS lesions (248, 489).
ORF50 encodes a 691-aa nuclear polypeptide (Fig. 3) that is
highly posttranslationally modified, including extensive phos-
phorylation (311, 312). Transient transfections demonstrate
that Rta directly but selectively transactivates KSHV promot-
ers that are expressed with later kinetics during reactivation
(89, 97, 238, 310–312, 477, 524, 525, 537). The C terminus of
Rta contains four repeats of alternating acidic and bulky hy-
drophobic amino acids, a primary structure conserved with
many eukaryotic transcriptional activation domains (311), and
is sufficient to strongly activate transcription when targeted to
promoters with a heterologous DNA binding domain (311,
454, 524) (Fig. 3). Deletion of the activation domain generates
a truncated mutant of Rta that forms mixed multimers with
196 DOURMISHEV ET AL. MICROBIOL.MOL.BIOL.REV.

wild-type Rta and functions as an ORF50-specific dominant
negative inhibitor of transactivation (311). Furthermore, ex-
pression of the truncated Rta in transfected BCBL-1 (PEL)
cells, in the context of physiologic levels of endogenous ORF50
expressed from the viral template, potently suppresses sponta-
neous reactivation from latency and strikingly suppresses viral
replication induced by TPA, sodium butyrate, and ionomycin
(311). The dominant negative Rta truncation mutant thus dem-
onstrated that (i) transcriptional transactivation by ORF50/Rta
is not only sufficient but also essential for viral reactivation and
(ii) multiple reactivation signals that function using different
biochemical mechanisms all converge at ORF50/Rta and re-
quire downstream transactivation by Rta to successfully re-
activate the virus (311). Similarly, methotrexate treatment of
PEL cells inhibits the expression of Rta, the activation of genes
downstream of Rta, and viral reactivation (119).
ORF50/Rta binds to several viral promoters in a sequence-
specific fashion (89, 126, 295, 310, 476, 477; S. Lynch, K. Dris-
coll, D. Palmeri, and D. M. Lukac, unpublished observation),
yet direct DNA binding by Rta is not sufficient to specify a
promoter as a target of ORF50/Rta-mediated transactivation;
instead, specificity appears to be governed by combinatorial
interactions with cellular proteins. Rta binds directly to a pal-
indromic sequence shared by the ORF57/Mta and K8/K-bZIP/
RAP promoters, and the N-terminal 272 aa of Rta is sufficient
for binding in vitro (310) (Fig. 3). However, genetic and bio-
chemical experiments demonstrate that transactivation of the
Mta promoter by Rta requires a direct interaction with the
sequence-specific DNA binding protein RBP/Jk (also known
as CBF-1 and CSL); transactivation of this promoter is en-
hanced by DNA binding of Rta to its element (295, 310). RBP-
Jk-binding sites also mediate Rta-driven activation of the pro-
moters of KSHV ORF6/single-stranded DNA binding protein
and thymidine kinase (295; Lynch et al., unpublished). Simi-
larly, in vitro experiments demonstrate that the DNA binding
domain of Rta (aa 1 to 272) (310) is sufficient to bind to a
heterologous element shared by the viral PAN and K12/kapo-
sin A promoters (477, 478), yet fusion of this polypeptide to a
heterologous activation domain is not sufficient to transacti-
vate the kaposin promoter in vivo (89). Instead, activation
requires aa 1 to 490 of Rta in this context (89), a region of Rta
that probably interacts with other cellular DNA binding factors
(Fig. 3). Furthermore, autoactivation of the proximal ORF50/
Rta promoter by Rta (127, 194, 454) requires an interaction
with the cellular protein octamer-1 (but not octamer-2) and an
intact octamer element that is located approximately 200 bp
upstream of the ORF50 transcription start site (440).
