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Virology 328 (2
Structure–functional analysis of human immunodeficiency
virus type 1 (HIV-1) Vpr: role of leucine residues on
Vpr-mediated transactivation and virus replication
Dineshkumar Thotalaa, Elizabeth A. Schafera, Biswanath Majumdera, Michelle L. Janketa,
Marc Wagnera, Alagarsamy Srinivasanb, Simon Watkinsc, Velpandi Ayyavooa,*
aDepartment of Infectious Diseases and Microbiology, University of Pittsburgh, Pittsburgh, PA 15261, United StatesbDepartment of Microbiology and Immunology, Thomas Jefferson University, Philadelphia, PA 19107, United States
cCenter for Biologic Imaging, University of Pittsburgh, Pittsburgh, PA 15261, United States
Received 13 July 2004; accepted 13 July 2004
Abstract
HIV-1 Vpr has been shown to transactivate LTR-directed expression through its interaction with several proteins of cellular origin
including the glucocorticoid receptor (GR). Upon activation, steroid receptors bind to proteins containing the signature motif LxxLL,
translocate into the nucleus, bind to their response element, and activate transcription. The presence of such motifs in HIV-1 Vpr has
prompted us to undertake the analysis of the role of specific leucine residue(s) involved in Vpr–GR interaction, subcellular localization and
its effect on Vpr–GR-mediated transactivation. The individual leucine residues present in H I, II, and III were mutated in the Vpr molecule
and evaluated for their ability to interact with GR, transactivate GRE and HIV-1 LTR promoters, and their colocalization with GR. While Vpr
mutants L42 and L67 showed reduced activation, substitutions at L20, L23, L26, L39, L64, and L68 exhibited a similar and slightly higher
level of activation compared to Vprwt. Interestingly, a substitution at residue L22 resulted in a significantly higher GRE and HIV-1 LTR
transactivation (8- to 11-fold higher) in comparison to wild type. Confocal microscopy indicated that Vpr L22A exhibited a distinct
condensed nuclear localization pattern different from the nuclear/perinuclear pattern noted with Vprwt. Further, electrophoretic mobility shift
assay (EMSA) revealed that the VprL22A–GR complex had higher DNA-binding activity when compared to the wild type Vpr–GR complex.
These results suggest a contrasting role for the leucine residues on HIV-1 LTR-directed transactivation dependent upon their location in Vpr.
D 2004 Elsevier Inc. All rights reserved.
Keywords: Glucocorticoid receptor; Transactivation; Replication
Introduction
Human immunodeficiency virus type-1 (HIV-1) Vpr is a
14-kDa virion-associated non-structural protein. While Vpr
has been shown to be important for viral replication in non-
dividing cells (Balliet et al., 1994; Connor et al., 1995;
Heinzinger et al., 1994; Subbramanian et al., 1998),
increased viral replication in T-cell lines as well as activation
0042-6822/$ - see front matter D 2004 Elsevier Inc. All rights reserved.
doi:10.1016/j.virol.2004.07.013
* Corresponding author. Department of Infectious Diseases & Micro-
biology, University of Pittsburgh, 130 DeSoto Street, Pittsburgh, PA 15261.
Fax: +1 412 383 8926.
E-mail address: [email protected] (V. Ayyavoo).
in latently infected cells have also been reported (Levy et al.,
1995; Nakamura et al., 2002). Vpr transactivates HIV-1 LTR
and increases virus replication in target cells (Agostini et al.,
1996; Cohen et al., 1990; Wang et al., 1995). Recently, we
and others have shown that Vpr, either in the context of virus
infection or as exogenous protein, transactivates HIV-1 LTR
and upregulates viral replication before the synthesis of Tat
in infected cells. Also, Vpr and Tat together transactivate
HIV-1 LTR in an additive manner (Hrimech et al., 1999;
Sawaya et al., 2000; Vanitharani et al., 2001). Specifically,
HIV-1 Vpr-mediated transactivation is shown to occur
through steroid receptors and other coactivators (Kino et
al., 1999; Sherman et al., 2000; Vanitharani et al., 2001).
004) 89–100
D. Thotala et al. / Virology 328 (2004) 89–10090
Steroid receptors belong to a super family of ligand-
dependent transcription factors that are known to interact
with other proteins containing the signature motif, LxxLL
(Feng et al., 1998; Heery et al., 1997). Upon binding to their
ligands, these receptors translocate into the nucleus and
activate/repress transcription depending upon the signal
(Northrop et al., 1992; Paliogianni and Boumpas, 1995;
Schmidt et al., 1994). HIV-1 Vpr has been shown to interact
with glucocorticoid receptor (GR) and p300/CBP and
activate transcription through the glucocorticoid response
element (GRE) (Felzien et al., 1998; Kino et al., 2002a).
