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Current Pharmaceutical Design, 2003, 9, 1803-1815 1803
1381-6128/03 $41.00+.00 © 2003 Bentham Science Publishers Ltd.
Proteolytic Events of HIV-1 Replication as Targets for Therapeutic
Intervention
J. Tözsér1,* and S. Oroszlan2
1Department of Biochemistry and Molecular Biology, Faculty of Medicine, Debrecen University, H-4012 Debrecen,
Hungary and 2HIV Drug Resistance Program, National Cancer Institute at Frederick, Frederick, MD 21701, USA
Abstract: Acquired immunodeficiency syndrome (AIDS) is a worldwide epidemic caused by infection with HIV, a
human retrovirus. Proteolysis occurs at many points of the retroviral life-cycle, and these events can be considered as
targets for chemotherapy. The most well-known proteolytic action in the retroviral life-cycle is the processing of the Gag
and Gag-Pro-Pol polyproteins with the virally encoded protease at the late phase of viral infection. Protease inhibitors,
together with reverse transcriptase inhibitors, are important components of the drug combinations currently used to treat
HIV patients. The current combination therapy substantially reduced morbidity and mortality in HIV-infected patients.
However, these drugs do not allow viral eradication, therefore their long-term use is required, allowing the development
of resistance in a large portion of patients. Furthermore, several adverse metabolic side effects have been observed
associated with the therapy. Thus, new approaches are required to eradicate HIV infection, which may include targeting of
the potential early-phase function of the viral protease, and other crucial proteolytic events of the viral replication, such as
the ubiquitin-dependent proteolytic degradation of the unfolded viral proteins as well as the inhibition of envelope protein
processing.
Key Words: HIV-1, life-cycle, proteolysis, viral protease, envelope protein, ubiquitin, proteasomal degradation.
1. INTRODUCTION
Several anti-retroviral drugs are available for clinical use
to treat AIDS patients, and the most commonly used
treatment involves the combination of inhibitors of two
replication enzymes of human immunodeficiency virus type
1 (HIV-1), reverse transcriptase (RT) and protease (PR). The
use of these drugs in combination (a treatment termed highly
active antiretroviral therapy, HAART) substantially reduces
morbidity and mortality in HIV-infected patients (Reviewed
in [1]). Unfortunately this combination therapy is expensive,
therefore it is not readily available for patients in developing
countries, and it does not allow viral eradication. Due to the
lack of virus elimination, long-term use of these drugs is
required, allowing the virus to mutate into drug-resistant
forms in a large portion of patients. The existence of organ
sanctuaries (e.g., brain, testis) and long-lived infected cells
carrying proviral DNA contribute to the limits of eradicating
the virus (for a review see [2]). Most of the problems
associated with the HIV therapy are the consequence of the
necessarily long-term use of the drugs. Besides the
appearance of resistance several metabolic side effects exist,
including hypersensitivity, lipodystrophy syndrome, insulin
resistance and cardiovascular diseases (for reviews see [3-
5]).
Therefore it is essential to find drugs targeting the same
enzymes but with improved characteristics, or drugs targeted
to alternative steps of the viral replication cycle. Most of the
steps of the retroviral life-cycle could be targeted, and being
*Address correspondence to this author at the Debrecen University, H-4012
Debrecen, P. O. Box 6, Hungary. E-mail: tozser@indi.biochem.dote.hu
explored to provide novel anti-retroviral drugs. The aim of
this review is to summarize the essential proteolytic events in
viral replication, as well as the current status and future
prospects of the use of protease inhibitors.
2. OVERVIEW OF THE RETROVIRAL REPLICA-
TION CYCLE
To help the understanding of the proteolytic target points
in HIV infection, a short description of the retroviral life-
cycle is presented below (Fig. 1). In the early phase of the
cycle, the virus enters the host cell by membrane fusion or
receptor-mediated endocytosis (step 1). Then reverse
transcription occurs in the entering capsid structure, in which
the plus-strand RNA genome is converted to a double-
stranded DNA by RT (step 2). The error-prone nature of the
RT typically results in one mutation/genome in this step;
therefore, the virus is considered as a quasi-species. The high
mutation rate, together with the high recombination capacity
of the virus, allows the development of resistance towards
any kind of inhibitors targeting viral enzymes or proteins.