Numerous other protein-protein interactions modulate trans-
activation mediated by ORF50/Rta. KSHV Rta binding pro-
tein (K-RBP) is a ubiquitously expressed cellular protein that
contains homology to Krueppel-associated box-zinc finger pro-
teins and synergizes with Rta in transactivation of the promot-
ers of KSHV Mta, K8/K-bZIP/RAP, K6/vMIPI, and Rta itself
(525). K-RBP interacts directly with a DNA binding fragment
of Rta, but their combinatorial transactivation mechanism has
not yet been described. The cellular Jun protein cooperates
with ORF50/Rta through a mutual interaction with the coac-
tivator CBP (208). A DE target of ORF50/Rta, the posttran-
scriptional activator protein ORF57/Mta (Fig. 1), uses an
uncharacterized mechanism to synergize with Rta in a pro-
moter-specific fashion (259). Repression of Rta-mediated ac-
tivation has also been demonstrated for the E1A protein by
competition for CBP and for HDAC1, which binds to a central
region of Rta (208).
Since Rta is essential for viral reactivation (311), it follows
that all, or a subset, of its direct targets of transactivation will
also be essential for lytic reactivation of KSHV. The observa-
tion that Rta directly transactivates the promoters of many of
the candidate pathogenic genes of KSHV, including K12/
kaposin, vIL-6, vMIP-I, vIRF-1, vGPCR, and K1 (55, 97, 119,
126, 507, 525), suggests that the ability of these proteins to
abrogate normal cellular growth control and physiology is es-
FIG. 3. Primary structure-function map of KSHV ORF50/Rta protein. This schematic shows a linear representation of the amino acid content
and predicted structural motifs of the ORF50/Rta protein. The amino acid numbering is as published in reference 436. The approximate position
of each functional domain is shown by a black bar, with amino acid boundaries indicated by numbers, corresponding to the function shown in the
column on the right (see the text for references). Abbreviations: ⫹⫹⫹, basic amino acid rich; LZ, putative leucine zipper; ST, serine/threonine-
rich; hyd/DE/hyd, repeats of hydrophobic and acidic amino acids, as in reference 311 and described in the text.
V
OL. 67, 2003 KSHV MOLECULAR GENETICS 197

sential for optimizing the cellular and anatomical milieu for
viral replication. Likewise, activation of cellular IL-6 (125) by
Rta and the ability of Rta to block p53-mediated apoptosis by
competing for binding to CBP (209) confirm that Rta also
targets cellular pathways of growth control. Concordant with
the orchestration of the KSHV lytic cycle by Rta and patho-
genic progression, Chen et al. demonstrated that the promoter
controlling the expression of ORF50/Rta is repressed by meth-
ylation and is demethylated by inducing signals; clinically, the
ORF50/Rta promoter in a latent KSHV carrier was highly
methylated, while it was virtually unmethylated in most pa-
tients with MCD, PEL, and KS (96).
Thus, lytically infected cells may serve not only as reservoirs
of infectious virus but also as reservoirs of viral proteins that
influence cell proliferation in a paracrine fashion. This pre-
sents a paradox, since (as discussed above) host cells that are
stimulated to reactivate KSHV succumb to virus-mediated lysis
during productive replication. However, a slow or inefficient
lytic cycle, allowing a prolonged period of viral delayed-early
gene expression prior to viral cell lysis, would be consistent
with such a model. Indeed, reactivation of KSHV in one cul-
tured endothelial cell model displays just such a slow lytic
phase (106). Nonproductive, abortive reactivation might also
be expected to enhance cell growth.
Putative reservoirs of KSHV lytic gene expression may thus
function to recruit and stimulate the proliferation of immune
cells (by paracrine factors like the vMIPs, vIL-6, and viral
OX2) at sites of viral replication, potentially to increase the
availability of target cells permissive for viral replication. Re-
cruitment and stimulation of target cells to enhance their per-
missiveness has been documented for other herpesviruses such
as human and murine cytomegalovirus (261, 438) and equine
herpesvirus type 1 (509). Supporting this hypothesis, the addi-
tion of inflammatory cytokines to cultured human peripheral
blood leukocytes from KSHV-positive patients is essential for
maintenance of persistent viral infection in these cells (155,
347, 468). The spontaneous reactivation of the virus in a char-
acteristic small percentage of cells, evident in every KS speci-
men, as well as infection of PELs and endothelial cells, is also
consistent with the requirement for low-level lytic-gene expres-
sion in maintenance of the infected-cell population. Translated
to the clinical setting, this may represent a pathogenic mech-
anism by which KSHV initiates or maintains the aggressive
inflammatory infiltrate that characterizes KS lesions.
