JOURNAL OF VIROLOGY, Apr. 2006, p. 3694–3700
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Vol. 80, No. 8
Induction of G2Arrest and Binding to Cyclophilin A Are Independent
Phenotypes of Human Immunodeficiency Virus Type 1 Vpr
Orly Ardon, Erik S. Zimmerman, Joshua L. Andersen, Jason L. DeHart,
Jana Blackett, and Vicente Planelles*
Division of Cellular Biology and Immunology, Department of Pathology, University of Utah School of Medicine,
Salt Lake City, Utah 84132
Received 26 November 2005/Accepted 25 January 2006
Cyclophilin A (CypA) is a member of a family of cellular proteins that share a peptidyl prolyl cis-trans
isomerase (PPIase) activity. CypA was previously reported to be required for the biochemical stability and
function (specifically, induction of G2arrest) of the human immunodeficiency virus type 1 (HIV-1) protein R
(Vpr). In the present study, we examine the role of the Vpr-CypA interaction on Vpr-induced G2arrest. We find
that Vpr coimmunoprecipitates with CypA and that this interaction is disrupted by substitution of proline-35
of Vpr as well as incubation with the CypA inhibitor cyclosporine A (CsA). Surprisingly, the presence of CypA
or its binding to Vpr is dispensable for the ability of Vpr to induce G2arrest. Vpr expression in CypA?/?cells
leads to induction of G2arrest in a manner that is indistinguishable from that in CypA?cells. CsA abolished
CypA-Vpr binding but had no effect on induction of G2arrest or Vpr steady-state levels. In view of these results,
we propose that the interaction with CypA is independent of the ability of Vpr to induce cell cycle arrest. The
interaction between Vpr and CypA is intriguing, and further studies should examine its potential effects on
other functions of Vpr.
Cyclophilin A is a member of a family of cellular proteins
that shares a peptidyl prolyl cis-trans isomerase (PPIase) activ-
ity. To date, 16 cyclophilin genes and numerous cyclophilin
pseudogenes have been identified in the human genome (11,
26, 32). The PPIase activity of cyclophilins appears to be im-
portant for the maintenance of proper protein conformation
through cis-trans interconversion of N-terminal peptide bonds
aminoterminal to proline (10, 31). This enzymatic activity con-
tributes to cyclophilin involvement in cell signaling, mitochon-
drial function, molecular chaperone activity, RNA splicing, stress
responses, gene expression, and regulation of kinase activity (10,
Cyclophilins owe their name to their ability to bind to the
immunosuppressive drug cyclosporine A (CsA) with high affinity
(17). CsA binding to CypA potently inhibits CypA isomerase
activity. The immunosuppressive effects of CsA result from
binding of the CypA/CsA complex to calcineurin, resulting
in calcineurin inhibition (20, 24).
A role for CypA in the life cycle of primate lentiviruses
emerged in 1993 with the isolation of CypA as a yeast two-
hybrid partner of human immunodeficiency virus type 1
(HIV-1) core protein, p24 (26). CypA binding to p24 is nec-
essary for the infectivity of HIV-1, and blocking this binding,
either through site-directed mutations in p24 or competitive
inhibitors such as CsA, severely impairs the infectivity of
HIV-1 particles (reviewed in reference 14). It is thought that
the binding of CypA to p24 is a necessary event for the efficient
uncoating of viral particles following viral entry (25). Recent
evidence indicates that the effects of CsA on HIV-1 replication
are not solely derived from CsA inhibition of CypA binding to
p24 in target cells, as treating HIV-1-producing cells with CsA
also reduces the infectivity of progeny viral particles, albeit in
a CypA-independent fashion (9, 18, 30).
HIV-1 Vpr exerts several deleterious effects when expressed
in human cells, including induction of cell cycle arrest in G2
and apoptosis. Vpr induces a DNA damage-like signal that
triggers known downstream checkpoint responses involving
certain cell cycle-related kinases and phosphatases, such as
ATR, Chk1, Wee1, and Cdc25 (5, 15, 19, 21, 22, 28, 29, 33, 37).
