Indian J Med Res 121, April 2005, pp 270-286
270
Upon infection by human immunodeficiency virus
type 1 (HIV-1), host cells react with various innate,
cellular and humoral immune responses to counteract
the viral invasion. Limited and transient restriction of
viral infection is normally achieved. However, HIV
ultimately overcomes these antiviral responses
resulting in successful viral infection and replication.
Expression of several HIV-1 regulatory and
accessory genes such as vif, vpu and tat is known to
regulate some of the host cell innate immune
responses to maximize viral infection or
replication1-3. For example, a host innate antiviral
response mediated by APOBEC3G was recently
found to sabotage HIV reverse transcription through
cytidine deamination within minus DNA
strand4-7.Targeting viral replication by DNA
Key words Apoptosis - cell cycle G2/M arrest - disease progression - HIV-1 - host-pathogen interaction - host immune responses  -
nuclear transport - Vpr - viral pathogenesis
HIV-1 viral protein R (Vpr) & host cellular responses
Richard Yuqi Zhao*+, Michael Bukrinsky** & Robert T. Elder+
*Department of Pathology, University of Maryland School of Medicine, Baltimore, MD 21201,
**Department of Microbiology and Tropical Medicine, The George Washington University, Washington,
DC 20037 & +Children’s Memorial Research Center, Northwestern University Feinberg School of
Medicine, Chicago, IL 60614, USA
Accepted February 21, 2005
During infection of host cells by HIV-1, active host-pathogen interactions take place. The final
balance between these interactions determines the efficiency of viral infection and subsequent
disease progression. HIV-infected cells respond to viral invasion with various antiviral strategies
such as innate, cellular and humoral immune antiviral defense mechanisms. On the other hand,
the virus has also developed tactics to suppress these host cellular responses. Among the many
viral offensive strategies, viral protein R (Vpr) plays a particularly active role. Vpr involved in
nuclear transport of the viral pre-integration complex, activation of viral transcription, induction
of cell cycle G2/M arrest and apoptosis of the host cells. However, specific roles of these Vpr
activities in viral pathogenesis and their contribution to disease progression are not fully
understood. HIV-1 defective for some or all of these Vpr activities have been associated with
slow disease progression in some patients. With regard to the host responses to  vpr gene expression,
studies show that Vpr is specifically targeted by CD8 T-lymphocytes during acute viral infection
and that the host innate immune response may also play a crucial role in suppressing the effects
of Vpr on various cellular activities. The effect of host cellular responses to  vpr gene expression
and its roles in nuclear transport, cell cycle G2/M regulation and induction of apoptosis are
discussed in this review. Strategies with potential application for future antiviral therapies
directed at suppressing Vpr activities are described.
271
deamination seems to be one of the major host innate
responses defending against retroviral infections. On
the other hand, HIV-1 accessory protein Vif prevents
APOBEC3G from entering the HIV-1 virion during
viral assembly, thus ensuring viral replication in target
cells8,9.
HIV-1 viral protein R (Vpr) is another accessory
protein. It can be found in the serum of HIV-1 infected
patients and in the cerebrospinal fluid (CSF) of AIDS
patients with neurological pathologies10,11. Because
Vpr is a highly conserved viral protein, it presents a
good target for host antiviral responses. However,
little is known at present about Vpr-host cell
interactions or whether Vpr is involved in suppressing
host antiviral responses as Vif does. Numerous
reports certainly indicate a dynamic interaction
between Vpr and host cellular responses. We will
briefly summarize those findings here and will
examine the potential contribution of Vpr to viral
pathogenesis and disease progression.
HIV-1 Vpr is a virion-associated viral gene
product with an average length of 96 amino acids,
and a calculated molecular weight of 12.7 kDa.
However, it typically appears as either a 14 kD or
15 kDa band due to post-translational modifications.
Vpr is a highly conserved viral protein among HIV,
simian immunodeficiency viruses (SIV) and other
lentiviruses12,13. Besides lentiviruses, its protein
sequence shares no strong homology with any of
the known proteins. A tertiary structure of Vpr
proposed on the basis of nuclear magnetic resonance
(NMR) analysis consists of an ? -helix-turn-? -helix
domain in the amino-terminal half from amino acids
17 to 46 and a long ? -helix from 53 to 78 in the
carboxy-terminal half14,15. These three ? -helices are
folded around a hydrophobic core in a structure
which allows interaction of Vpr with different
cellular proteins16.
Vpr displays several distinct activities in host cells.
These include cytoplasmic-nuclear shuttling 17,
induction of cell cycle G2 arrest18 and cell killing19.
These three Vpr-specific activities were shown to
be functionally independent of each other20, 21 and
have been demonstrated in a wide variety of
eukaryotic cells ranging from human to yeast,
indicating that Vpr most likely affects highly
conserved cellular processes.
In this review, we describe our current
understanding of the host-Vpr interactions and the
potential roles of Vpr activities in viral pathogenesis
and disease progression.