HIV-1 Vpr is a pleiotropic protein, with diverse functions
that include cell cycle arrest, apoptosis, nuclear import of
the preintegration complex, transcriptional activation/repres-
sion, and interaction with viral and several cellular proteins
(Levy et al., 1993; Subbramanian et al., 1998; Zhao et al.,
1994). Structure–function studies have indicated that
nuclear localization, virion incorporation, cell cycle arrest,
and transcriptional regulation are mediated by different
functional domains of Vpr (Di Marzio et al., 1995;
Mahalingam et al., 1997; Sawaya et al., 2000; Singh et
al., 2001). Vpr-mediated cell cycle arrest is linked to an
increase in virus production (Goh et al., 1998; Levy et al.,
1995; Yao et al., 1998); however, Vpr–GR transcriptional
coactivation function is reported to be independent of cell
cycle arrest (Kino et al., 2002b; Sherman et al., 2000).
Previous studies have shown that Vpr mimics glucocorti-
coids and induces similar functions through its interaction
with GR (Ayyavoo et al., 1997). GR is a member of the
steroid hormone receptor family of transcription factors that
regulates a large number of genes in a hormone or ligand-
dependent manner. Under normal conditions, GR is present
in the cytosol as an inactive complex with two molecules of
heat shock protein 90 (HSP 90). Upon binding of the
ligands, GR dissociates from the HSP molecules and
translocates into the nucleus, binds to the consensus
palindromic sequences known as GRE and activates/
represses transcription based on the stimuli (Cadepond et
al., 1992; Li et al., 2002).
Recent structural studies utilizing NMR and CD spectro-
scopy of the synthetic full-length Vpr showed three helical
domains and three turn regions with slightly different
boundaries in comparison to the structures noted with the
N- and C-terminal fragments of Vpr (Morellet et al., 2003;
Wecker et al., 2002). The structural features of the protein
include a well-designed g-turn (14–16)-a helix (17–33)—
turn (34–36) followed by an a helix (40–48)—loop (49–
54)-a helix (55–83) domain and a flexible C-terminal
sequence. Vpr contains multiple leucine-rich domains
(LxxL) in addition to the LxxLL motif in the C- and N-
terminal region of the protein. The predicted amino acid
sequence analysis of Vpr from viruses of different clades
has shown conserved leucine motifs in helices I and III,
suggesting an important role for these domains in patho-
genesis. Vpr, being a virion-associated molecule, supports
the notion that Vpr might be available as a viral protein
during early infection (before the appearance of Tat and
Rev) to enhance gene expression by directly binding to the
viral and cellular promoters (Sawaya et al., 2000; Vanithar-
ani et al., 2001; Zhao et al., 1994). Though previous studies
have addressed the Vpr-GR coactivator functions, these
studies pertain to the mutational analysis of residues 64–68
corresponding to the LxxLL motif. To precisely map and
understand the role of leucine residues present in the a-
helices, we have constructed mutants of Vpr using site-
specific mutagenesis. All the leucine mutants were analyzed
for their ability to transactivate a reporter gene under the
control of GRE and HIV-1 LTR. Next, we evaluated
whether the subcellular localization of Vpr altered GR
translocation as well as its effect on GRE and HIV-1 LTR
transactivation in the absence of hormone stimulation. The
results generated here suggest that the leucine residues can
be functionally categorized into three groups: alterations of
residues can lead to a reduced, minimal or no effect, or an
enhanced level of transactivation in comparison to the wild-
type Vpr. Interestingly, the Vpr mutant L22A translocated
GR with a distinct condensed nuclear localization pattern
resulting in a significantly higher transactivation effect when
compared to Vprwt. Additionally, the increased DNA-
binding activity of VprL22A-GR directly correlates with
Vpr coactivator function.
Results
Generation and characterization of leucine mutants of Vpr
Structure analysis by NMR and CD spectroscopy show
that Vpr contains three helical domains with a basic amino
acid-enriched C-terminus. These helical domains are rich in
leucine residues with classical steroid signature motifs
(LxxLL) in the helical domains. Steroid receptors interact
with proteins through these classical motifs. To identify the
specific leucine domains involved in Vpr-GR interaction,
we have substituted individual leucine (L) residues in helix
I, II, and III to alanine (A) by site-directed mutagenesis (Fig.
1). To enable the detection of Vpr, sequences corresponding
to an eight amino acid Flag epitope (DYKDDDDK) were
fused in frame to the 3V end of the Vpr coding sequence. To
confirm that all of these Vpr mutants expressed a stable
protein in vivo, 293T cells were transfected with pVpr
expression plasmids. Cells were lysed 48 h after transfection
and subjected to immunoblot analysis with the M2 mono-
clonal antibody. Protein expression analysis indicated that
all mutant Vpr molecules showed an appropriate band
corresponding to 14 kDa size protein similar to wild type
Vpr (Fig. 1B). Additionally, the specificity of the antibody
was further confirmed by using the lysate of vector
(pcDNA3.1) transfected 293T cells, which did not recognize
any band. These results indicate that the steady state level of
all the single leucine mutant Vpr molecules is similar to that
of wild-type Vpr.