Recent evidence suggests that Vpr interacts with the chroma-
tin in a unique manner, which results in activation of the G2
checkpoint without causing double-strand breaks (22). Vpr is
packaged into virus particles, and its expression has been
shown to induce a plethora of other effects in target cells,
including transactivation of the viral promoter, modulation of
the accuracy of the reverse transcription process, induction
of apoptosis, and disruption of nuclear envelope integrity
(reviewed in references 4, 23, and 35).
The Vpr gene product is a small 96-amino-acid protein. The N
terminus of Vpr contains four conserved proline residues (posi-
tions 5, 10, 14, and 35). These proline residues were shown to
undergo cis-trans isomerism to varying degrees in studies that
The activity of CypA was shown to be required for the biochem-
ical stability and function (specifically, induction of G2arrest) of
with CypA and activity of Vpr (34).
In the present study, we examine the impact of CypA inter-
action on Vpr-induced G2arrest and Vpr levels of expression.
In agreement with the studies by Zander et al. (34), we find
that Vpr can be coimmunoprecipitated with CypA and that this
interaction is disrupted by mutation of the proline-35 residue.
However, substitution of proline-35 by alanine or asparagine
* Corresponding author. Mailing address: Department of Pathology,
University of Utah School of Medicine, 30 N 1900 East, SOM 5C210,
Salt Lake City, UT 84132. Phone: (801) 581-8655. Fax: (801) 587-7799.
results in stable proteins that fail to bind to CypA and are still
capable of inducing cell cycle arrest. Cells in which CypA had
been genetically ablated are sensitive to Vpr-induced G2ar-
rest. In addition, incubation with CsA abolished the Vpr-CypA
interaction but failed to inhibit Vpr-induced G2arrest. We
propose that the ability of Vpr to interact with CypA is inde-
pendent of its ability to induce cell cycle arrest.
MATERIALS AND METHODS
Cells and drug treatments. Human embryonic kidney (HEK) cell line 293FT
(Invitrogen, Carlsbad, Calif.) cells were cultured in Dulbecco’s modified Eagle’s
medium with 10% fetal bovine serum (FBS), 1% L-glutamine solution. One
milligram of G418 per milliliter was added when maintaining the cells. CypA?/?
Jurkat cells (7) and CypA?cells were cultured in RPMI 1640 medium (Invitro-
gen, Carlsbad, CA) supplemented with 10% FBS, 2 mM L-glutamine (Invitro-
gen). Exponentially growing HeLa cells were cultured in Dulbecco’s minimal
essential medium (Invitrogen) supplemented with 10% fetal bovine serum and
1% penicillin-streptomycin-L-glutamate (Invitrogen). Where indicated, cells
were incubated with 2.5 ?M cyclosporine A (CsA; Sigma Aldrich, St. Louis, MO)
for 24 h.
Lentivirus vectors. Lentivirus vectors were produced by transient transfection
of HEK293FT cells. For defective lentivirus vector production, plasmids pHR-
GFP and pHR-VPR and the indicated mutants were cotransfected with
pCMV?R8.2?Vpr (3) and pHCMV-VSVG (2) by calcium phosphate-mediated
transfection (36). Virus supernatants were collected at 48, 72, and 96 h post-
transfection. Harvested supernatants were cleared by centrifugation at 2,000 rpm
(828 ? g). Cleared supernatants were concentrated by ultracentrifugation at
25,000 rpm (115,889 ? g) for 1.5 h at 4°C. Concentrated virus was allowed to
resuspend overnight at 4°C, and the suspension was frozen at ?80°C for storage.
Vector titers were measured by infection of HeLa cells as described below,
followed by flow cytometric analysis of cells that were positive for the reporter
molecule, green fluorescent protein (GFP). Vector titers were calculated with
the equation [(F ? C0)/V] ? D, where F is the frequency of GFP-positive cells
found by flow cytometry, C0is the total number of target cells at the time of
infection, V is the volume of inoculum, and D is the virus dilution factor. The
virus dilution factor used for titrations was 10. The total number of target cells
at the time of infection was 1 ? 106. Infections were performed at a multiplicity
of infection (MOI) of 2 with 10 ?g of Polybrene/ml for 2 h.