HIV-1 Vpr and host cellular responses
All regulatory and accessory HIV-1 viral proteins
are being targeted by HIV-1-specific CD8-positive
cytotoxic T-lymphocytes (CTLs)22. However, Vpr is
being preferentially targeted by the CD8+ T-
lymphocytes as compared with other viral proteins,
at least during the acute phase of the viral
infection23,24, suggesting a salient role of Vpr during
the early phase of infection. Consistently, some of
the cellular proteins, such as heat shock proteins
(HSPs), respond quickly to viral infection or vpr gene
expression, which suggests another level of host innate
immune response25,26 (our unpublished data). For
example, HSP27 and HSP70 mRNA transcription
appeared as early as 3-8 h following HIV infection.
We now know that some of the small heat shock
proteins, such as HSP27 or HSP70, exert effective
protective effect against some or all of the Vpr
activities27-29. However, responsive elevations of
HSPs to HIV-1 infection are transient as the HSP27
and  HSP70 mRNA transcripts were significantly
downregulated by 24 h after viral infection,
concomitant with the first appearance of the full
length genomic HIV-1 mRNA30. This observation
implies an active interplay between HIV viral proteins
and HSP27 or HSP70. Indeed, an active and
antagonistic interaction was seen between Vpr and a
yeast homologue of HSP2731. Vpr suppresses antigen-
specific CD8-mediated CTL and Th1 immune
responses32. Consistent with this notion, Rhesus
macaques infected with HIV-2 lacking vpr gene had
increased antibody titres compared to monkeys
infected with the wild-type virus33. Although molecular
mechanisms underlying suppression of CTL and
antibody production by Vpr are presently unknown,
it was surmised that Vpr may prevent antibody
production against the virus by inhibiting T-cell clonal
expansion through suppressing T-cell proliferation and
inducing cell cycle G2/M arrest34. Evidence also
ZHAO et al: HIV-1 VPR
272 INDIAN J MED RES, APRIL  2005
suggests that Vpr may suppress host inflammatory
responses, which present another level of the host
immune responses to viral infections35. Vpr inhibits
host inflammatory responses by down regulating pro-
inflammatory cytokines (TNF?  and IL-12) and
chemokines (RANTES, MIP-1?  and MIP-1? ) in a
manner similar to glucocorticoids36,37; Vpr additionally
suppresses host inflammatory response by inhibiting
nuclear factor kappa B (NF? B) activity through the
induction of I? B37.
Therefore, there appears to be at least two levels
of host responses to vpr gene expression; one is the
cellular immune response mediated by CD8+ CTLs;
and another innate immune responses involving some
of the cellular chaperone proteins. Conversely, Vpr
counteracts those host immune responses. It prevents
T-cell proliferation, suppresses host inflammatory
responses including production of cytokines and
chemokines. Vpr may also have additional
mechanisms to counterbalance some of those innate
reactions, such as heat shock proteins, that have
specific suppressive activities against Vpr. These
specific host responses to Vpr and the counteracting
effect by Vpr strongly suggest a very dynamic
interaction between vpr gene expression and host
reactions. Future studies should reveal to what extent
these interactions contribute to the success of viral
infection and will determine the best way to exploit
those specific host responses to design strategies
aimed at suppressing Vpr.
Potential association of Vpr activities with viral
pathogenesis and disease progression
The importance of Vpr in viral pathogenesis has
been addressed in a number of earlier studies of
SIVmac219  infection in Rhesus monkeys. However,
these experiments produced conflicting results and
interpretations, which were further complicated by
the presence of two genes in SIVmac219 homologous to
the  vpr gene of HIV-1. These two genes,  vpr and
vpx, are thought to have arisen by duplication of the
ancestral vpr gene13, although phylogenetic analysis
of various lentiviruses later suggested that non-
homologous recombination may have played a role in
the evolution of vpr and vpx genes12,38. It is also
believed that the functions of the single vpr of HIV-
1 have been divided among the vpr and vpx genes of
SIV39.  In one study, Rhesus monkeys infected with
the SIVmac219 defective in vpr had low viral burden
and no disease progression while monkeys infected
with the wild-type virus or those Vpr-defective viruses
spontaneously reverted to the wild-type exhibited high
viral burden and rapid disease progression 40. In
contrast, in two other studies 41,42, no significant
differences in disease progression were found
between the vpr-deficient SIVmac219 and the parental
wild-type virus, and all of the monkeys developed
AIDS41,42. However, in the experiment most relevant
to infection by HIV-1 with the single  vpr gene,
monkeys infected with SIVmac219 defective in both the
vpr and vpx genes had severely attenuated infections
with much lower viral burden and no evidence of
disease progression41,43. At least a 100-fold decrease
in pathogenic index was found in the infectivity of
these mutant viruses in comparison with the wild-type
viruses, confirming the role of Vpr in viral
pathogenesis43.
Vpr seems to be required for in vivo replication
of the virus. An earlier study in Rhesus monkeys
showed rapid reversion of a single base pair mutation
in the stop codon of the vpr gene40. The requirement
for Vpr in vivo by HIV-1 was further illustrated in
chimpanzees and an accidentally infected laboratory
worker, who was initially infected with a Vpr-
defective HIV-1 viral laboratory strain IIIB44. The
vpr  gene in the viral strain IIIB has a frame shift
mutation at codon 73, which results in a truncated
Vpr protein with only the first 72 amino acids.