Fig. 1. (A) Schematic representation of HIV-1 Vpr leucine mutants: Vpr leucine mutants were generated by site directed mutagenesis. Vpr wild type represents
the vpr gene derived from pNL43 proviral DNA and the change in residues at a particular position is marked for each mutant. LxxL motifs are underlined and
the helical domains are marked as I, II, and III HD above the amino acid sequence. (B) Immunoblot analysis of expression of Vpr mutant molecules: 293T cells
were transfected with 5 Ag of Vpr expression plasmids with a Flag tag using Lipofectamine. Forty-eight hours posttransfection cells were lysed and equal
amounts of protein containing total cell lysates were resolved in a 12% SDS-PAGE and immunoblotted with M2-Flag monoclonal antibody (1:250 dilution).
Vpr mutant constructs representing the residues are marked on top of each lane. The vector represents 293T cells transfected with the pcDNA3.1 vector
backbone.
D. Thotala et al. / Virology 328 (2004) 89–100 91
Transcriptional activation of GRE and HIV-1 LTR-driven
reporter gene by HIV-1 Vpr mutants
To precisely determine the role of specific leucine
residues involved in GRE transactivation, we cotransfected
Vpr mutant plasmids with the pGRE-luciferase reporter
construct in HeLa cells, which express GR endogenously.
Forty-eight-hour posttransfection, cells were lysed and cell
lysates were normalized for transfection efficiency by h-gallevel and assessed for luciferase activity. The fold difference
was calculated considering the activity observed with Vprwt
as 1 (Fig. 2A). Individual substitution mutations at L42 and
L67 significantly (P b 0.01) reduced the GRE-luciferase
activity when compared to Vprwt, whereas mutations at L23,
L39, and L68 exhibited similar activity and L20, L26, and
L64 slightly higher (N5-fold in case of L26 and N3 in case of
L64) levels of transactivation activity. Interestingly, the
mutation at L22 increased the activity by 8- to 11-fold over
wild type. Next, we addressed the question regarding the
requirement of GR for Vpr-mediated transactivation using
CV-1 cells, which are negative for GR expression. CV-1
cells were transfected with pVpr expression plasmid and
pGRE-directed luciferase reporter activity was measured.
CV-1 cells transfected with pVpr and pGRE plasmids alone
did not show any transcriptional activity (data not shown),
whereas cotransfection of the GR expression plasmid with
the reporter and Vpr expression constructs resulted in high
luciferase activity similar to the levels noted in HeLa cells
(Fig. 2B). The mutation at L22 consistently showed the
highest activity both in HeLa and CV-1 cells; however, the
presence of GR was necessary for Vpr-mediated GRE-
activation in CV-1 cells.
In an effort to assess the relevance of the transactivation
function of Vpr for virus replication, HeLa cells were
cotransfected with pVpr mutant molecules and the pHIV-1
LTR-luciferase reporter construct and were monitored for
luciferase activity (Fig. 2C). HIV-1 Vprwt plasmid increased
the transactivation activity by 4- to 6-fold when compared to
the vector backbone-transfected cells. Considering the Vprwt
activity as 1, mutations in L42 and L67 completely
abolished the activity, whereas L22A showed a 17-fold
higher activity than Vprwt. It is interesting to note that L22A
showed high transcriptional activity on both HIV-1 LTR and
GRE promoters in HeLa cells. To confirm Vpr-mediated
HIV-1 LTR transactivation is through the GRE domain in
LTR sequences, we transfected CV-1 cells with HIV-1 LTR
and Vpr mutants with or without the GR expression
plasmid, and assessed the reporter activity (Fig. 2D). Results
indicated that HIV-1 LTR-mediated transactivation by Vpr
plasmids is slightly lower (data not shown) in the absence of
GR, whereas cotransfection of pGRa plasmid increased the
transactivation efficiency similar to that observed in HeLa
cells (RLU). These results differ from the GRE-mediated
transactivation, where presence of GR is absolutely neces-
sary suggesting that HIV-1 Vpr might bind to other
promoter elements present in the HIV-1 LTR. However,
Fig. 2. Effect of HIV-1 Vpr leucine mutants on pGRE and HIV-1 LTR transactivation: HeLa cells were transfected with HIV-1 LTR luciferase or GRE-
luciferase reporter plasmid in the presence and absence of Vpr mutants using Lipofectamine. Forty-eight hours posttransfection cells were lysed and luciferase
activity was measured. Fold activation was calculated as one, representing Vpr wild type activity. Panel A, HeLa cells transfected with pGRE-luciferase and
pVpr mutant molecules; Panel B, CV-1 cells transfected with pGRE-luciferase, pGR and pVpr mutants; Panel C, HeLa cells transfected with pHIV-1 LTR-
luciferase and pVpr mutant plasmids; Panel D, CV-1 cells transfected with pHIV-1 LTR-luciferase, pGR and pVpr mutant plasmids. The fold activation SD was
derived from three independent experiments. * designates the P value of b0.01.