Following the reverse transcription, a preintegration complex
(PIC) is formed, composed of the genomic DNA and some
of the protein components of the entering capsid. The PIC
enters the nucleus (step 3). This step is typically a passive
process in case of most retroviruses, since they can infect
only dividing cells, in which the nuclear envelope is
disassembled; however, in the case of HIV-1 and other
retroviruses belonging to the lentiviral subfamily, an active
transport of the PIC allows the infection of nondividing cells.
The integration of the viral DNA into the host genome (step
4) is mediated by the integrase (IN), which is an essential
part of the PIC. The late-phase of the retroviral life-cycle
1804 Current Pharmaceutical Design, 2003, Vol. 9, No. 22 Tözsér and Oroszlan
starts with the transcription of viral DNA into various RNA
forms by the host RNA polymerase II (step 5). A significant
fraction of newly synthesized mRNA reaches the cytoplasm
unspliced, to serve as template for the Gag and Gag-Pro-Pol
polyproteins (step 6), and to be packaged into virions as
genomic RNA. A singly spliced RNA serves as a template
for the envelope protein (Env) synthesis in the endoplasmic
reticulum and Golgi. The Env protein is processed by a
cellular enzyme into a surface protein (SU) and a transmem-
brane protein (TM), and exported to the plasma membrane,
to the site of assembly. Various singly and multiply spliced
RNAs serve as templates for the synthesis of accessory
proteins, such as Tat, Rev, Vif, Nef, and Vpr. The Gag and
Gag-Pro-Pol polyproteins assemble at the membrane where
the Env proteins are concentrated (step 7); then the
assembled "immature" particle buds from the membrane
(step 8), followed by "maturation" to a morphologically
distinct form with a condensed core (step 9).
3. PROTEOLYTIC EVENTS IN THE LATE-PHASE
OF RETROVIRAL REPLICATION
3.1. Function and Properties of the Virally Encoded
Protease
3.1.1. Polyprotein Processing by the Retroviral Protease
In the last stage of the late phase of viral replication, the
Gag and Gag-Pro-Pol polyproteins are assembled together
with the envelope proteins and the viral genomic RNA at the
plasma membrane of the infected cell, and budding yields
“immature” virions with a “doughnut-shaped” capsid
structure (Fig. 1). The PR cleaves the viral polyproteins at a
limited number of sites (Fig. 2A), producing a condensed,
cone-shaped core of “mature” infectious HIV-1 (Fig. 1). This
function of the PR is essential for virus replication. Recent
reports suggest that redox regulation may be involved in PR
activation and subsequent “maturation” [6-8]. As shown in
Fig. 2A, the PR is coded on the pro gene and is part of the
Gag-Pro-Pol polyprotein synthesized by ribosomal
frameshifting (Reviewed in [9]). The relatively low
frequency of frameshifting assures that the amount of
replication enzymes in the virions is only about 5-10 % of
those of the structural proteins encoded by the gag gene. The
cleavage sites where the polyproteins are processed are given
in Fig. 2B. For comparison, cleavage sites verified within
some viral proteins as well as cleavage sites in encapsidated
cellular proteins, are also shown here.
3.1.2. Properties and Specificity of the Retroviral Protease
The structure of the HIV-1 PR was determined more than
a decade ago, and currently it is the most represented
structure in the protein structural databanks (for reviews see
[10,11]). The HIV-1 PR is a homodimeric aspartic protease
of 99-residue-long subunits, and in the absence of a ligand, it
is almost perfectly symmetrical. It has two flap regions that
are flexible in the absence of the ligand, but they are closed
Fig. (1). Replication cycle of HIV-1. The cycle starts by the interaction of the envelope protein with the receptor and coreceptor on the
surface of susceptible cell, leading to fusion and entry of the cone-shaped core, or alternatively, the virus may enter into the cell by receptor-
mediated endocytosis (step 1). Reverse transcription (step 2) generates a double-stranded DNA copy of the RNA genome. The provirus is
transported into the nucleus (step 3) and integrated (step 4) into chromosomal DNA. In the late phase of the cycle, the viral DNA is
transcribed by the cellular RNA polymerase II (step 5). Unspliced RNA is used as template for the synthesis of structural proteins and
replication enzymes (step 6), while the Env precursor protein is synthesized from spliced RNA in the endoplasmic reticulum and Golgi.
Virion proteins and progeny RNA assemble at the plasma membrane (step 7), and progeny “immature” virus is released by a process of
budding (step 8) followed by PR-mediated “maturation” (step 9) into infectious virus.