However, the mutant vpr gene in the virus initiating
the infection reverted to the wild-type Vpr both in
this human subject and experimentally infected
chimpanzees44. These results clearly demonstrate the
necessity of Vpr in vivo.
Even though Vpr sequence is one of the most
conserved regions in HIV genome, with estimated
similarities of 87 per cent between different viral
strains45,46, Vpr sequence variations were inevitably
found in viruses infecting patients showing fast or slow
disease progression.  Earlier efforts in studying the
role of Vpr in disease progression were mostly based
on nucleotide sequence analyses47-51. Even though
such analysis is useful in detecting gene deletions or
273
insertions, it cannot be used to predict functional
changes derived from amino acid substitutions. As a
result, different conclusions were drawn with regard
to potential contribution of Vpr to disease progression.
A synonymous or non-synonymous amino acid
substitution in Vpr could dramatically affect Vpr
functions as shown in a number of mutagenesis
studies52-55. A potential problem associated with these
mutagenesis studies is, however, that the vpr mutants
were often artificially created and therefore may not
represent the profile of naturally occurring mutations.
An alternative method to rapidly determine
multiple Vpr activities, such as cell cycle G2 arrest,
nuclear localization and induction of cell death, is the
use of fission yeast (Schizosaccharomyces pombe)
model system, which allows simultaneous
determination of these three Vpr activities using an
inducible gene expression system20,56,57. These three
Vpr activities observed in fission yeast are very similar
to that observed in mammalian cells58-60. By using
the yeast model system, these Vpr activities were
shown, as expected, to be highly conserved in many
of the viral laboratory strains and also in viruses
isolated from patients progressing to AIDS61.  In
contrast, a significant proportion (23-50%) of Vpr in
viruses isolated from a non-progressing mother-child
pair was functionally defective in all three Vpr
activities61,62. Variation analyses of the protein
sequences indicated that these Vpr proteins carried
unique amino acid substitutions that frequently
interrupted highly conserved domains, which include
a N-terminal ? -turn-?  helix and an epitope sequence
that is preferentially targeted by the CTL immune
response61. Association of slow disease progression
with Vpr defect was also found in another HIV-
infected non-progressor63. The vpr gene isolated from
this patient contained both premature stop codons and
an unusual Q3R polymorphism, which significantly
impairs the ability of Vpr to confer cytopathicity but
has no effect on the efficiency of viral replication.
Together, these data support the idea that functional
Vpr might be one of the viral factors contributing to
disease progression. However, these three reported
non-progressors are thus far the only HIV-infected
patients known to carry functionally defective Vpr.
Whether these are unique cases or a common trend
remains to be determined. Based on protein sequence
analysis of Vpr deposited in the database, a high
frequency of R77Q Vpr mutations were reported to
associate with patients with slow disease
progression64. Although the authors convincingly
demonstrated that this Vpr mutation, when isolated
from an HIV-1 viral strain HxBRU, impaired the
ability of Vpr to induce apoptosis, it was not present
in viruses isolated from some of those long-term non-
progressors61,65. Therefore, association of this
mutation with slow disease progression remains
unproved65,66. It also remains open how strong a
correlation is between Vpr functional defects and
slow disease progression of HIV-infected patients.
Induction of cell cycle G2/M arrest
To ensure accurate transmission of the genetic
information, eukaryotic cells have developed an
elaborate network of checkpoints to monitor the
successful completion of every cell cycle step and to
respond to cellular abnormalities such as DNA
damage and replication inhibition as they arise during
cell proliferation. Two of the best characterized
G2/M checkpoints, DNA damage and DNA
replication67-70 were first characterized in detail by
genetic analysis in fission yeast. The G2 to M
transition is controlled in fission yeast by the
phosphorylation status of Tyr15 on Cdc2, the cyclin-
dependent kinase which regulates the cell cycle in all
eukaryotic cells71. In fission yeast, Tyr15 is
phosphorylated by the Wee1 and Mik1 kinases to hold
the cell in G2, and rapid dephosphorylation by the Cdc25
phosphatase triggers the G2 to M transition71-74.
The DNA damage checkpoint is activated by
ionizing radiation or ultraviolet light, and activation of
this checkpoint leads to inhibitory phosphorylation of
Cdc2 at Tyr15 by a multi-step pathway75,76. The early
genes in the pathway, which include Rad1, Rad3,
Rad9, Rad17, Rad26 and Hus1, are thought to sense
the DNA damage and lead to phosphorylation of the
Chk1 protein77. In response to double strand DNA
breaks (DSBs) induced by ionizing radiation, for
examples Rad17 acts as a checkpoint-specific loading
factor, which responds to the DNA damage by loading
a 9-1-1 protein complex onto the sites where DNA is
damaged78,79. The 9-1-1 protein complex is also
known as the checkpoint clamp complex (CCC),
which is composed of Rad1, Rad9 and Hus179. In
addition, the Rad3-Rad26 protein complex binds to
ZHAO et al: HIV-1 VPR
274 INDIAN J MED RES, APRIL  2005
sites of DNA damage independently of the 9-1-1
protein complex. The independent binding of these
two protein complexes to DNA damage to initiate
the DNA structure checkpoint is believed to protect
the cell against inappropriate checkpoint
activation68,79,80. Activation of Chk1 is mediated by
Crb2, which may bridge Rad3 and Chk1 81-83. The
activated Chk1 kinase then directly phosphorylates
the Cdc25 phosphatase84. The phosphorylated Cdc25
binds Rad24/25 protein, and this complex is
transported out of the nucleus to render Cdc25
inactive85. The activated Chk1 also regulates the Mik1
kinase to inhibit Cdc286. DNA damage thus initiates
a Chk1-mediated protein phosphorylation cascade
ending in the inactivation of Cdc25 phosphorylase and
activation of Mik1 kinase to increase inhibitory
phosphorylation of Tyr15 on Cdc2.