D. Thotala et al. / Virology 328 (2004) 89–10092
presence of GR enhances Vpr-mediated transactivation of
both autologous and heterologous promoters.
Role of the leucine residues in Vpr–GR interaction
GR has been shown to interact with Vprwt directly to
enhance the coactivator effect. To determine the specific
leucine residue involved in GR interaction, a GST-pull
down assay was performed using 35S-labeled in vitro
translated mutant Vpr proteins and recombinant GR that
was expressed in Escherichia coli BL-21 cells and purified
using GST beads. As a control, GST beads as well as in
vitro translated vif gene product were used (Fig. 3A).
Results indicated that GST-GR pulled all our leucine mutant
Vpr molecules, whereas the GST control did not show any
interaction. Additionally, the use of in vitro translated Vif
product was not pulled by GST-GR suggesting that the pull
down assay shows specificity. Next, to ensure that our in
vitro translated Vpr mutants expressed the appropriate size,
we loaded 1/10th of the in vitro translated product in SDS-
PAGE and it was then autoradiographed (Fig. 3A-input).
Results from these analyses suggest that the individual
leucine substitution of Vpr did not alter the Vpr–GR
interactions and that the observed interaction is very
specific.
Since the pull-down assay utilizes a prokaryotic
expressed protein, we wished to confirm this interaction in
eukaryotic cells. HeLa cells were transfected with phGRa
and Vpr mutant expression plasmids, and immunoprecipi-
tation was performed followed by Western blot analysis
(Fig. 3B). Transfected cell lysates were immunoprecipitated
with anti-GR antibody, resolved on SDS-PAGE, and
immunoblotted for Vpr using M2-flag specific antibody
(Fig. 3B). An interaction between Vpr mutants and GR was
observed as noted in the in vitro GST-GR pull down assay,
whereas vector-transfected cells did not show any specific
bands at 14 kDa size. Similarly, cell lysates were immuno-
precipitated with anti-Vpr antibody (M2-Flag) and immu-
noblotted with anti-GR antibody. Results indicate that cells
transfected with pVpr expression plasmids pulled down GR,
whereas vector-transfected cell lysate did not show any
detectable band (data not shown) suggesting that single
point Vpr leucine mutant did not interfere with GR
interaction and this interaction is specific.
Effect of subcellular distribution of Vpr mutants on GR
translocation
GR-mediated transactivation is generally correlated with
the translocation of GR from the cytoplasm to the nucleus
followed by binding to GRE sequences (DeFranco, 2002;
Htun et al., 1996). Upon expression, HIV-1 Vprwt localizes
at the nuclear membrane/nucleus of the cells (Kamata and
Aida, 2000). To examine whether the subcellular distribu-
tion of Vpr alters GR translocation and its ability to bind to
GRE sequences, we transfected HeLa cells with pVprwt and
assessed the localization pattern of Vpr and GR by indirect
immunofluorescence using Vpr- and GR-specific antibodies
(Fig. 4A). Vprwt and GR colocalized at the nuclear rim and
nucleus of the Vprwt-transfected cells (a–d), whereas control
Fig. 3. (A) In vitro interaction of Vpr and GR by GST pull-down assay:
Bacterially expressed GST or GST-GR were bound to glutathione-
sepharose beads and incubated with in vitro-translated Vpr mutants protein
labeled with [35S]methionine in 250 Al binding buffer (20 mM Tris–Hcl, pH
7.8, 100 mM NaCl, 10% Glycerol, 1 mM dithiothreitol, 1 mM EDTA, 1
mM phenylmethanesulfonyl fluoride, 1 mM leupeptin, 1 mM pepstatin).
Beads were washed extensively with binding buffer and bound proteins
were eluted by boiling them in reducing sample buffer, followed by SDS-
PAGE and autoradiography. Pull: GST-GR represents in vitro translated
Vpr mutants pulled using GST-GR beads; Pull: GST represents in vitro
translated Vpr mutants pulled using GST beads; and INPUT, represents in
vitro translated Vpr and Vpr products used in pull-down assays. (B) In vivo
interactions between GR and Vpr mutants: HeLa cells were transfected with
GR and Flag-tagged Vpr mutant expression plasmids. Immunoprecipitates
containing GR and Vpr were prepared with anti-GR antibody and applied to
a 12% SDS-PAGE gel. Immunoprecipitated complex was transferred to a
membrane and immunoblotted with anti-Flag M2 antibody followed by
chemiluminescence’s detection. All the mutants are designated on top of the
lane with their respective changes. Vector lane represents lysates from
pcDNA 3.1 vector-transfected cells.