Proteolytic Events of HIV-1 Replication Current Pharmaceutical Design, 2003, Vol. 9, No. 22 1805
Fig. (2). Schematic representation of the HIV-1 genome (A) and sequences of HIV protease cleavage sites (B).
The location of open reading frames is indicated. The RNA encapsidation signal (ψ) is shown near the 5' end. The proteins encoded by the
pro and pol genes are synthesized by frameshifting mechanism from genome-sized RNA template to yield Gag-Pro-Pol polyproteins. Gag and
Gag-Pro-Pol polyproteins are processed at the last step of viral life-cycle by the viral PR at the sites indicated by arrows above the boxes. Env
is also synthesized as a precursor (SU + TM) and processed at the indicated site by a cellular protease.
The Gag polyproteins are processed into matrix (MA), capsid (CA), nucleocapsid (NC), and three smaller fragments and proteins designated
according to their molecular weight (e.g. p1, a one kDa peptide), while cleavage of Gag-Pro-Pol polyproteins also yields transframe protein
(TF), protease (PR), reverse transcriptase (RT) which is active as p66/p51 having RNase H (RH) domain in the larger protein, and integrase
(IN). Cleavages within these proteins which may occur in the early-phase of viral life-cycle are indicated by arrows below the boxes.
Classification of cleavage sites was done based on the sequence around the site of cleavage.
1806 Current Pharmaceutical Design, 2003, Vol. 9, No. 22 Tözsér and Oroszlan
down on the bound inhibitors as well as substrates (Fig. 3).
The activity of the PR is mediated by the active site aspartate
residues, which are held in a rigid position by a network of
hydrogen bonds called the “fireman’s grip”. The enzyme has
an acidic pH optimum, but its actual value depends on the
substrate [12].
Fig. (3). Ribbon diagram of the active protease dimer with a bound
ligand. The ligand is shown in ball-and-stick representation.
It was already recognized in the early stage of HIV PR
studies that understanding of its specificity is crucial for the
design of efficient inhibitors. Numerous studies have been
done on the specificity of the HIV-1 PR (For reviews see
[13-16]). It is a special feature of the PR, that it is not
possible to give a consensus substrate sequence, even though
the enzyme is fairly specific. Retroviral protease cleavage
sites are currently classified into two groups [17-19]. Type 1
c le ava ge s ite s have a roma tic re sidue a nd Pro, and type 2 s ite s
have hydrophobic res idues (e xc luding Pro) a t the s ite of
c le ava ge (Fig. 2B). The P2 a nd P2’ pos itions (nota tion is
a cc ording to [20]) a re als o critica l in determining the
s pe cific ity [18,19]. In type 1 clea vage s ites of primate
lentivirus e s, like H IV-1, there is a pre ferenc e for A sn a t P2
a nd be ta -branche d hydrophobic res idue (V a l or Ile ) a t P2’,
w hile in type 2 clea vage s ites the P2 pos ition is typic a lly beta
bra nc hed a nd the P2’ re sidue is G lu or G ln (Fig. 2B ). The type
1 c le a va ge site is ve ry importa nt for se veral re as ons . No other
protease, except pepsin, is known to act at the imino side of a
Pro residue. Indeed, the uniqueness of Pro in that position
was recognized even before the discovery of the PR [21] and
it was suggested early that inhibitors based on the this type
of cleavage site should be specific against the retroviral PRs
[22]. Proline residues, especially after Tyr or Phe in the
sequence (as the case in type 1 cleavage sites) have a
relatively high probability of forming the cis isomer rather
than the trans isomer of the preceding peptide bond [23] and
molecular modeling studies indicated that a trans rather than
the cis isomer provided a better fit into the substrate-binding
pocket [19]. Based on these findings we inferred that
isomerization at the proline of retroviral cleavage sites may
be involved in the triggering of maturation events [19].
Subsequently the conformational selectivity of the HIV-1 PR
towards the trans isomer of the cleaved peptide bond was
demonstrated by NMR and kinetic studies [24,25].