HIV-1 production and infections. HIV-1 molecular clones pNL4-3 and pNL4-
3-VprX were transfected into 293FT cells by calcium phosphate transfection as
described for lentiviral vector production. Twenty-four hours after transfection,
virus-producing 293FT cells were cocultured with 1 ? 107MT-2 human T-cell
leukemia virus-transformed CD4?T cells for 5 h. MT-2 cells were then cultured
alone until approximately 75% of cell blasts exhibited syncitia. Virus-containing
supernatants were then cleared of cell debris by centrifugation at 828 ? g for 10
min. Viral stocks were then frozen at ?80°C. Cells were infected by spin infection
FIG. 1. CypA is not required for Vpr-mediated cell cycle arrest. (A) Cell cycle analysis of CypA?and CypA?/?Jurkat cells infected with
pHR-VPR, encoding Vpr and GFP, or the control vector, pHR-GFP, encoding GFP only. Vector-infected cells were analyzed 24 h postinfection
for DNA content and, separately, for GFP expression, using flow cytometry with the Modfit software package (Verity Software House, Inc.,
Topsham, Maine). Relative G1, S, and G2/M distributions as well as infection rates are indicated. “Percent infected” indicates the percentage of
GFP-positive cells. The results are representative of two different experiments. (B) CypA expression in Jurkat cells. Lysates of CypA?/?and
CypA?were subjected to Western blot analysis with anti-CypA antibody to verify the lack of CypA expression in CypA?/?cells.
VOL. 80, 2006INDEPENDENT PHENOTYPES OF HIV-1 Vpr3695
as follows: 1 ? 106cells were diluted in viral stocks with 10 ?g/ml Polybrene and
centrifuged at 1,700 ? g for 2 h at 25°C, after which cells were resuspended in
normal growth medium and incubated at 37°C in 5% CO2.
Cell lysis and immunoprecipitation assays. HEK293FT cells were transfected
using calcium phosphate-mediated transfection. Twenty-four hours posttransfec-
tion, cells were detached using trypsin, washed, and lysed in lysis-and-immuno-
precipitation (IP) buffer (20 mM Tris, pH 7.5, 100 mM NaCl, 0.5% NP-40, 0.5
mM EDTA) containing protease inhibitors (Complete tablets; Roche, Indianap-
olis, IN) for 10 min on ice. Lysates were centrifuged at 2,000 rpm for 10 min and
supernatants were collected. One microliter of rabbit anti-CypA (undiluted se-
rum; EMD Biosciences, San Diego, CA) antibody was added, and tubes were
incubated with an end-over-end mixing at 4°C for 1 h. Protein A/G agarose
(Santa Cruz Biotechnology, Santa Cruz, CA) was added to immunoprecipitates
and incubated overnight with an end-over-end mixing at 4°C. Immunoprecipi-
tates were washed five times with IP buffer, followed by boiling in XT sample
buffer (Bio-Rad, Hercules, CA).
Western blotting. Cell lysates or IP eluates were boiled for 5 min prior to being
loaded on Criterion XT bis-tris gels (Bio-Rad, Hercules, Calif.) for electro-
phoretic separation. Proteins were transferred to Immobilon polyvinylidene
difluoride membranes (Millipore, Bedford, MA) by a semidry transfer method
(Bio-Rad) and then blocked for 45 min at room temperature in blocking solution
(5% skim milk and 0.1% Tween 20 in phosphate-buffered saline [PBS]). Rabbit
primary antibodies against CypA (1:10,000; EMD Biosciences, San Diego, CA),
actin (1:1,000; Santa Cruz), or mouse anti-hemagglutinin (HA) (1:1,000; Covance,
Berkeley, CA) were applied at indicated dilutions and incubated at 4°C overnight.