D. Thotala et al. / Virology 328 (2004) 89–100 93
cells exhibited a weak cytoplasmic GR distribution and
when exposed to 10�6 AM of dexamethasone, GR trans-
located into the nucleus (e–h). Since dexamethasone is a
high affinity ligand for GR and participates in the trans-
location of GR into the nucleus, we compared the local-
ization pattern of Vpr and GR in the absence of additional
hormone exposure using charcoal-stripped serum containing
medium (a–d). It is interesting to note that HIV-1 Vprwt and
GR colocalized at the nuclear rim as well as in the nucleus.
Structure–function analysis of Vpr has shown that in
comparison to wild type Vpr, individual substitution
mutations in Vpr (especially in the helices) altered the
subcellular distribution (Kamata and Aida, 2000; Mahalin-
gam et al., 1997). First to examine the sub-cellular local-
ization of Vprwt and leucine mutants in the absence of
endogenous GR, we transfected CV-1 cells with Vpr
expression plasmids and performed indirect immunofluor-
escence using M2-Flag antibody (Table 1). Results indicate
that Vprwt, L42, and L67 exhibit nuclear membrane
distribution, whereas, L22, L23, L26, and L68 exhibited a
cytoplasmic pattern. Mutants L20, L39, and L64 showed
both cytoplasmic and perinuclear expression. Next, to
evaluate how the subcellular distribution of Vpr mutants
impacted GR localization, CV-1 cells were cotransfected
with pVpr leucine mutants and pGR and assessed for the
subcellular distribution of Vpr and GR (Suppl. Figs. 1A and
B; Table 1). Vpr mutants L23, L26, and L68 predominantly
exhibited a cytoplasmic pattern, whereas Vprwt, L20, L64,
and L67 localized in the nuclear membrane and cytoplasm
upon expression. Vpr L22A displayed a distinct nuclear
distribution in the presence of GR expression (Fig. 4B). Vpr
mutant L22A and GR colocalized into the nucleus in a
condensed form. To further establish the pattern of L22A
and GR, we performed confocal microscopy using cells
transfected with L22A and GR plasmids (Fig. 4C). Our
results indicate a distinct condensed pattern of L22A and
GR suggesting that L22A and the GR coactivator complex
might have a different localization pattern in the nucleus.
Additionally, it is interesting to note that none of this
complex localizes within the nucleolus compartment.
Together, these data suggest that the increase in GR-
VprL22A-mediated transactivation might be due partly to
the ability of GR and L22A to colocalize into the nucleus.
DNA binding affinity of GR–Vpr complex
Vpr has been shown to exert its transactivation effect
with GR through GRE sites, gel shift assay was performed
using GRE consensus sequences to assess the DNA binding
of Vpr–GR complex (Tseng et al., 2001). Cell lysates
derived from HeLa cells transfected with pVprwt resulted in
a specific complex with the labeled GRE sequences (Fig.
5A). This DNA–protein complex induced by Vpr was
similar to the complex induced by dexamethasone (1 AM),
whereas cell lysate from vector-transfected cells did not
show any distinct band at the corresponding position. Next
to test whether presence of both Vpr and GR is required for
GRE-binding, we transfected CV-1 cells with pVpr alone or
pVpr and pGR and extracted the lysates and performed
electrophoretic mobility shift assay (EMSA). Results
indicate that lysate from pVpr-transfected cells did not
show any binding, whereas Vpr–GR complex containing
lysates showed specific GRE binding (data not shown),
suggesting that Vpr–GR complex is required for GRE
binding activity in the absence of hormonal stimulation.
HIV-1 LTR and GRE reporter assays indicated that Vpr
leucine mutants exhibited differential transactivation. For
example, mutant L22A exhibited higher transactivation
activity and a distinct nuclear localization pattern. These
results suggest that GR-VprL22A might have significant
DNA-binding ability. To measure the ability of GR-L22A to
bind to the GRE domain in comparison to Vpr wild type, we
employed a competition gel-shift mobility assay using p32-
labeled double-stranded GRE oligonucleotides (Fig. 5B).
