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JOURNAL OF VIROLOGY, Sept. 2011, p. 9637–9640 Vol. 85, No. 18
0022-538X/11/$12.00 doi:10.1128/JVI.05074-11
Copyright © 2011, American Society for Microbiology. All Rights Reserved.
NOTES
The TRIM5␣ Genotype of Rhesus Macaques Affects Acquisition of
Simian Immunodeficiency Virus SIVsmE660 Infection after
Repeated Limiting-Dose Intrarectal Challenge
䌤
Matthew R. Reynolds,
1
* Jonah B. Sacha,
2,3
Andrea M. Weiler,
1
Gretta J. Borchardt,
1
Chrystal E. Glidden,
1
Neil C. Sheppard,
4
Francesca A. Norante,
1
Philip A. Castrovinci,
1
Jacqueline J. Harris,
5
Henry T. Robertson,
5
Thomas C. Friedrich,
1
Adrian B. McDermott,
6
Nancy A. Wilson,
1
David B. Allison,
5
Wayne C. Koff,
6
Welkin E. Johnson,
7
and David I. Watkins
1,8
AIDS Vaccine Research Laboratory, 555 Science Dr., Madison, Wisconsin 53711
1
; Vaccine and Gene Therapy Institute, Oregon Health and
Science University, 505 NW 185th Ave., Beaverton, Oregon 97006
2
; Oregon National Primate Research Center, Oregon Health and
Science University, 505 NW 185th Ave., Beaverton, Oregon 97006
3
; Vaccine Research, Worldwide Research and Development,
Pfizer Inc., 10777 Science Center Drive, San Diego, California 92121
4
; Department of Biostatistics, Section on Statistical Genetics,
University of Alabama at Birmingham, Birmingham, Alabama 35294
5
; International AIDS Vaccine Initiative, New York,
New York 10038
6
; New England Primate Research Center, Department of Microbiology and Molecular Genetics,
Harvard Medical School, Southborough, Massachusetts
7
; and Department of Pathology and
Laboratory Medicine, University of Wisconsin—Madison, Madison, Wisconsin
8
Received 10 May 2011/Accepted 27 June 2011
It has recently been shown that polymorphism at the rhesus macaque TRIM5 locus can affect simian
immunodeficiency virus (SIV) replication. Here we show that TRIM5 alleles can also affect acquisition of
SIVsmE660. Animals coexpressing the TRIM5
TFP
and TRIM5
CypA
alleles took significantly longer to become
infected with SIVsmE660, but not SIVmac239, after repeated limiting-dose intrarectal challenge than did
animals expressing other TRIM5 allele combinations. Our results indicate that the TRIM5 alleles can be a
barrier to productive infection and that this should be taken into account when designing acquisition studies
using SIVsmE660 or related viruses.
Lentiviruses and their hosts have been interacting and
adapting to each other for millions of years. Humans and
nonhuman primates, like other mammals, express cellular re-
striction factors to inhibit viral replication (6). These restric-
tion factors offer protection against a broad range of viruses
and represent an important barrier to zoonotic transmissions.
Recently, three major classes of retroviral restriction factors
have been identified: APOBEC3, tripartite motif protein 5
alpha (TRIM5␣), and tetherin (12, 16, 19, 20). Each of these
factors targets a different step in the virus life cycle to suppress
viral replication. To counteract these restriction factors, pri-
mate lentiviruses have developed a series of accessory proteins
that, in part, circumvent their actions and allow viruses to
efficiently replicate (11).
Polymorphic alleles at the rhesus macaque TRIM5 locus cor-
relate with a 1.3- to 3-log reduction in simian immunodeficiency
virus (SIV) replication (7, 10). The mechanism by which TRIM5␣
affects viral replication is unclear, but it appears to act by binding
incoming viral capsids and mediating premature viral uncoating
(18). A recent study also suggests a role for TRIM5 mediating the
early, innate immune response to retroviral infection (14). Rhesus
macaque TRIM5 is polymorphic, and the encoded protein in-
cludes a TRIM5-cyclophilin A (CypA) chimera in which CypA
replaced the TRIM5␣ binding domain (7, 10, 13, 17, 19). While
there are multiple TRIM5 alleles, they can be categorized into
three functional allelic classes based on variations in their C-ter-
minal domains and their relative effects on SIVsmE543-3 repli-
cation: TRIM5
CypA
, TRIM5
TFP
, and TRIM5
Q
(7). Combinations
of these alleles result in six possible functional TRIM5 genotypes.