A schematic diagram of the substrate binding site of
HIV-1 PR with modeled interaction of the bound residues of
the MA↓CA cleavage site is given in Fig. 4A. The substrate
binds to the enzyme in an extended beta conformation, and it
is anchored by several hydrogen bonds. Substrate-based
inhibitors bind in a very similar manner. The enzyme
recognizes at least seven substrate residues, from P4 to P3’
(Fig. 4A). Based on detailed specificity studies as well as
HIV-1 PR-inhibitor crystal structures, there appear to be a
very strong sequence context dependence of the specificity
of PR. In other terms, the interactions between the ligand
side chains and respective binding pockets are not additive;
various types of ligand side chain interactions complicate
them [26]. The internal substrate side chain interactions that
affect the preference at a given subsite can be classified into
three types (Fig. 4B). In a cis interaction the substrate side
chains positioned at the same side of the substrate main
chain influence the positions of each other; for example, a
beta-branched side chain at P2 diminishes the place available
for the P4 side chain. In a trans interaction sequential
residues being at the opposite site of the substrate main chain
influence each other’s positions. For example, a bulky side
chain (like Tyr) at P1’ reduces the space available for the P1
and P2’ side chains by pushing the peptide backbone of the
substrate toward those sites. In a coupled interaction a substi-
tution of a side chain causes a sequential rearrangement of
other substrate side chains and could therefore influence the
preference for a distant site. Such interactions can cause the
effect of the P3’ side chain on P2 [26]. The strong s equence
context-depe ndenc e should be taken into ac count in the de sign
of protea se inhibitors : a mutation in a subs tra te binding subs ite
of the PR indirec tly c ould influence the s pe cific ity of the othe r
binding s ite s.
As discussed above, HIV-1 PR cleaves the Gag and Gag-
Pro-Pol proteins at various positions during viral maturation.
Although only one, a typical type 2 viral HIV-1 PR
maturation cleavage site, the CA↓p2 site, contains charged
residue at the P2-P2’ region, a P2' glutamate, this residue is
highly conserved in HIV-1 [27]. The cleavage at this site is
accelerated by lower pH, and this was suggested to play a
regulatory role in the viral protein processing [28].
Crystallographic structures of HIV proteases have been
reported with inhibitors having P2’ Glu residues [29,30]. In
these structures the P2’ Glu showed two weak hydrogen
bond interactions with the amides of Asp 29 and Asp 30 of
the enzyme. The proximity of the Glu side chain to the side
chain of Asp 30 suggested that they share a proton [30]. The
presence of glutamate at the P2' position of various peptide
substrates was found to be responsible for the pH effect [31].
Interestingly, the cleavage site within the N-terminal domain
of p24 contains Glu residue at both the P2 and P2' positions
(Fig. 2B). The presence of two glutamates in this site
enhanced its sensitivity to pH [32]. Unlike the P2 and P2'
glutamates, Glu at the P3 and P3’ positions (as in the
cleavage site in the C-terminal domain of CA) did not result
in such an effect [32, our unpublished results]. It is of
interest to note, that unlike in the Gag and Gag-Pro-Pol
cleavage sites, cellular protein cleavage sites frequently
Proteolytic Events of HIV-1 Replication Current Pharmaceutical Design, 2003, Vol. 9, No. 22 1807
contain charged residues, especially Glu at P2’ [13]. The
reason for the markedly different subsite preference in viral
and cellular proteins of the HIV-1 PR is not known.
3.1.3. HIV Protease Inhibitors and Development of
Resistance Against Them
The names and structures of HIV PR inhibitors currently
used in therapy are shown in Fig. 5. A common feature of
these compounds is that they are peptidomimetics; they
mimic the substrates of the PR. The enzyme-inhibitor
interactions, similar to the enzyme-substrate interactions, are
primarily hydrophobic ones. Typically these inhibitors
contain a phenyl residue at the P1 position. Another common
feature of these inhibitors is that they contain a nonhydro-
lyzable transition state mimic, like a hydoxyethylamine
group at the site corresponding to the cleavable bond in the
Fig. (4). Schematic representation of the substrate binding subsites of the HIV-1 protease (A) and type of internal substrate side chain
interactions (B).
The relative size of each subsite is indicated approximately by the area enclosed by the curved line around each substrate side chain.
According to the nomenclature, residues of the ligand (substrate or inhibitor) interacting with the protease are designated as P1, P2, P3 etc.
from the scissile bond towards the N-terminus of the substrate, and P1’, P2’, etc. towards the C-terminus. The respective substrate binding
subsites are designated as S1, S2, etc. The relative location of residues forming the subsites (see Fig. 6) is also shown. Some of the mutations
occurring in resistance are indicated as wild type residue/residue found in resistant viruses. Amino acid residues of the second subunit of the
protease dimer are indicated by a prime. The substituted residues are represented by open, dashed-lined objects in panel B, and the induced
conformation of the other side chains is represented by open, thick-lined objects.