The DNA replication checkpoint is activated by
treatment with hydroxyurea, which inhibits DNA
replication, and this checkpoint also controls the G2 to
M transition through inhibitory phosphorylation of
Cdc276. Parts of this DNA replication checkpoint are
shared with the DNA damage checkpoint as Rad1,
Rad3, Rad9, Rad17, Rad26 and Hus1 are required for
both checkpoints in fission yeast87. The same 9-1-1
and Rad3-Rad26 checkpoint protein complexes may
associate with the DNA replication complex 79.
However, the DNA replication checkpoint acts
primarily through phosphorylation of Cds1 kinase,
which is mediated by another protein Mrc188,89. There
is a minor contribution from the Chk1 kinase, and either
kinase is sufficient by itself to give cell cycle arrest
when DNA synthesis is inhibited90. Activated Cds1
kinase inactivates Cdc25 through the same mechanism
as Chk1 and may also activate the Wee1 and Mik1
kinases, which phosphorylate Tyr15 of Cdc267,90.
Cell cycle G2/M controls, which were often initially
defined in yeast, are highly conserved, and most of
Table. Human homologues of fission yeast cell cycle control genes
Fission yeast Human Putative activity
Mitotic regulators:
Cdc2 CDK1 Cyclin B-dependent kinase
Cdc13 Cyclin B B-type cyclin
Wee1 WEE1 Tyrosine kinase
Mik1 — Tyrosine kinase
Cdc25 CDC25A/B/C Tyrosine phosphatase
DNA damage and replication checkpoints:
Rad1 hRAD1 Nuclease
Rad3 ATM/ATR Protein kinase
Rad9 hRAD9 3’-5’ exonuclease
Rad17 hRAD17 Unknown
Rad24/25 14-3-3 Binds to phosphorylated ser
Hus1 hHUS1 A PCNA-related protein
Chk1 CHK1 Serine/Threonine Kinase
Cds1 CHK2 Serine/Threonine Kinase
Crb2 BRCA1 Unknown
Mrc1 CLSPN Unknown
Cellular proteins involved in Vpr-induced G2 arrest:
PP2A PP2A Protein phosphatase 2A
Paa1 A A regulatory subunit
Pab1 B B regulatory subunit
Ppa2 C C catalytic subunit
Ppa1 C C catalytic subunit
Wos2 P23 Inhibitor of Wee1
—, not found
275
the genes required for the G2/M checkpoints have
human homologues (Table). In general, these
homologues have similar, although not always
identical, roles in the control of the human cell cycle.
There is a tendency for multiple, partially redundant
checkpoints in human cells compared to simpler
checkpoints in yeast probably reflecting the more
complex requirements for cell cycle control in
multicellular eukaryotes. For example, the single rad3
gene in fission yeast is required for both the DNA
damage and replication checkpoints and activation of
the  chk1 and cds1 checkpoint kinases68,79,80.  In
human cells, there are two homologues of rad3, ATM
and ATR. The primary role of ATM is in the DNA
damage checkpoint and activation of  CHK2, the
human homologue of cds1, whereas the primary role
of  ATR is in the DNA replication checkpoint and
activation of CHK191,92.  Similarly, there is only one
Cdc25 tyrosine phosphatase that dephosphorylates
Cdc2 in fission yeast. In human cells, there are three
CDC25 homologues, CDC25A, CDC25B and
CDC25C, and each of them can be phosphorylated
by CHK193. All three of these phosphatases have been
shown to be involved in the control of the G2/M
transition, even though their specific roles in this
process have not yet been well characterized94-96. The
p53 gene is an example of an additional level of cell
cycle control in human cells. The p53 transcription
factor, which has no homologues in yeast, has multiple
roles including regulation of apoptosis and the cell
cycle with an essential role in the G1 DNA damage
checkpoint 97.  It also has important roles in the G2
damage checkpoint. It inhibits Cdc2 through activation
of Gadd45, p21, and 14-3-3? . In addition, it is involved
in regulatory feedback loops with ATM/ATR and
CHK198. The conservation of checkpoints even
extends to the regulatory mechanisms as illustrated
by the negative regulation of Cdc25 by relocation to
the cytoplasm from the nucleus in both fission yeast
and human cells. This relocation in both organisms is
dependent on 14-3-3 proteins85,99.