A previous study showed that cell lines expressing TRIM5
TFP
or
TRIM5
CypA
alleles were resistant to infection with the molecular
clone SIVsmE543-3 but not with SIVmac239 (7). Cells expressing
TRIM5
Q
alleles were susceptible to both viruses. These in vitro
results were recapitulated in vivo in a cohort of animals infected
with SIVsmE543-3. Animals with two resistance-conferring
TRIM5 alleles (TRIM5
TFP/TFP
or TRIM5
TFP/CypA
) had a 2.0- to
3.0-log reduction in chronic-phase plasma virus replication in
comparison to animals with the TRIM5
Q/Q
genotype. Further-
more, animals with one resistance-conferring allele (TRIM5
TFP/Q
or TRIM5
Q/CypA
) had intermediate plasma viral loads. A less
dramatic effect on plasma virus replication was also seen in a
cohort of SIVmac251-infected animals by using similar TRIM5
groupings (10).
We first investigated whether the biological isolate
SIVsmE660 might harbor TRIM5␣-resistant variants. Previous
* Corresponding author. Mailing address: AIDS Vaccine Research
Laboratory, 555 Science Dr., Madison, WI 53711. Phone: (608) 890-
0842. Fax: (608) 265-8084. E-mail: mrreynol@wisc.edu.
䌤
Published ahead of print on 6 July 2011.
9637
studies identified amino acids in Gag p27
CA
affecting TRIM5-
mediated restriction (2, 7, 9). Since two different stocks of
SIVsmE660 were used in our studies, we employed ultradeep
pyrosequencing as previously described (1) to determine
whether there existed any variation that might result in
TRIM5␣-resistant quasispecies. Briefly, we used four overlap-
ping reverse transcription-PCR amplicons of approximately 2.5
kb spanning the entire SIVsmE660 genome. The reverse tran-
scription-PCR products were then randomly fragmented using
modified transposons (Nextera; Epicentre Biotechnology) and
pyrosequenced. At average nucleotide depths of 493 for
SIVsmE660 stock 17480 and 819 for SIVsmE660 stock 17571,
we found no nonsynonymous variation above 1% from the
SIVsmE543-3 regions previously identified to affect rhesus ma-
caque TRIM5-mediated restriction (Fig. 1) (7). This suggested
that an animal’s TRIM5 genotype could impose a bottleneck
for productive infection following limiting-dose intrarectal
SIVsmE660 challenge.
Only a few human immunodeficiency virus (HIV) strains
cross mucosal barriers to establish infection in humans (4).
Investigators have been using repeated limiting-dose SIV chal-
lenge of rhesus macaques to mimic HIV transmission and have
shown that only one to three viral species similarly initiate
infection in monkeys (5, 21). Under these conditions, it is
possible that the TRIM5 genotype of an animal might play a
role in whether an animal becomes productively infected with
SIV. To address this issue, we retrospectively analyzed a cohort
of 62 Indian rhesus macaques at the Wisconsin National Pri-
mate Research Center (WNPRC) that were not vaccinated
with SIV antigens to determine if TRIM5 genotype affected
the rate at which they became productively infected with
SIVsmE660. We genotyped our animals based on the following
three allelic classes: TRIM5
CypA
, TRIM5
TFP
, and TRIM5
Q
.We
established the TRIM5 genotypes of the animals either by
sequencing genomic DNA as previously described (7) or by
sequence-specific priming PCR (PCR-SSP) assays. To distin-
guish TRIM5
TFP
and TRIM5
Q
alleles by PCR-SSP, we took
advantage of unique polymorphisms in the TRIM5 SPRY/
B30.2 domain and used previously published thermal cycling
conditions and reagents (3). The TRIM5
TFP
and TRIM5
Q
PCR-SSP reactions share a forward primer (5⬘-AGA GAG
CTA ACA GAT GCC CG-3⬘) designed to anneal to both of
the TRIM5 variants, while the reverse primers targeted unique
sites within the TRIM5
TFP
and TRIM5
Q
alleles to confer spec
-
ificity (TFP allele reverse primer, 5⬘-ACA ATG AAA GGA
GCA AAA GGA GTA TGT G-3⬘; Q allele reverse primer,
5⬘-CAC AAT GAA AGG AGC AAA AGG AGT ATG TA-
3⬘). Primers were used at a 0.3 M working concentration. The
TRIM5
CypA
allele was genotyped using a previously published
protocol which utilized PCR followed by NsiI digestion (13).
To confirm that the consistency of the PCR-SSP genotyping
was equivalent to that of genomic DNA sequencing, we tested
a subset of animals by both methods and obtained identical
results.