Blots were washed three times in TPBS (0.1% Tween 20 in PBS) for 5 min, each
time at room temperature. Secondary horseradish peroxidase-conjugated goat
anti-mouse or anti-rabbit immunoglobulin G antibodies were applied for 45 min
at room temperature. Blots were washed again three times in TPBS before
protein detection with enhanced chemiluminescence reagent (Amersham,
Cell cycle analysis. At given times postinfection, Jurkat cells were collected,
washed with fluorescence-activated cell sorting (FACS) buffer (2% FBS, 0.5 mM
EDTA, and 0.02% sodium azide in PBS), fixed with 2% paraformaldehyde in
PBS, and permeabilized with 0.1% Triton X-100 in PBS for 15 min. Cells were
washed again with FACS buffer, incubated in DNA staining buffer (10 ?g of
propidium iodide/ml and 11.25 kU of RNase A/ml in FACS buffer) for 15 min,
and analyzed by FACScan flow cytometry for GFP expression or DNA content
FIG. 2. CypA is not required for Vpr-induced cell cycle arrest in the context of HIV-1 infection. Vpr was introduced by infection with
replication-competent HIV-1NL4-3. The isogenic, Vpr-negative mutant, HIV-1NL4-3vprX, was used as a negative control. Infections of CypA?and
CypA?/?cells were analyzed at 4 (A) and 7 (B) days postinfection by simultaneous intracellular p24 and DNA content staining with propidium
iodide. The p24-positive (infected) and -negative (uninfected) cells from the cultures were analyzed by flow cytometry for DNA content after being
gated based on p24 status. Relative G1, S, and G2/M distribution as well as infection rates are indicated.
3696ARDON ET AL. J. VIROL.
(Beckton Dickinson, Franklin Lakes, N.J.). For detection of HIV-1 p24 antigen-
expressing cells, we used a previously described protocol (6, 12). Briefly, 1 ? 106
cells were washed twice in flow cytometry buffer and permeabilized using the
Cytofix/Cytoperm kit (Pharmingen BD, San Jose, CA). The permeabilized cells
were resuspended in 100 ?l of intracellular staining buffer and incubated at 4°C
with 5 ?l of human anti-HIV p24 monoclonal antibody (obtained from the AIDS
Research and Reference Reagent Program, Division of AIDS, National Institute
of Allergy and Infectious Diseases, National Institutes of Health; monoclonal
antibody to HIV-1 p24, clone 71-31, was from Susan Zolla-Pazner ). The cells
were washed twice in intracellular staining buffer, resuspended at 1 ? 107cells/ml
in intracellular staining buffer, and incubated at 4°C with 10 ?l fluorescein
isothiocyanate-conjugated F(ab?)2goat anti-human immunoglobulin G (Caltag,
Burlingame, CA) for 30 min. The antibody-labeled cells were washed twice with
intracellular staining buffer. The cells were then resuspended in DNA staining
buffer as described above. Cell cycle profiles were modeled by using ModFit
software (Verity Software, Topsham, ME).
RESULTS AND DISCUSSION
Vpr induces cell cycle arrest in G2independently of the
presence or absence of CypA. Our previous studies have shown
that infection with a lentiviral vector (pHR-VPR-IRES-GFP, re-
ferred to as pHR-VPR in this study) (29) that encodes a Vpr-
IRES-GFP tandem cistron faithfully recapitulates the effects of
Vpr, including induction of cell cycle arrest (37), apoptosis (5),
and transactivation of the viral long terminal repeat (29). To test
whether the presence of CypA is necessary for induction of cell
cycle arrest, we infected CypA?/?(7, 34) or unmodified Jurkat
cells (herein referred to as CypA?) with pHR-VPR or a control
vector, pHR-GFP (29). Vector-infected cells were analyzed 24 h
postinfection for DNA content and, separately, for GFP expres-
sion using flow cytometry (Fig. 1A). CypA?/?Jurkat cells exhib-
ited cell cycle arrest (68% cells in G2/M; 65% GFP?cells) com-
parable to that of CypA?Jurkat cells (77% cells in G2/M; 70%
GFP?cells). The CypA expression status of the cells was verified
by Western blotting (rabbit anti-CypA; EMD Biosciences, San
Diego, CA) and confirmed the lack of CypA expression in
CypA?/?cells (Fig. 1B).