1808 Current Pharmaceutical Design, 2003, Vol. 9, No. 22 Tözsér and Oroszlan
substrates. Saquinavir was the first approved HIV-1 PR drug.
It is based on a type 1 cleavage site, in which the Pro was
replaced by a saturated isoquinoline ring. Indinavir and
nelfinavir also mimic the type 1 cleavage site; ritonavir was
developed from a symmetric molecule, while amprenavir is a
sulfonamide compound. Lopinavir is the first second-
generation PR inhibitor. Its core is identical to that of
ritonavir, and it was designed to diminish the interactions of
the inhibitor with Val 82, a residue that is frequently mutated
in drug-resistant strains of the virus [33]. It is used in
combination with a smaller amount of ritonavir.
Resistance develops against PR inhibitors. In most of the
cases, mutations occur in the PR gene (Fig. 6A). While most
of the natural sequence variations (that is residues found in
different HIV-1 clones in the absence of PR inhibitor
treatment) are outside of the substrate binding sites, several
of the mutations conferring resistance involve residues of the
substrate binding subsites; therefore, they are expected to
alter the specificity and catalytic power of the enzyme.
Recent studies showed, that in patients receiving HIV PR
inhibitors, not only is the PR mutated, but mutations were
also observed at the NC↓p1 and p1↓p6 Gag cleavage sites
(Fig. 6B). These mutations were first described in in vitro
studies, but later they were also found in patients undergoing
indinavir therapy [34-36]. These mutations occurred together
with mutations in the PR gene [36]. Evolution of PR
cleavage sites other than NC↓p1 and p1↓p6 in the internal
(P2-P2') positions is limited, and mutations are rarely
observed even upon drug treatment [37]. Cleavage at these
sites appears to be a rate-limiting step in polyprotein
processing [34,38]. Peptides representing these sites have the
Fig. (5). Name and structure of HIV-1 protease inhibitors currently used in antiretroviral therapy.
N
H
NN
H
O
O
O
H2N
N
OH
O NH
Saquinavir
Invirase
Ro 31-8959
N
N
NH
N
OH
O
OH
NHO
Indinavir
Crixivan
MK-639
L735,524
NNH
NN
H
S
O
OH
NO
OH
O
S
N
Ritonavir
Norvir
ABT-538
N
HN
OH
O
OH
SNHO
Nelfinavir
Viracept
Ag-1343
O
O N
H
O
NSOO
NH2
OH
Amprenavir
Aguerase
VX-478
HN N
H
N
O
O OH
N
H
O
O
Lopinavir
Aluviran
ABT-378
Proteolytic Events of HIV-1 Replication Current Pharmaceutical Design, 2003, Vol. 9, No. 22 1809
lowest specificity constants (kcat/Km) among all HIV-1
cleavage sites [39,40]. Furthermore, there is a significant
sequence polymorphism at these sites (Fig. 6B), which also
may have an impact on virion infectivity [27]. Recently we
have studied the hydrolysis of oligopeptides representing
these cleavage sites with representative mutations found as
natural variations or that arise as resistant mutations [41].
Wild-type and five drug-resistant PRs with mutations within
or outside the substrate binding site were tested. While the
natural variations showed either increased or decreased
susceptibility of peptides toward the PR, the resistant muta-
tions always had a beneficial effect on catalytic efficiency
[41]. Comparison of the specificity changes obtained for the
various substrates suggested that the maximization of the van
der Waals contacts between substrate and PR is the major
determinant of specificity: the same effect is crucial for
inhibitor potency. Resistant mutants of HIV-1 PR must
possess sufficient proteolytic activity (kcat/Km) to support
viral replication by correctly cleaving Gag and Gag-Pro-Pol
precursors. The order of Gag cleavage is also important for
infectivity [42]. Unlike our findings with the MA↓CA and
CA↓NC cleavage sites [19,31], the natural NC↓p1 and
p1↓p6 sites do not appear to be optimized for rapid
hydrolysis. Therefore, it is possible to increase the cleavage
rate by mutation at these sites when the PR activity is
diminished due to the accumulation of PR mutations. Hence,
mutation of these rate-limiting cleavage sites can partly
compensate for the reduced catalytic activity of drug-
resistant mutant HIV-1 proteases.