We next investigated whether the TRIM5 genotype affected
acquisition of SIVsmE660. Rhesus macaques used in this anal-
ysis were intrarectally inoculated on a weekly or biweekly basis
with initial limiting doses ranging from 225 to 450 50% tissue
culture infective doses (TCID
50
) for SIVsmE660-17571 (52
animals) and 800 TCID
50
for SIVsmE660-17480 (10 animals)
as previously described (15, 21). We performed a Kaplan-
Meier analysis to test whether any of the TRIM5 genotypes
differed in the number of challenges required to achieve pro-
ductive infection. We discovered that animals having the
TRIM5
TFP/CypA
genotype took significantly more exposures to
get infected than animals expressing the other TRIM5 geno-
types (log rank test; chi-square ⫽ 15.74; df ⫽ 5; P ⫽ 0.008)
(analyses performed with GraphPad Prism version 4.0c) (Fig.
2A). Of note, four animals with the TRIM5
TFP/CypA
genotype
were the only animals to remain uninfected at the conclusion
of intrarectal challenges, two animals after the seventh chal-
lenge and two after the eleventh challenge. We therefore fur-
ther explored the resistance to infection with SIVsmE660 of
the TRIM5
TFP/CypA
animals in comparison to the other geno
-
types. We compared the numbers of challenges needed to
achieve productive infection per TRIM5 genotype by using the
parametric generalized gamma model because it was deter-
mined to be the best model in comparison to nonparametric
models by Akaike information criterion scores for examining
dosages that increased over time. For this analysis, we desig-
nated the inoculation after intrarectal challenges were stopped
for the uninfected animals as the infecting dose (e.g., we des-
ignated the animals remaining uninfected after 11 inoculations
as being infected with the 12th inoculation). Animals with the
TRIM5
TFP/CypA
genotype took significantly longer to produc
-
tively infect with limiting doses of SIVsmE660 than each of the
remaining TRIM5 genotypes (P ⬍ 0.0001) (Fig. 2B).
Since the TRIM5 genotype affects acquisition of infection
with SIVsmE660, we sought to determine if it has the same
effect on another SIV strain commonly used in challenge stud-
ies, the molecularly cloned SIVmac239. Kirmaier et al. previ-
ously established that SIVmac239, unlike SIVsmE543-3, is re-
sistant to all of the TRIM5 allelic classes in vitro, which is likely
due to amino acid substitutions in the viral capsid that prevent
or inhibit TRIM5-mediated restriction (7). However, it is still
possible in the setting of a limiting-dose challenge in which
only a few viruses likely cross the mucosal barrier and establish
infection that TRIM5␣ might have an effect on acquisition. We
therefore examined a cohort of 38 naïve animals repeatedly
challenged with limiting doses of SIVmac239 (produced in
vitro with rhesus macaque peripheral blood mononuclear cells
[PBMC]) housed at the WNPRC and at the University of
FIG. 1. Sequence of SIVsmE660 Gag p27
CA
known to affect TRIM5-
mediated restriction. Partial sequences of the core nucleocapsid protein
amino acids 72 to 101 of the two stocks of SIVsmE660 analyzed in this
study are aligned against that of the closely related molecular clone
SIVsmE543-3. The putative amino acids affecting restriction mediated by
TRIM5
TFP
(
74
LPA
76
) and TRIM5
CypA
(R
83
) are highlighted in red. Non
-
synonymous variations from the SIVsmE543-3 sequence are color coded
by frequency. X indicates a mixed amino acid sequence.
9638 NOTES J. V
IROL.
Pittsburgh. Of note, a few animals included in this cohort
were repeatedly challenged with either 30 or 300 TCID
50
SIVmac239, which in retrospect was probably an extremely
limiting dose of the virus, because it required between 8 and 21
challenges to achieve infection. We also had two animals with
either the TRIM5
TFP/TFP
or TRIM5
TFP/Q
genotype that did not
become infected after 13 limiting-dose challenges. Despite
this, we detected no significant difference in the rates of ac-
quisition of SIVmac239 for the different TRIM5 genotypes
when we performed the Kaplan-Meier analysis (P ⫽ 0.67) (Fig.
3). However, due to the relatively small number of animals in
this analysis, it is possible that TRIM5 has a more limited effect
on acquisition of SIVmac239 that cannot be discerned in our
study.