The HIV-1 Vpr protein induces cell cycle G2 arrest
through inhibitory phosphorylation of Cdc2 both in
fission yeast and human cells, suggesting that Vpr
affects a conserved cellular process. Specifically Vpr
induces hyperphosphorylation of fission yeast Cdc2 or
human CDK1, the human homologue of Cdc218,56,100.
It exerts its inhibitory effect through T14A and Y15F
of CDK1 and Y15F of Cdc2, as expression of
nonphosphorylatable Cdc2 mutants, T14A Y15F of
CDK1 and Y15F of Cdc2, prevents Vpr-induced G2
arrest18,101. Furthermore, Vpr inhibits Cdc25
phosphatase102,103 and activates Wee1 kinase102,104 to
promote phosphorylation of Cdc2/Cdk1 during induction
of G2 arrest.  Consistent with the roles of Wee1 and
Cdc25 in Vpr-induced G2 arrest, proteins that are
involved in regulation of Cdc25 or Wee1 have also been
identified to either augment or alleviate Vpr-induced
G2 arrest. Fission yeast Wos2, which is a human p23
homologue and aWee1 inhibitor105, has been shown to
be a multicopy Vpr suppressor102. A Cdc25 inhibitor
rad2585, which is the human 14-3-3 homologue
enhances Vpr-induced G2 arrest when overproduced
in fission yeast102. Recent studies further showed that
Vpr binds to CDC25C and 14-3-3 in human cells106,107.
Given that the DNA checkpoints and Vpr both
induce G2 arrest through inhibitory phosphorylation
of Cdc2 which is regulated by Wee1 and Cdc25, Vpr
might induce G2 arrest through a checkpoint pathway.
This possibility has been evaluated in fission yeast by
expressing vpr in mutant fission yeast strains defective
in early and late steps of the checkpoint pathways.
None of the early checkpoint-specific mutants (rad1,
rad3, rad9 and rad17) showed a significant effect
on the induction of G2 arrest by Vpr 101,102,108.
Furthermore, mutations in both chk1 and cds1, which
are thought to be the last steps specific for the
checkpoint67,84,90, also do not block Vpr-induced G2
arrest102,108. Therefore, Vpr does not appear to use
the DNA-damage or DNA-replication checkpoint
pathways to induce G2 arrest in fission yeast.
Early data in human cells tended to support the
conclusion that Vpr does not induce G2 arrest through
the DNA damage checkpoint pathways. Vpr still
induced G2 arrest in cells from patients with ataxia
telangiectasia (AT)103. These AT cells are mutant for
the ATM gene, which is a human homologue of fission
yeast Rad3, and they do not arrest in G2 in response
to DNA damage109-111. However, recent reports show
that Vpr activates ATR and a second human
homologue of fission yeast Rad3, and other steps in
this checkpoint pathway such as Rad17, Hus1,
BRCA1 and Y-H2AX112,113. These studies suggest
ZHAO et al: HIV-1 VPR
276 INDIAN J MED RES, APRIL  2005
that Vpr induces G2 arrest through either a cellular
response to DNA replication stress or to a signal that
“mimics” DNA damage. Expression of vpr does not
increase gene mutation frequency114 and or changes
radiosensitivity of the checkpoint defective mutant102,
which argues against the possibility that Vpr actually
causes DNA damage. It is thus reasonable to think
that other signals other than actual DNA damage
triggers DNA damage-like cellular responses. These
cellular responses could include the nuclear herniation
caused by Vpr115 or cellular stress responses to vpr
gene expression27-29. Since ATR and CHK1 are
primarily responsive to changes in DNA replication,
an alternative possibility is that Vpr may interfere with
DNA replication. This possibility is certainly supported
by a number of reports showing that Vpr induces
genomic instability, formation of micronuclei and
aneuploidy116,117. All of these changes in DNA
structures could be perceived as replication stresses,
which would trigger cell cycle arrest.
Considering that G2/M DNA checkpoints are
highly conserved between mammalian and fission
yeast cells (Table), it is unclear at the moment why
human ATR and CHK1 are activated by Vpr but  rad3
(the fission yeast homologue of ATR/ATM) or chk1/
cds1 (CHK1/CHK2) double deletion in fission yeast
does not block Vpr-induced G2 arrest 101,102. One
factor possibly contributing to the observed activation
of ATR in mammalian cells is that retroviral integration
appears to activate ATR118, and the experiments
showing activation of ATR by Vpr were done with
lentiviral vectors which might therefore activate ATR
to some extent independently of Vpr. In addition, it
was noticed that activation of ATR and CHK1 only
accounts for only part of the G2 arrest induced by
Vpr112. Other as yet unidentified molecular
mechanism(s) may explain at least half of the G2 cell
population induced by Vpr. Interestingly, Roshal et
al119 showed that treatment of Vpr-producing
mammalian cells with caffeine completely blocks
Vpr-induced G2 arrest. Caffeine is part of the
methylxanthine family, and similar to the caffeine
effect, another methylxanthine pentoxifylline (PTX)
also inhibits Vpr-induced G2 arrest in mammalian
cells34. Similarly, both PTX and caffeine suppress
Vpr-induced G2 arrest in fission yeast117,120. Since
PTX or caffeine inhibits Vpr-induced G2 arrest in
fission yeast where the classic DNA checkpoints
apparently play no role, these observations suggest
molecular mechanism other than the classic DNA
checkpoints may be involved in activation of ATR and
regulation of Cdc25 and Wee1.