Furthermore, many of the cytokines known to reactivate
KSHV in PEL cells, including oncostatin M, hepatocyte
growth factor, IFN-␥, IL-6, HIV-1 Tat, and an unidentified
soluble factor released from HIV-1-infected cells (43, 88, 224,
339, 347, 476, 510), are expressed in KS lesions (reviewed in
reference 154) and are also required as growth factors for
spindle cells (66, 153, 341, 368), suggesting that reactivation of
KSHV from latency may be coregulated with stimulation of
target cells.
PERSPECTIVES AND FUTURE DIRECTIONS
Although ORF50/Rta can autoactivate its own expression
(127, 194, 440, 454), little is known about the direct effect of
the other physiologic reactivation signals on either the induc-
tion of ORF50 expression or its subsequent function during
reactivation. Functional interactions of ORF50/Rta with nu-
merous heterologous proteins have been demonstrated in un-
infected cells, but their relevance to viral reactivation and
replication remain unevaluated. The expression or regulation
of the binding partners of ORF50/Rta will probably provide a
significant regulatory step in the success or efficiency of reac-
tivation of the virus to productive replication. Such regulation
could impact the potential for Rta expression in the absence of
productive replication, the induction of “sublytic” gene expres-
sion programs during reactivation, the period of DE growth-
deregulatory gene expression prior to lysis, or the uniformity of
latently infected cell populations to permissively support reac-
tivation.
Does ORF50/Rta integrate all of the viral “reactivation”
signals that induce lytic gene expression and/or viral replica-
tion? Chatterjee et al. recently demonstrated that IFN-␣ di-
rectly induces vIL-6 from latent KSHV in an IE fashion (95);
the lack of any other detectable viral gene expression suggests
that lytic genes can be induced in the absence of ORF50/Rta.
Therefore, some signaling pathways can bypass ORF50 to ac-
tivate DE genes, providing a mechanism by which viral growth-
stimulating genes can be induced independently of productive
reactivation, and cell lysis. The response of Rta expression or
function to a particular signal might thus determine the fate of
an individual infected cell to serve exclusively as either a res-
ervoir of progeny virus or lytic growth factors.
A second provocative aspect of KSHV biology is the con-
servation of multiple viral genes with the potential for coun-
teracting the IFN response of the host. Even more provocative
is that the virus has also conserved positive modulators of the
host IFN response, suggesting that a subset of IFN-inducible
cellular activities are essential for viral replication and may
promote permissive infection. Thus, the broad induction of
many IFN-responsive genes following de novo infection of
endothelial cells by KSHV (359, 406) probably is not attribut-
able solely to the classic mammalian antiviral response. In-
deed, two viral proteins, one latent and one lytic, actively
stimulate interferon target genes. Microarray analyses of the
global effects of LANA-1 expression in B cells demonstrated
that 5 of 15 transcripts up-regulated at least twofold were from
IFN-stimulated genes, in the absence of detectable effects on
the IFN-␣ or IFN-␤ itself (421). Likewise, the lytic switch
protein ORF50/Rta functions as a ligand-independent activa-
tor of STAT3 by directly binding to it, recruiting it to the
nucleus, stimulating its dimerization, and activating the tran-
scription of STAT-driven reporter genes (210). In fact, IFN-␥
alone has proviral effects on KSHV replication, demonstrated
by the induction of lytic reactivation of latent KSHV by the
addition of IFN-␥ to the growth media of cultured PEL cells
(43, 88, 339) and the promotion of maintenance of persistent
KSHV infection by IFN-␥ addition to primary explanted PBLs
(155, 347, 468). In agreement, early attempts to treat KS pa-
tients with IFN-␥ were associated with progression of disease
(172, 271), suggesting that this cytokine promoted viral repli-
cation in infected people.