Since lentiviral vectors allow expression of high levels of
heterologous proteins, it is possible that ectopic expression of
Vpr in the previous experiment may have overcome a potential
restriction (such as biochemical instability of Vpr or an inactive
conformation) derived from the lack of CypA. To validate the
above results in a more relevant expression system, we per-
formed similar experiments in which Vpr was introduced by
infection with replication-competent HIV-1NL4-3(1). As a neg-
ative control, we utilized the isogenic, Vpr-negative mutant
HIV-1NL4-3vprX, which contains a frameshift mutation that
completely inactivates Vpr (5, 19, 27). We performed infec-
tions of CypA?and CypA?/?cells at a multiplicity of infection
(MOI) of 0.5. Infected cultures were analyzed at 96 h by flow
cytometry after combined staining for intracellular p24 and
DNA content. The p24-positive and -negative cells from each
infection were then electronically gated and analyzed for cell
cycle distribution (Fig. 2). Infections of CypA?/?cells were
routinely low (between 0.5% and 2.5% at 96 h postinfection)
and viruses failed to spread in these cells, consistent with a
known requirement for CypA in target cells during the uncoat-
ing step (9, 18, 30). In contrast, infections of CypA?cells were
at a high level (between 25% and 60% at 96 h postinfection)
and showed viral spread (data not shown). Despite the low
frequency of infected cells in CypA?/?cultures, it was possible
to use electronic gating to analyze the cell cycle of infected and
uninfected cells separately. As expected, CypA?cell infection
with HIV-1NL4-3, but not with HIV-1NL4-3VprX, resulted in a
dramatic increase of the G2/M peak after 96 h (Fig. 2A; 63.2%
and 29.2% cells in G2/M, respectively). The lack of CypA in
CypA?/?cells did not affect the sensitivity of cells to G2arrest
by infection with HIV-1NL4-3(67.3% cells in G2/M). After 7
days of infection, the percentages of HIV-1NL4-3-infected cells
exhibiting G2/M DNA content were 80.1% and 60.8% for
CypA?and CypA?/?cells, respectively (Fig. 2B).
The above results indicate that Vpr expression is associated
with induction of G2arrest in a manner that is indistinguish-
able between CypA?and CypA?/?cells, whether Vpr is ex-
pressed from a lentiviral vector or from infectious, full-length
HIV-1. The apparent discrepancy between our results and
those reported by Zander et al. (34) prompted us to reexamine
the Vpr-CypA interaction. Vpr binding to CypA was previously
reported (8, 34), and this interaction was postulated to be
required for Vpr stability and for its ability to induce G2arrest
(8). Bruns et al. indicated that the proline residue at position
FIG. 3. Coimmunoprecipitation of Vpr with CypA. (A) 293FT cells
were transfected with the indicated lentiviral vectors, and a transfec-
tion efficiency of 95% was recorded. Twenty-four hours posttransfec-
tion, cells were lysed and anti-CypA antibody was added to lysates.
Coimmunoprecipitation with protein A/G agarose beads was carried
out at 4°C overnight. Samples were then washed, boiled, and analyzed
by sodium dodecyl-sulfate polyacrylamide gel electrophoresis and
Western blotting. HA-tagged Vpr was detected using anti-HA anti-
bodies. CsA was added to a final concentration of 2.5 ?M following
transfection of cells with pHR-VPR. The blot shown in the upper
panel was stripped and reprobed with anti-CypA antibody to ensure
that comparable amounts of CypA had been immunoprecipitated in
lanes 1 to 6 (lower panel). Cell lysate inputs were analyzed, prior to
immunoprecipitation, for levels of HA-Vpr, CypA, and actin, as indi-
cated. (B) Vpr steady-state levels in CypA?/?Jurkat cells are not
compromised by mutation of proline-35 or incubation with CsA (lanes
1 to 5). The steady-state level of Vpr protein was examined in parallel
in Jurkat CypA?cells (lane 6).