The additional molecular mechanism might involve
protein phosphatase 2A (PP2A). Although this protein
phosphatase has no known role in the DNA
checkpoints, it has an important role in Vpr-induced
G2 arrest. Okadaic acid is a specific inhibitor of
PP2A, and okadaic acid was shown to inhibit Vpr-
induced G2 arrest both in human100 and fission yeast
cells56.  Further evidence for an important role of
PP2A comes from PP2A mutant strains. PP2A is
composed of three subunits, one catalytic (C) and
two regulatory (A and B) subunits. When vpr was
expressed in a strain with a deletion for a catalytic
subunit (ppa2) or a regulatory subunit ( pab1) of
PP2A, Vpr-induced G2 arrest was reduced 108,121.
Other evidence supporting involvement of PP2A in
Vpr-induced G2 arrest comes from other viral proteins
with effects on cell cycle G2/M controls. PP2A
appears to be a common viral target since other
viruses such as simian virus 40 (SV40), polyoma virus,
human T lymphotrophic retrovirus and adenovirus
affect the enzymatic activity of at least a subset of
PP2A proteins122. Even though these viruses are not
otherwise related, they all seem to have adapted a
similar strategy to affect cellular processes by direct
interaction with PP2A. Similar to the Vpr effects,
both adenoviral E4orf4123-125 and HTLV Tax protein
induce cell cycle G2 arrest 118. These two viral
proteins both bind to PP2A and affect its enzymatic
activity123,126. Interestingly, similar to Vpr, Tax-
induced G2 arrest is reversible by caffeine 118. Further
examinations indicated that Tax binds to CHK2 in
Jurkat T-cells 118 but it complexes with CHK1 in other
T-cells127. Taken together, it is possible that a
concerted cellular mechanism interlinks PP2A and
ATR/CHK1 in the cellular response to  vpr gene
expression during the induction of G2 arrest.
Nuclear transport of viral preintegration
complex
Ability to replicate in non-dividing cells (terminally
differentiated macrophages and incompletely
277
activated CD4+ T lymphocytes) is the characteristic
feature of HIV-1 which determines to a large extent
its high replicative capacity and pathogenesis. To
replicate in non-dividing cells, HIV-1 needs to
transport its genomic DNA [in the context of the viral
pre-integration complex (PIC)] from the cytoplasm
into the nucleus of a target cell. Vpr is believed to be
among the main regulators of HIV-1 nuclear
import17,128.
Proteins engaging in nuclear transport typically
contain a classical nuclear localization sequence
(cNLS)129,130, which is a short amino acid region rich
in basic amino acids (lysines and arginines) and binds
to the adaptor protein importin ? . The complex of
cNLS-importin ?  then binds to the receptor importin
?  through the importin ? -binding region on importin
? . Importin ?  interacts with components of the nuclear
pore complex (NPC) as an essential part of the
nuclear translocation process. The NPC is a large
structure composed of 50 to 100 proteins called
nucleoporins, which contains a central 10 nm aqueous
channel through which proteins are actively
transported.  Directionality of this translocation
process is ensured by Ran.  A high concentration of
RanGTP inside the nucleus stimulates binding of
RanGTP to cNLS-importin ? -importin ?  complex and
disassembles it to release the protein carrying the
cNLS into the nucleoplasm. Importin ?  and importin
? -RanGTP are then exported out of the nucleus to be
reused in another round of nuclear transport. This
model for cNLS translocation is partially based on
work done in budding yeast, and the high degree of
conservation is demonstrated by functional
complementation of many budding yeast mutants in
nuclear transport proteins by human homologues131.
Most studies have reported that Vpr expressed
without other viral proteins localizes predominantly
to the nuclear envelope in human, fission yeast and
budding yeast20,132,133.  Two hypotheses (not
necessarily mutually exclusive) for the mode of action
of Vpr in HIV-1 nuclear import have been proposed:
(i) that Vpr targets the HIV-1 PIC to the nucleus via
a distinct, importin-independent pathway134,135; or (ii)
that Vpr modifies cellular importin-dependent import
machinery136,137. The first model was based on the
observation that in the in vitro nuclear import assay
Vpr can enter nuclei in the absence of soluble import
factors135.  Consistent with this concept, Vpr was
shown to induce dynamic disruptions in the nuclear
envelope115 which may serve as entry points for
isolated Vpr and for the PICs.
However, based on our results and reports from
several other laboratories, we favour a hypothesis that
Vpr uses a modification of the importin ?  pathway to
enter the nucleus138,139. Vpr was shown to bind to
importin ?  both from human and budding yeast, but
the binding site is different from the binding site for
cNLS133,136,137. Vpr also binds to nucleoporins, thus
performing activity normally attributed to importin
? 133,140,141, suggesting that Vpr might mediate binding
of the PIC to the nuclear pore. However, another
study showed that importin ?  was necessary for
nuclear localization of Vpr and that importin  ? ,
importin ? , cNLS and Vpr form a ternary complex136.