Conversely, IFN-␣ has antiviral effects on KSHV infections
in PEL and explanted PBL models (88, 346), as well as in
clinical settings (274), affirming that only specific IFN-induced
products can be usurped by the virus to its advantage. For
example, IRF7, a critical protein in the cellular response to
198 DOURMISHEV ET AL. MICROBIOL.MOL.BIOL.REV.

IFN-␣, is one of the most strongly induced transcripts follow-
ing de novo KSHV infection (406), but the virus expresses
ORF45, an IE gene product that blocks IRF-7 phosphorylation
and nuclear accumulation (543). vIL-6 also plays a novel role
in the ability of KSHV to escape the antiviral effects of IFN-␣
(95).
Remarkably, the virus has conserved four lytic and latent
antagonistic homologs of IRFs. This seeming redundancy in
anti-IFN functions may reflect the requirement for the virus to
temper the IFN response so that it does not exceed the thresh-
old at which it is beneficial for replication. Alternatively, the
individual viral modulators of the IFN pathway may have been
conserved to target very specific regulators of the response,
stimulating the functions beneficial for the virus while blocking
the harmful ones. Supporting this hypothesis, K9/vIRF-1 is
both an antagonist of cellular IRFs and well as a transcrip-
tional activator and is essential for efficient lytic gene expres-
sion during reactivation (293). A key question that remains,
however, concerns the mechanism(s) by which specific IFN-
induced cellular genes have a proviral effect. One clue may
come from models of mouse cytomegalovirus infection, in
which a G
1
cell cycle arrest following IFN induction is essential
for viral DNA replication (219, 434).
Many of the questions concerning the requirement for latent
and lytic gene expression in KSHV pathogenesis will be ad-
dressable with newly described systems for de novo infection of
endothelial cells (106, 130, 278, 358). Combining such systems
with tenable genetic approaches for generating viral mutations,
such as bacmid clones of full-length KSHV (61, 123, 541), will
allow precise determination of gene functions during infection
by using knockouts and deletions of viral genes and critical cis
elements, as well as knockins to replace wild-type viral genes
with mutants and variants. The contribution of the latent and
lytic cycles to pathogenesis in endothelial cells, the effects of
expression of the pathogenic latent and DE genes in the con-
text of viral infection, and the determination of genetic re-
quirements and gene expression kinetics during de novo infec-
tion and establishment of persistence, can all be realized.
The PEL models remain the most robust systems for under-
standing KSHV persistence and reactivation from latency.
Since most PEL cells are dually infected with latent KSHV and
EBV (reviewed in reference 138), this suggests that B-cell
reservoirs of KSHV may also be EBV infected in vivo. Fur-
thermore, KSHV LANA-1 can modulate the expression pro-
gram of the EBV latent genes: it potently activates the pro-
moter of the EBV LMP-1 gene (202) (a constitutive signaling
and transformation protein [reviewed in reference 256]) but
reduces the expression of EBV EBNA-1 (the LANA-1 or-
tholog) and represses EBV EBNA-2 activation of a second
latency promoter (273). Likewise, the KSHV lytic switch pro-
tein Rta binds to RBP-Jk (295), a key cellular target of the
EBV latent transforming program (256), and in dually infected
PEL cells, KSHV and EBV can be selectively induced from
latency (342, 490). As additional functional mechanisms of the
KSHV proteins are revealed, interesting insights into cross-
regulation of the two viruses could contribute to a greater
understanding of the unique KSHV-associated diseases.
Little is known about primary infection by KSHV and the
progression of KS in the absence of coinfection with HIV.
Future prospective studies of African children, including dis-
ease progression (Kaplan-Meier analyses) and subtype-specific
infection, should be revealing. In the West, the risk factors for
infection in the 3 to 10% of the general population who are
seropositive are currently not understood, nor are the geo-
graphic risk factors that stratify the Mediterranean popula-
tions. These insights are crucial for reducing the risk of KSHV
transmission, infection, and disease in humans, while the mo-
lecular studies and animal models will continue to reveal the
unique mechanisms of this pathogen and targets for treatment.
ACKNOWLEDGMENTS
Research in the laboratory of D.M.L. is supported by the New Jersey
Commission on Cancer Research, American Foundation for AIDS
Research, the Ruth Estrin Goldberg Memorial for Cancer Research,
and the American Cancer Society.
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