VOL. 80, 2006INDEPENDENT PHENOTYPES OF HIV-1 Vpr3697
35 of Vpr was essential for the interaction with CypA (8). In
order to directly examine the interaction between CypA and
Vpr, we performed coimmunoprecipitation studies with wild-
type Vpr as well as with three Vpr mutants. Vpr(P35N) (8) and
Vpr(P35A) disrupt proline-35. Vpr(R80A) is unable to induce
G2arrest or apoptosis (5, 13). 293FT cells were transfected
with pHR-VPR, Vpr mutants, or GFP. Twenty-four hours
posttransfection, cells were lysed and anti-CypA antibody was
used for immunoprecipitation. Immunoprecipitates were then
analyzed by sodium dodecyl-sulfate polyacrylamide gel electro-
phoresis followed by Western blotting, using an anti-HA antibody
that recognizes an amino-terminal hemagglutinin epitope present
in all Vpr vector constructs.
In agreement with the previous finding, we found that Vpr
efficiently coprecipitated with CypA (Fig. 3A, lane 5). In con-
trast, both Vpr(P35N) and Vpr(P35A) were impaired in their
abilities to coprecipitate with CypA, as evidenced by extremely
faint Vpr bands (Fig. 3A, lanes 3 and 4). Vpr(R80A) also failed
to coprecipitate with CypA, as indicated by the absence of a
detectable Vpr band (Fig. 3A, lane 2). The blot shown on the top
panel of Fig. 3A was then stripped and reprobed with anti-CypA
antibody in order to verify that equal amounts of CypA had been
immunoprecipitated in experiments 1 through 6 (Fig. 3A, second
blot). Analysis of the steady-state levels of Vpr by Western
2? to 5?) revealed that all Vpr mutants were expressed at compa-
rable or higher levels than wild-type Vpr.
The above data indicate that the inability of Vpr mutants to
coprecipitate with CypA did not stem from a decrease in the
mutant protein steady-state levels, which would have suggested
FIG. 4. Induction of G2arrest by Vpr and Vpr(P35N) and failure of CsA to inhibit Vpr function. CypA?(A) or CypA?/?(B) Jurkat cells were
mock infected or infected with pHR-GFP, pHR-VPR, or pHR-VPR(P35N) and then subdivided into two cultures, which were incubated in the
presence or absence of 2.5 ?M CsA, respectively. Cells were analyzed 48 h postinfection for DNA content and, separately, for GFP expression
using flow cytometry. Relative G1, S, and G2/M distributions as well as infection rates are indicated.
3698ARDON ET AL.J. VIROL.
a loss of protein stability. In addition, the fact that Vpr(R80A)
failed to coprecipitate with CypA indicates that residues out-
side the Vpr (residues 1 to 40) domain (8) are also important
for binding to CypA and that the presence of proline-35 is not
sufficient for such binding.
To further explore the specificity of Vpr-CypA interaction,
we performed parallel coimmunoprecipitation experiments in
which cells were cultured in the presence of 2.5 ?M CsA. CsA
binds to CypA and competitively inhibits the interaction be-
tween p24 Gag and CypA (9, 18, 30). CsA incubation did not
impair the steady-state levels of Vpr in the cells, as evidenced
by Western blotting of input lysate (Fig. 3A, third blot from the
top, compare lanes 5? and 6?). CsA incubation, however, abol-
ished the interaction between CypA and Vpr (Fig. 3A, top blot,
compare lanes 5 and 6).
Expression of wild-type or Vpr mutants in CypA?/?Jurkat
cells further demonstrated that CypA is dispensable for the
steady-state levels of Vpr expression (Fig. 3B, lanes 2, 4, and
5). CypA?/?Jurkat cells presumably contain all cyclophilins
other than CypA. Thus, it is formally possible that Vpr may
interact with other cyclophilins, and this, in turn, may compen-
sate for the absence of CypA. Since CsA inhibits all known
cyclophilins, we asked whether incubation of CypA?/?cells
with CsA would affect the levels of Vpr expression. As shown
in Fig. 3B (compare lanes 2 and 3), CsA did not appreciably
affect the steady-state level of Vpr. The Vpr expression level in
CypA?/?cells was similar to that obtained in CypA?cells (Fig.