Thus, Vpr appears to bind to a previously unknown
site on importin ?  142, and this complex in turn binds
to importin ?  which mediates transport through the
nuclear pore. The effect of Ran-GTP binding to
importin  ?  on the ternary complex has not been
reported and it is not understood why Vpr is frequently
observed to localize at the nuclear envelope, although
this may be related to described above binding of Vpr
to nucleoporins. One study found Vpr to be at the
inside of the nuclear envelope suggesting that Vpr is
transported through the pore but then stops at the
inside of the nuclear pore rather than being released
into the nucleoplasm133.
In the nuclear transport of the viral PIC, Vpr
appears to cooperate with cNLSs present on other
HIV-1 proteins, in particular matrix protein (MA).
There has been disagreement over the role and
identity of cNLS in the nuclear transport of PIC, and
at least one reason for this disagreement is the
presence of two cNLS in MA143.  Deletion of either
MA cNLS does not prevent nuclear translocation of
PIC in macrophage cells144,145, but when both are
deleted, nuclear transport of the PIC no longer occurs
even when Vpr is present143. Thus, Vpr does not
function as an independent nuclear transport factor.
However, when one or both MA cNLS are present
in the PIC, nuclear transport occurs efficiently only
in the presence of Vpr133,136,137,146, suggesting Vpr is
ZHAO et al: HIV-1 VPR
278 INDIAN J MED RES, APRIL  2005
an important player in the nuclear transport of PIC.
Vpr increases the binding of the MA cNLSs to
importin ? 137, and this increased affinity may at least
in part account for Vpr’s activity in HIV-1 nuclear
transport.  In general, the major role of Vpr may be
to stimulate the nuclear import of unusually large
complexes carrying relatively weak NLSs, as the
effect of Vpr on nuclear import of the model substrate,
serum albumin coupled to cNLS, decreased with an
increase in the number of coupled cNLS copies 136.
One interesting implication of a conserved Vpr-
binding site present both on human and budding yeast
importin ?  is that this binding site plays some important
cellular function in nuclear transport and that a cellular
protein may bind to this site. Agostini et al142 have
identified this cellular protein as Hsp70, a highly
conserved heat shock protein, which competes with
Vpr for binding to importin ? . Hsp70 can in fact
replace Vpr in the nuclear transport of PIC and similar
to Vpr also strengthens the binding of MA cNLS to
importin  ? 142. One cellular role of this Vpr/Hsp70
binding site thus appears to be in strengthening the
interaction of a weak cNLS with importin  ? .
Therefore, one possible role for Hsp70 may be to
stimulate efficient translocation of large complexes
through the nuclear pore, similar to the role of Vpr in
the nuclear import of the HIV-1 PIC.
Induction of apoptosis
A major pathway for the induction of apoptosis by
Vpr is through the mitochondria. This intrinsic
pathway for apoptosis is initiated by mitochondrial
outer membrane permeabilization (MOMP)147. The
release of proteins from the space between the inner
and outer mitochondrial membranes ultimately leads
to apoptosis. Cytochrome c is particularly important
in this process since it combines in the cytoplasm with
Apaf-1 to activate procaspase 9, the initiating caspase
for the intrinsic pathway. Activated caspase 9 in turn
activates the downstream caspases such as caspase
3 which carry out many of the apoptotic events147.
Vpr is thought to lead to MOMP by virtue of
binding to ANT (adenine nucleotide transporter)
protein of the inner mitochondrial membrane148-150.
After crossing the outer mitochondrial membrane,
possibly through VDA (the Voltage Dependent Anion
Channel), binding of Vpr to ANT leads to
depolarization of the inner mitochrondrial membrane,
swelling of the inner mitochondria and ultimately
MOMP with release of the apoptosis factors. Among
the considerable evidence supporting this model are
depolarization of the inner mitochondrial membrane
by Vpr in intact cells, depolarization of isolated
mitoZHAO  et al: HIV-1 VPRchondria by purified
Vpr, strong binding between Vpr and ANT shown by
several methods, reduced cell killing when ANT levels
are decreased148-150 and activation of caspase 9 by
Vpr151,152.
While there is considerable evidence supporting the
idea that Vpr induces apoptosis through MOMP, there
are other reports that do not readily fit into this model
and which raise the possibility that Vpr kills cells
through other redundant pathways. The localization of
Vpr raises one question about the MOMP model since
Vpr has been consistently reported to be in the nucleus
or at the nuclear membrane20,53,132,133,153,154 rather than
in the mitochondria150. It may be that only a small
fraction of Vpr molecules localizes to the mitochondria,
which is sufficient to induce apoptosis and methods
used to visualize Vpr may have overlooked this small
amount. However, the predominant nuclear localization
of Vpr and the association of nuclear localization with
cell killing in Vpr mutants20,154 suggest that Vpr located
in the nucleus may sometimes play a role in initiating
cell killing.
Other observations seemingly inconsistent with the
MOMP model concern the activation of caspases by
Vpr.  While activation of caspase-9 with no activation
of caspase-8 supports the role of MOMP in the
induction of apoptosis by Vpr151, there have been
other conflicting reports that Vpr does activate
capase-864,155. Caspase-8 activation is thought to be
a hallmark of the extrinsic pathway for apoptosis
induction by death receptors such as FAS and TNF 156.