Different fold excess of cold GRE oligonucleotide (ranging
from 2.5- to 4-fold) was used. Competition assays revealed
Fig. 4. (A) Expression and cellular colocalization of GR and pVpr mutant molecules: HeLa cells were transfected with Flag tagged pVprwt and pGR expression
plasmids and grown in a glass chamber slide. Forty-eight hours posttransfection cells were fixed and incubated with anti-GR and anti-Flag antibodies. Cells
were washed five times with PBS and incubated with secondary antibody conjugated to TRITC or FITC for 90 min at room temperature. Cells were washed
five times in PBS before mounting with Vectashield containing DAPI. Fluorescence was visualized with a Nikon microscope equipped with a digital camera
and images were captured as Photoshop files. Vprwt (a–d), represents cells cotransfected with pVprwt and phGRa expression plasmids. Panel Dex (e–h),
represents HeLa cells transfected with pcDNA3.1 vector control and phGRa expression plasmids and treated with 10 nM of dexamethosone and stained with
anti-GR antibody. (B and C) Immunofluorescence and Confocal visualization of VprL22A and GR colocalization in HeLa cells: HeLa cells were transfected
with Flag-tagged pVprL22A and phGRa expression plasmid and stained with anti-Flag and anti-GR antibody as before. B represents the immunofluorescence
studies and Panel C represents the confocal image analysis.
D. Thotala et al. / Virology 328 (2004) 89–10094
that Vpr wild type could be competed off with 2-fold excess
of cold GRE oligonucleotide (Lane 4), whereas a 4-fold
higher concentration was required to compete the GR-L22A
mutant (Lane 10), suggesting that nuclear extract containing
GR-VprL22A might have higher DNA-binding affinity to
GRE. This could partly explain the increase in Vpr–GR-
mediated transactivation in the case of VprL22A mutant.
Correlation of Vpr-mediated transactivation to virus
replication
To functionally correlate the transactivation effect of Vpr
in the context of virus replication, we performed a single
round infection assay using virus particles containing Vprwt
and Vpr mutants molecules by complementing pNL43 R-E-
plasmid with Vpr mutants and HxB2-Env plasmids. We
chose the single round infection system to precisely
quantitate the effect of virion-associated Vpr on HIV-1
replication. The ability of Vpr mutants to incorporate into
the virus particles was determined as described (Singh et
al., 2001) and presented in Table 1. Vpr mutant L39 and
L42 was not included in the single round infection due to
their deficiency in virion incorporation. The quantitation of
virus particles released into the media of the infected cells
by p24 antigen assay was used for evaluation. Virus
particles containing Vpr L22A showed maximum virus
Fig. 5. (A) Electro Mobility Shift Assays: EMSAwas performed using labeled GRE oligonucleotide with HeLa cell nuclear extract. Lane oligo, labeled GRE
oligonucleotide in the absence of nuclear extract; Lane vector, GRE labeled oligonucleotide in the presence of the nuclear extract of HeLa cell transfected with
the vector plasmid; Lane Vpr-wt, GRE-labeled oligonucleotide in the presence of the nuclear extract of HeLa cells transfected with pVpr wt and phGRa
expression plasmids; Lane Dex, HeLa cell lysates with over expression of phGRa and stimulated with 10 nM dexamethasone. (B) Competition assays:
competition of GRE binding to HeLa cell nuclear extracts by molar excess (1- to 4-fold) of the cold GRE (lanes 3–10). Lane 1, labeled GRE in the absence of
nuclear extract; lane 2, labeled oligonucleotide in the presence of nuclear extract of cells alone; Lanes 3–6, nuclear extracts from cells transfected with pVprwt
in the presence of labeled GRE and 1-, 2-, 3-, and 4-fold excess of cold GRE oligonucleotide, respectively; and Lanes 7–10, nuclear extract from cells
transfected with pVprL22A in the presence of labeled GRE and 1-, 2-, 3-, and 4-fold excess of cold GRE oligonucleotide, respectively. Arrow indicates the
major complex formed.
D. Thotala et al. / Virology 328 (2004) 89–100 95
replication (5 ng/ml) followed by Vpr wild type (4 ng/ml)
on day 5, whereas mutants L23A and L68A resulted in
reduced levels (b2.5 ng/ml) of replication (Fig. 6). Similar
results were observed in multiple experiments using
PBMCs isolated from different healthy donors (data not
shown). Similarly, using the single round infection assay
with Vpr mutant incorporated virion molecules, we infected
CEM-GFP cells containing a plasmid encoding the green
fluorescent protein (humanized S65T GFP) driven by the
HIV-1 long terminal repeat and measured the virus
Table 1
Functional characteristics of HIV-1 Vpr mutants
Vpr mutants Expression Cell
cycle arrest
Virion
incorporation
Vpr wt ++ +++ yes
L20A ++ ++ yes
L22A ++ ++ yes
L23A + � yes
L26A ++ � yes
L39G ++ +++ no
L42G ++ � no
L64A ++ ++ yes
L67A ++ ++ yes
L68A ++ +/� yes
N.D., not determined.
infection by measuring the GFP in flow cytometry analysis
(data not shown). Vpr L22A mutant incorporated virus
showed an increase in number of cell number as well as
MFI compared to Vprwt incorporated virus particles,
suggesting that L22A increase virus replication during
early infection. These results indicate that an increase in
viral replication kinetics by Vpr is directly correlated with
the ability to transactivate HIV-1 LTR and GRE. However,
it is important to note that this affect is only due to the
virion-associated Vpr mutant molecules that are presented
Subcellular distribution of
Vpr in the absence of GR
Subcellular distribution of
Vpr in the presence of GR
Nuclear membrane Nuclear membrane
Cytoplasm + nuclear
membrane
Cytoplasm + nuclear
membrane
Cytoplasm Nucleus
Cytoplasm Cytoplasm
Cytoplasm Cytoplasm
Cytoplasm + nucleus N.D.