3.2. Processing of the Envelope Protein
Viral surface glycoproteins are involved in both binding
and penetration of the virus into the target cells (reviewed by
[43,44]). Like the Gag and Gag-Pro-Pol, these glycoproteins
are also synthesized as precursors, and sorted through the
secretory pathway of the infected cells, where they are
cleaved at a highly conserved basic consensus sequence
(Arg-Xaa-Lys/Arg-Arg-↓-Xaa) (for a review see [45]). This
cleavage leads to the formation of the mature surface (SU)
and transmembrane (TM) glycoproteins that remain
Fig. (6). Sequence variation of HIV-1 protease (A) and the Gag region around p1 (B). The protease sequence of the HXB2 clone is given, the
conserved regions characteristic of retroviral proteases are indicated by bold, larger letters. Residues of the protease, which are involved in
ligand binding, are underlined. Residues occurring as natural variations are indicated by lower case letters in both sequences, while those
which have also been found as mutations occurring in drug resistance are in bold. Residues occurring only in drug resistant mutants are
indicated by capital bold letters.
1810 Current Pharmaceutical Design, 2003, Vol. 9, No. 22 Tözsér and Oroszlan
associated by noncovalent interactions. The SU recognizes
the host-cell receptors, and then the envelope complex
undergoes a conformational change that leads to exposure of
the N-terminal fusion peptide of the TM, which then
promotes virus-cell fusion in the classical infection route.
The cleavage and maturation of the envelope glycoproteins
are required for infectivity [46]. This processing is
performed by enzymes belonging to the Ca2+-dependent
prohormone convertase (PC) family, including furin, PC7,
PACE4 (paired amino acids-cleaving enzyme 4), and PC5
(reviewed in [45]). Calcium-independent enzymes may also
be involved in this process [47,48]. So far two types of
inhibitors have been developed to inhibit the Env cleavage.
An oligopeptide-chloroalkylketone derivative [49] and an
inhibitor derived from serine protease inhibitors (serpins) by
mutagenesis [50]. Due to the high variability of HIV and the
inevitable appearance of resistant mutants against the drugs
targeting the viral proteins, it may be beneficial to utilize
drugs targeting the cellular proteins and enzymes involved in
the life-cycle.
An additional cleavage within the V3 loop of the SU was
reported [51], and the enzymes were later identified as T-cell
membrane-associated serine protease, tryptase TL2 [52], and
urokinase-type plasminogen activator in case of
macrophages [53]. The significance of the V3 cleavage in
infection is not completely understood, it is proposed to
enhance the fusion process [53].
3.3. The Role of Ubiquitin and Ubiquitin-Dependent
Proteasomal Degradation in Assembly
Ubiquitin is a small, highly conserved protein that has
several functions in the eukaryotic cells. The classical role of
ubiquitination is to target the tagged proteins for degradation
by a multisubunit ATP-dependent protease termed the
proteasome. Ubiquitination is also emerging as a signal for
trafficking inside the cell. The first report that retroviral
particles contain ubiquitin was in 1988 when it was detected
in HIV as previously seen with murine leukemia virus
(MuLV) particles [54]. Free, and late assembly domain-
conjugated (p6-conjugated in HIV-1) ubiquitin was also
found in simian immunodeficiency virus (SIV), MuLV and
EIAV [55,56], while avian leukosis virus contains only free
ubiquitin [57]. Although these late assembly domain-
containing proteins appear to be monoubiquitinated, which is
not the typical protein tagging for degradation, it has been
shown, that the use of proteasome inhibitors, which deplete
the free ubiquitin pool by entrapping ubiquitins in
conjugated forms, inhibit HIV-1 budding and hence viral
replication [58]. On the other hand, EIAV budding was not
inhibited by proteasome inhibitors [56]. Interestingly, the
proteasome inhibitors also reduced Gag maturation in HIV-
1, thus influencing the processing by PR [58]. One possible
mechanism is the buildup of defective Gag translation
products that otherwise would be rapidly degraded by the
proteasome [58]. A large portion (even one-third) of newly
synthesized cellular proteins cannot fold properly, and are
degraded by the proteasome, and the same could be valid for
the viral proteins [59]. Kisselev and Goldberg [60] reviewed
several type of inhibitors developed against the proteasomes.
Ritonavir and saquinavir, inhibitors used against HIV-1 PR
also can inhibit proteasomes [61,62]. However, since the
proteasomal degradation has a protective function in the
early-phase, use of proteasomal inhibitors may have dubious
effects on HIV replication.