It has also been reported that a fragment of Vpr is
able to induce cell death without caspase activation157,
and even that Vpr induces a necrotic type of cell death
in neurons158. The observation that Vpr is able to kill
fission yeast cells56, where caspases play at most a
minor role in cell death159, also suggests that there
may be a caspase- and mitochondria- independent
pathway for cell killing by Vpr.
279
A prospective role of HIV-1 in future antiviral
therapy
Evidence described in this review suggests that
Vpr plays a pivotal role in viral pathogenesis and
disease progression. Specifically Vpr activities are
linked to promotion of viral infection in non-dividing
macrophages and monocytes, activation of viral
transcription and replication, and depletion of CD4
T-lymphocytes. Therefore, strategies that can be used
to inhibit these adverse Vpr effects could potentially
alleviate the impact of the virus and benefit infected
patients. It is desirable then to identify Vpr-specific
inhibitors for the design of future anti-Vpr regimens.
A number of hexameric peptides with a di-tryptophan
motif were found by genetic selection in budding yeast
to suppress G2 arrest and apoptosis in T-cells160. So
far a fission yeast small heat shock protein 16 (Hsp16)
is the only protein known to specifically block all Vpr
activities27. However, its utility in suppressing Vpr-
mediated viral infection in macrophages/monocytes
or its potential beneficial effect for HIV-infected
patients has yet to be determined.
Vpr is a virion-associated protein that is packaged
in the viral particle in quantities similar to those of
the major structural proteins132. This unique property
of Vpr could potentially be used to target antiviral
proteins to the virus. A proposed strategy is to create
a chimeric protein that fuses Vpr with a functional
protein capable of interfering with the progeny virion
production161,162. Various Vpr fusion proteins have thus
far been created. In all case, Vpr fusion proteins are
efficiently incorporated into the viral particles,
confirming feasibility of Vpr-mediated virion
incorporation. Since HIV-1 protease is required for
the processing of HIV-1 viral precursor proteins in
order to generate infectious viral particles, (Gag and
Gag-Pol), one approach is to inhibit viral maturation
by sequestering protease162, i.e., to use a Vpr fusion
protein containing the HIV-1 protease cleavage sites.
A chimeric Vpr containing the cleavage sequences
from the junction of p24 and p2 completely abolished
viral infectivity162. A similar approach was used in
targeting dimerization of the protease163. A four amino
acid peptide from the C-terminal end of the protease,
which is the dimmer interface region, was fused to
Vpr. Incorporation of this chimeric protein results in
reduced viral infectivity163. The HIV-1 integrase was
targeted by fusing Vpr with a single-chain antibody
(scAb) or a single-chain variable antibody fragments
(SFvs) against the integrase164,165. Suppression of
progeny virion production was observed with both Vpr
fusions164,165. These results indicate that the
inactivation of progeny virions by the use of chimeric
Vpr fusion proteins represents a promising strategy
in developing future antiviral therapies.
The unique properties of Vpr could also be used
for other therapeutic purposes. The ability of Vpr to
arrest the cell cycle in G2 provides an opportunity to
explore its potential as a cell cycle ”G2 blocker” to
inhibit growth of the cancerous cells. Furthermore,
since mammalian cells residing in the G2 phase of
the cell cycle are most sensitive to radiation, Vpr-
induced G2 arrest could conceivably sensitize tumour
cells to radiation. The combination of gene and
radiotherapies based on Vpr and the radiosensitivity
of G2 cells may lead to the development of new
methods to treat cancer. Several earlier studies have
shown that expression of vpr can effectively suppress
tumour growth in vitro and in vivo166-169. Vpr also
induces apoptosis in various tumour cells regardless
of p53 status166,168. Interestingly, one report shows
that Vpr not only reduces tumor growth in a mouse
but also triggers sufficient immune responses that the
mouse is protected against a second oncogenic
challenge170.
Because Vpr is capable of suppressing various
host inflammatory responses, it also could potentially
be used as a therapeutic tool for treating diseases
with hyperinflammatory conditions such as rheumatoid
arthritis, sepsis and Crohn’s disease35. This strategy
may have advantages over other conventional anti-
inflammatory drugs since Vpr specifically inhibits pro-
inflammatory cytokines such as TNF?  and IL-12.
Moreover, it limits chemokine production and
suppresses NF? B-mediated transcription36,37. In
addition, Vpr was shown to be more effective in
reducing inflammatory cytokines than some of the
commonly prescribed anti-inflammatory drugs35
indicating that the use of Vpr as an anti-inflammatory
agent merits further investigation.
ZHAO et al: HIV-1 VPR
280 INDIAN J MED RES, APRIL  2005
Acknowledgment
       Authors acknowledge the financial support received from
the National Institutes of Health, USA.
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Reprint requests: Dr Richard Y. Zhao, Department of Pathology, University of Maryland School of Medicine
10 South Pine Street, MSTF 600, Baltimore, MD 21201-1192, USA
e-mail: RZhao@som.umaryland.edu