Our study highlights the complex nature of virus-host inter-
actions and the difficulty of modeling HIV transmission in
rhesus macaques. Our analysis also expands upon a recent
report showing that TRIM5␣ alleles can affect the acquisition
of SIVsmE660 infection by naïve and vaccinated rhesus ma-
caques (8). In this study, however, TRIM5
TFP
and TRIM5
CypA
were grouped together as “restrictive” alleles, and homologous
expression of these alleles resulted in delayed infection with
SIVsmE660. In our cohort of animals, we further subdivided
the genotypes to include TRIM5
CypA
as a separate allele and
only the heterozygous TRIM5
TFP/CypA
genotype made a signif
-
icant impact on the rate of acquisition of SIVsmE660 infection.
It is possible that since animals with the TRIM5
TFP/CypA
geno
-
type have two TRIM5␣ molecules that bind distinct regions of
FIG. 2. Acquisition of SIVsmE660 infection after repeated limiting-dose challenge is affected by TRIM5 genotype. (A) Kaplan-Meier curve
analysis of the effects different TRIM5 genotypes have on the rate of acquisition of SIVsmE660 infection after repeated limiting-dose intrarectal
challenge. The statistical significance of the rates of infection of the different TRIM5 genotypes was determined by log rank test. (B) A comparison
of the numbers of challenges needed to productively infect animals expressing the different TRIM5 genotypes with SIVsmE660. Significance
between the rates of infection of animals having the TRIM5
TFP/CypA
genotype and the other TRIM5 genotypes was determined by parametric
generalized gamma model.
FIG. 3. Acquisition of SIVmac239 infection after repeated limiting-dose intrarectal challenge is not affected by TRIM5 genotype. The
Kaplan-Meier curve analysis revealed no significant differences in the acquisition of SIVmac239 infection after repeated limiting-dose intrarectal
challenge among the TRIM5 genotypes. We performed a power analysis and determined that our cohort of SIVmac239 challenged animals had
73% power to detect an effect by the TRIM5
TFP/CypA
genotype on acquisition of infection similar to that observed for the cohort of SIVsmE660
challenged animals.
V
OL. 85, 2011 NOTES 9639
the viral capsid, they eliminate incoming virus more effectively
than animals having TRIM5␣ molecules targeting only one
region of the viral capsid, even for animals homozygous for
resistance-conferring TRIM5 alleles. It is also possible that the
TRIM5
TFP/TFP
and TRIM5
CypA/CypA
genotypes have a less dra
-
matic effect on the acquisition of infection, which would be-
come apparent in a larger cohort of animals. Our study also
indicates that under physiological conditions in which only a
few viruses are crossing the mucosal barrier, TRIM5␣ can
decrease the probability of productive infection occurring.
However, since restriction by TRIM5␣ is known to be satura-
ble, this effect may be subtle and overcome by high-dose mu-
cosal or intravenous exposures (19). In this regard, the previ-
ous studies identifying that TRIM5 genotypes differentially
affects SIVsmE543-3 and SIVmac251 replication did not note
an effect on acquisition of infection after intravenous inocula-
tion (7, 10). Our results suggest that animals entering repeated
limiting-dose SIV challenge studies should be typed for their
TRIM5 genotype, especially if SIVsmE660 or a related virus is
used. When possible, TRIM5
TFP/CypA
animals should be either
excluded from the study or balanced among test groups in a
manner similar to that for animals expressing protective major
histocompatibility complex (MHC) class I alleles.
We thank the veterinary staff at the Wisconsin National Primate
Research Center (WNPRC) for their assistance. We also thank Ben
Burwitz, Ben Bimber, Simon Lank, and David O’Connor for their
guidance in pyrosequencing and analyzing the SIVsmE660 stocks.
This research was supported by funds from the International AIDS
Vaccine Initiative, National Institutes of Health (NIH) grants R01
AI049120, R01 AI076114, R01 AI076114, R24 RR016038, R24
RR015371, and R37 AI076114 and contract HHSN266200400088C to
D.I.W., grant R01 AI083118 to W.E.J., grant P51 RR000167 to the
WNPRC, and grant RR00168 to the New England Primate Research
Center (NEPRC) from the National Center for Research Resources
(NCRR), a component of the NIH. This research was also supported
by Pfizer Inc.-sponsored research agreement 140-N-2076312 with
J.B.S. Additionally, this research was conducted at a facility con-
structed with support from Research Facilities Improvement Program
grant numbers RR15459-01 and RR020141-01.
This publication’s contents are solely the responsibility of the au-
thors and do not necessarily represent the official views of NCRR or
NIH.
We have no conflicting financial interests.
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9640 NOTES J. VIROL.
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