3B, compare lanes 2 and 6) when tested in parallel. Thus, we
conclude that the presence of CypA is not required for efficient
expression of Vpr. We also conclude that even though CsA
potently inhibits the Vpr-CypA interaction, this interaction is
dispensable for efficient expression of Vpr.
The ultimate goal of the present study was to establish the
requirement of CypA toward Vpr-induced G2arrest. The
availability of Vpr mutants defective for CypA binding, as
well as the pharmacological inhibitor CsA, allowed us to ask
whether binding to CypA could be dissociated from induction
of G2arrest. Expression of wild-type Vpr and Vpr(P35N) in-
duced dramatic G2arrest in CypA?cells as well as in CypA?/?
Jurkat cells (Fig. 4; 85.8% and 60.1%, respectively). Addition
of CsA to the cultures did not significantly change the G2arrest
levels (70.3% in CypA?and 56.7% in CypA?/?cells, respec-
tively). Vpr(P35N) also induced G2arrest (52.7%) in CsA, al-
though with slightly decreased efficiency compared with wild-type
Vpr. In CypA?/?, Vpr(P35N) induced levels of G2arrest (67.1%)
comparable to those of wild-type Vpr. Incubation of cultures with
CsA did not significantly affect G2arrest by Vpr(P35N) (45% in
CypA?and 65% in CypA?/?cells). Therefore, binding to CypA
is not required for the induction of G2arrest by Vpr.
The sharp discrepancies between our results and those re-
ported by Zander and colleagues could be reconciled, in part,
if CsA treatment induced the loss of stability of Vpr (and,
therefore, loss of function as well) in a CypA-dependent
manner. However, our findings demonstrate that although CsA
inhibits the interaction between Vpr and CypA, the steady-
state level of Vpr in the cells remains the same in CsA-treated
and untreated cells, and incubation with CsA does not affect
The concentration of CsA used in our experiments was 2.5
?M. We find that this concentration of CsA effectively inhibits
CypA-Vpr binding. This concentration is well below the 50-
?g/ml (equivalent to 41.6 ?M) CsA concentration used by
Zander and colleagues (34) when they observed loss of Vpr
stability in cells treated with this CypA inhibitor. We reason
that, since genetic removal of CypA failed to inhibit Vpr func-
tion or stability, the effects observed with 50 ?g/ml CsA may be
due to other effects of CsA which are CypA independent. For
example, high concentrations of CypA may be cytotoxic. A
precedent for the existence of an additional, ill-understood
effect of CsA stems from the observation that CsA inhibits the
infectivity of progeny HIV-1 virions in producer cells by a
mechanism that is independent of CypA inhibition (18, 30).
Since CsA is thought to inhibit all known cyclophilins, our
observations combining the presence of CsA and the genetic
elimination of CypA suggest that cyclophilins other than CypA
appear to also be dispensable for Vpr expression and induction
We conclude that while in vivo binding between Vpr and
CypA is clearly detectable, this binding is not necessary for the
stability of Vpr or its ability to induce G2arrest. The interac-
tion between Vpr and CypA is highly intriguing and should be
further investigated as a potential modulator of virus-host in-
We are thankful to Michael J. Blackwell and Sam Campbell for
excellent technical help and Warner Greene for useful discussions.
The following reagents were obtained through the NIH AIDS Re-
search and Reference Reagent Program, Division of AIDS, NIAID,
NIH: Jurkat T-cells CypA?/?from D. Braaten and J. Luban and
HIV-p24 human monoclonal antibody (71-31, catalog no. 530) from S.
This work was supported by NIAID grant AI49057 to V.P. J.L.A. is
supported by a Training Program in Microbial Pathogenesis, NIAID
T32 AI055434. E.S.Z. is supported by NIH Genetics Training Grant
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