4. PROTEOLYTIC EVENTS IN THE EARLY PHASE
OF RETROVIRAL REPLICATION
Unlike the late-phase of retroviral replication, the early
phase is much less understood, mostly due to the difficulties
in finding proper techniques and detection systems, since
only a small fraction of the entering virions is infectious. It
has been a paradigm in retrovirus research that unlike most
enveloped viruses including some retroviruses using the
receptor-mediated endocytotic route, HIV enters the cell
utilizing a pH-independent direct fusion of the virion and the
cell membrane (Reviewed in [44]). However, the distinction
between entry of pH-independent and pH-dependent viruses
is not as simple as it was originally thought. Several cases of
pH-independent viruses that appear to require endocytosis
for productive infection have now emerged [63].
Amphotropic and ecotropic murine leukemia viruses also use
the endocytic route, but the infection is pH independent [64].
Although HIV was shown to be able to enter the cells by
clathrin-coated vesicles [65] and a large fraction of HIV-1
can be taken up by the cell via endocytosis [66,67], it was
shown that this pathway usually does not result in productive
infection, presumably due to the inability of the virion to
escape from the endosome [66,67]. However, it has been
recently demonstrated that endocytotic entry of HIV-1 into
cells can lead to viral integration and gene expression [68].
The use of direct fusion and the endocytotic route depends
on the type of virus producing cells, target cells and viral
isolates [68]. Primary macrophages mostly take up HIV-1 by
macropinocytosis, a receptor-independent process [69].
Following uptake, a large part of the macropinocytosed
virions was degraded, likely because of
endosomal/lysosomal fusions [69] but productive infection
also occurred. This can be explained by the envelope-
mediated fusion of some HIV-1 virions with the vesicular
membrane. The infection route used by the virion may have
a profound effect on the proteolytic events and proteolytic
enzymes involved in the early phase of viral replication.
4.1. Potential Early Phase Proteolytic Events Mediated
by the Viral Protease
While the late-phase function of the virally coded
protease is well established, its function in the early phase is
controversial and much debated. It was first demonstrated for
equine infectious anemia virus (EIAV) [70] and later for
HIV [71] that the PR is part of the core structure, which
enters the infected cells. In both the receptor-mediated
endocytotic and macropinocytotic routes, the incoming core
is surrounded by an acidic environment, which has an
optimal pH for the retroviral protease.
4.1.1. Cleavage of Proteins of the Host Cell by the Viral
Protease
It has been observed, that the HIV PR is capable of
cleaving cytoskeletal and sarcomeric proteins including
vimentin, desmin, actin, myosin and tropomyosin [72,73]. It
has been proposed that the processing of vimentin is
Proteolytic Events of HIV-1 Replication Current Pharmaceutical Design, 2003, Vol. 9, No. 22 1811
important in the early phase of viral infection [74].
Interestingly, while the actin cleavage was regulated by
polymerization/depolymerization [73], the vimentin cleavage
can be regulated by phosphorylation: only the
dephosphorylated form of the protein is a substrate of the PR
[75]. The journey of the core towards the nucleus requires
interaction with the actin microfilaments [76]; therefore
proteolysis of the components of the actin microfilament
may be an important step in regulation of this process. The
cleavage of other cellular proteins, like the transcription
factor NF-κB and eukaryotic initiation factor eIF4GI, may
rather be important in the late phase of viral replication, by
affecting gene transcription and translation, as well as the
cytotoxicity of the virus [77,79].
4.1.2. Cleavage of Viral and Virion-Packaged Cellular
Proteins by the Viral Protease
Besides the mature PR, the core of HIV-1 contains
several viral proteins, including capsid (CA), nucleocapsid
(NC), reverse transcriptase (RT), RNase H, integrase (IN),
Vpr and Nef [71]. Cellular proteins were also detected in
virions including the cellular peptidyl prolyl isomerase,
cyclophilin A (Cyp A) that is incorporated into the virion by
binding to CA and its presence enhances viral infectivity
[80,81]. Actin, and various actin-binding proteins have also
been detected [82]. The fate of these proteins after entering
the cells is mostly unknown, but many were already
fragmented in the virions, and the involvement of the PR at
least in degrading elongation factor-1 alpha (EF1α) was
demonstrated [82]. During in vitro incubation of EIAV cores
in the presence of a chelating agent (EDTA) the NC was
further processed in situ into smaller fragments by the
incorporated PR [70,83]. To extend our initial studies of
EIAV NC cleavage, we used short synthetic peptides derived
from the highly homologous HIV-1 and HIV-2 NC as
substrates for PR. Based on these studies we predicted that
the cleavage site located in the proximal (N-terminal zinc
finger) of HIV-1 NC should be between Phe and Asn (see
Fig. 2B), shifted one residue downstream compared to the
EIAV cleavage site [84]. This was later confirmed using NC
protein as a substrate [85]. Among the other proteins of the
core, the HIV-1 RT, RNase H and Nef have been
demonstrated to be substrates of the HIV-1 PR [71,86,87].