Nuclear membrane N.D.
Cytoplasm + nuclear
membrane
Cytoplasm + nuclear
membrane
Nuclear membrane Nuclear membrane +
cytoplasm
Cytoplasm Cytoplasm
Fig. 6. Effect of virion-associated Vpr on virus replication during early
infection: Ficoll-Hypaque fractionated normal human PBMCs were
stimulated with PHA (5 Ag/ml) and infected with Vpr and Env trans-
complemented pNL43 R�E�. Cell free supernatants were collected at 24 h
intervals postinfection and assayed for the release of p24 antigen in the
medium. These experiments were repeated using PBMCs from different
seronegative donors and similar results were observed.
D. Thotala et al. / Virology 328 (2004) 89–10096
only in the virus particles. These results suggest that
presence of L22A Vpr mutant molecule in the virus
particles might increase virus replication during early
infection. Though we have observed an 8-fold increase in
transactivation assays, we did not see similar fold increase
in the single round virus replication assay. This could be
due to the presence of Vpr as virion-associated molecule s
and is absent in the proviral genome.
Discussion
AIDS pathogenesis is considered to be the result of the
interplay between the virus and the target cells of the host.
The cytopathic effects mediated by both the viral and
cellular genes have been suggested to be responsible for the
pathogenesis. Vpr has been implicated as one of the viral
genes contributing to AIDS pathogenesis along with HIV-1
env, tat, and nef. The features that qualify Vpr in this regard
are the following: (i) Vpr is incorporated into the virus
particles despite being a non-structural protein and the
amount of Vpr present in the virus particles is reported to be
in the range of few to ~2500 copies (Singh et al., 2001;
Tungaturthi et al., 2003), (ii) the presence of Vpr in cells
following virus infection, and (iii) the presence of free Vpr
(cell-free and virus-free) in the body fluids (serum and
CSF). Vpr has been reported to regulate multiple cellular
and viral functions. Vpr regulates both autologous and
heterologous promoters to enhance the transcription of viral
and cellular promoters. Additionally, Vpr-mediated cellular
effects have been linked to an increase in virus replication
and disease progression (Goh et al., 1998; Jowett et al.,
1995). Furthermore, the highly conserved nature of Vpr in
vivo suggests that Vpr plays an important role during early
infection and pathogenesis. Structure–function analyses of
Vpr have indicated that nuclear localization, cell cycle
arrest, and virion incorporation are mediated by distinct
domains of Vpr (Mahalingam et al., 1997). Recently, it has
been established that Vpr functions as a coactivator of
nuclear receptors through the signature motif LxxLL and
this function is distinct from its cell cycle regulation (Kino
et al., 1999; Sherman et al., 2000). Although recent mapping
studies suggest that the LxxLL domain in HIII of Vpr is the
interaction domain of nuclear receptors, there are also
related sequences in HI and HII. The role of these specific
amino acids involved in Vpr–GR interaction residues is
unclear. Here, we seek to identify the specific leucine
residues within the LxxLL domains involved in this
function by introducing individual leucine mutations. To
keep the structure and charge compatible we mutated the
leucine (L) residues into alanine (A) with the exception of
residues at L39 and L42, which were mutated to glycine
(G).
Our analysis of Vpr-mediated transactivation of GRE-
luciferase and HIV-1 LTR-luciferase revealed that leucine
residues are important for transactivation mediated by the
Vpr–GR complex. Disruption of leucine at these sites by
substitution with alanine/glycine significantly alters both
HIV-1 LTR and GRE-mediated transactivation in spite of
GR-Vpr mutants interaction. It is likely that the substitution
may interfere at the structure level. Such an effect could
result in disruption of specific residues that may participate
in Vpr’s structure, confirmation, and folding. Leucine
residues in the helical structure have been shown to
participate in protein–protein, protein–nucleic acid, and
protein–lipid interactions (Mahalingam et al., 1995; Tacke
et al., 1993). However, our results on GR and Vpr mutant
interactions show that GR physically interacts with all of the
Vpr mutants, suggesting that a single amino acid change in
these domains does not alter the binding. Additionally, it is
also possible that changes caused by a single leucine residue
could also compensated by other compensatory residues.