Furthermore, the PR itself undergoes self-degradation [88],
while Vpr, a nucleocytoplasmic shuttling protein of the PIC
[89], remains intact in the capsid [71]. We have observed
that the capsid protein is also a substrate of the PR in an
acidic environment, while the integrase, an essencial
component of the PIC is resistant towards proteolysis [32].
New cleavage sites in CA were identified (Fig. 2B), and one
of them showed a special pH sensitivity, as explained in
comparison with the Gag-Pro-Pol cleavage sites (see above).
The protease-mediated cleavage of viral and cellular proteins
of the core may be important for the formation of proper
preintegration complex.
4.1.3. Effect of HIV Protease Inhibitors in the Early Phase
of Viral Replication Cycle
Several research groups, including ours, found an
inhibitory effect of HIV-1 protease inhibitors on the early
phase of viral replication [90-94], while others did not
observe such an effect [95-97]. Action of the PR inhibitors in
the early-phase may be one factor contributing to the
enhanced in vivo antiviral potency of these drugs (for
example [98]). The different trafficking route of HIV-1
depending on the presence of receptor and virus isolate [68]
may be responsible at least in part for the conflicting results
obtained with PR inhibitors to study the role of PR in the
early phase of the viral life-cycle. Further studies are
required to clarify the role of the virally coded PR in the
early phase of viral replication. Furthermore, PR inhibitors
targeting the early-phase effect on the endocytotic route may
need somewhat different characteristics from the currently
used inhibitors. Those PR inhibitors which can efficiently
interfere with both the early and late phases of the viral life-
cycle would have a potentially great advantage: besides
protecting cells from infection, they can also influence the
chronically infected (integrated viral genome-containing)
cells, while the RT and IN inhibitors do not affect
chronically infected cells. However, at the present stage, we
cannot exclude the possibility that the PR-mediated
proteolysis of viral proteins in the early phase, if it occurs in
vivo, is not favoring the infection; rather it is a “suicidal”
effect.
4.2. Involvement of the Proteasome System in the
Cellular Defense Against HIV-1 Infection
Proteasomes are large multidomain complexes, which are
involved in the ATP-dependent degradation of most of the
intracellular proteins [99]. It has been shown that
proteasomal inhibitors, acting in the early phase of the life-
cycle, increased the efficiency of HIV infection, presumably
by preventing the degradation of the proteins of infectious
virions [100]. The substrates for proteasomal degradation
appear to be complexes containing largely completed reverse
transcription products, or fully formed preintegration
complexes [101].
ACKNOWLEDGEMENTS
We wish to thank Dr. Péter Bagossi and Gábor Zahuczky
for preparing figures and for valuable discussions. We are
grateful to Maritta P. Grau for editorial assistance. Our
research described in this review was sponsored in part by
the Hungarian Science and Research Fund (OTKA T 30092
and T43482).
ABBREVIATIONS
AIDS = Acquired immunodeficiency syndrome
CA = Capsid protein
Cyp A = Cyclophilin A
EF1α= Elongation factor-1 alpha
EIAV = Equine infectious anemia virus
HAART = Highy active antiretroviral therapy
HIV = Human immunodeficiency virus
IN = Integrase
MA = Matrix protein
MuLV = Murine leukemia virus
1812 Current Pharmaceutical Design, 2003, Vol. 9, No. 22 Tözsér and Oroszlan
NC = Nucleocapsid protein
PACE4 = Paired amino acids-cleaving enzyme 4
PC = Prohormone convertase
PIC = Preintegration complex
PR = Viral protease
RNase H = Ribonuclease H
RT = Reverse transcriptase
SIV = Simian immunodeficiency virus
SU = Surface protein
TM = Transmembrane protein
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