Previous studies by Kino et al. (2002a) and Sherman et al.
(2000) suggest that leucine mutations in these motifs abolish
the GR interaction. However, the Vpr mutant they used has
multiple leucine mutations within a single construct, thus
the discrepancy could be due the variation in these
constructs compared to ours. This could be due to the
changes in multiple leucine residues that might affect
structure and function. It is also possible that the adjacent
compensatory residues can rescue the loss of a single
leucine residue on Vpr–GR interaction. Though the Vpr–
GR interaction is not altered, changes in a leucine resides
such as L22 could alter other biophysical properties that
could result in altered subcellular distribution and DNA-
binding activity as observed.
Transcription of HIV-1 is mainly dependent on DNA-
binding proteins, transcription factors and viral transactiva-
tors. It is likely that Vpr transactivates HIV-1 LTR and/or
heterologous promoters through its interaction with GR and
therefore increases the ability of GR to bind to the
D. Thotala et al. / Virology 328 (2004) 89–100 97
transcriptions factors. In this regard, it has been shown that
Vpr also has DNA-binding ability that is linked to the C-
terminal basic residues (Hogan et al., 2003; Zhang et al.,
1998). It is not known whether Vpr binds directly to the
GRE sequence. Our results indicate that Vpr-mediated
DNA-binding activity requires GR, suggesting that Vpr-
interacting proteins might mediate similar functions. It is
interesting to note that a mutation in the L22 residue results
in significantly higher transactivation (6- to 10-fold more)
suggesting that L22A may not alter the Vpr–GR interaction
but instead enhance the Vpr–GR–GRE binding affinity. The
later is supported by the subcellular localization studies
where L22A and GR colocalize in the nucleus. Our study
focuses on the effect of Vpr–GR interaction in the absence
of dexamethasone. Though dexamethasone increases Vpr-
mediated GRE transactivation, hormones present in culture
media are sufficient for Vpr–GR interaction. Sherman et al.
(2001) observed similar results, whereas Kino et al. (2002a)
suggested that dexamethasone is necessary for Vpr–GR
binding. However, our results are in agreement with
Sherman et al. (2001) indicating that the presence or
absence of dexamethasone does not alter the ability of GR
to bind with Vpr mutant molecules.
Vpr is a multi-phenotypic protein, with distinct domains
to control-specific functions. Vpr-mediated cell cycle arrest
has been linked to virus replication (Goh et al., 1998;
Gummuluru and Emerman, 1999); however, recent studies
suggest that the transactivation by Vpr is not directly
correlated with cell cycle function (Sherman et al. 2001).
Transactivation of autologous/heterologous promoters and
cell-cycle arrest functions are regulated by distinct domains
of Vpr and are independent of each other (Sawaya et al.,
2000; Sherman et al., 2000). Our studies on L22A and
L39G (single point mutation) mutants positive for cell cycle
arrest function with opposing transactivation activity further
confirms that the transactivation activity of HIV-1 Vpr
through GR is independent of cell cycle arrest function in
the context of leucine residues. Additional studies using
mutants from other regions are needed to further confirm
this observation.
It is interesting to note that L22A has significant trans-
activation effects in the absence of other external stimuli such
as glucocorticoids suggesting that a mutation at L22 might
alter some other properties of Vpr such as altered localization
and/or DNA-binding ability. Confocal microscopic analysis
and further competition assays using GRE-binding by EMSA
further confirms that the increased transactivation of L22 is
due its ability to bind the GRE DNAwith GR. It is interesting
to note that many Vpr-GR induced functions can be mediated
in the absence of dexamethasone, further confirming our
previous studies of Vpr mimicking GC activity (Ayyavoo et
al., 1997). These studies support that Vpr functions can be
suppressed by a new class of antivirals targeting these
pathways. Further studies are needed to identify new targets
that could block virus replication without interfering with the
host cell functions.
Materials and methods
Cells
HeLa, CV-1, and 293T cells were grown in DMEM
supplemented with 10% FCS, 1% glutamine and 1%
penicillin-streptomycin. HeLa and HeLa-T4 cells were
obtained from NIH, AIDS reagent program. CV-1 cells
were obtained from Dr. Frank Jenkins at the University of
Pittsburgh and 239T cells were a generous gift from Dr.
Michelle Calos, Stanford University CA. CEM-GFP cells
were obtained through AIDS Research and Reference
Reagent Program, DAIDS, HIH from Dr. Jacaques Corbeil
and maintained as described (Gervaix et al., 1997). Blood
from HIV-1-negative healthy donors was used to isolate
peripheral blood mononuclear cells (PBMCs) by Ficoll-
Hypaque (Pharmacia, USA) gradient centrifugation. Puri-
fied PBMCs were resuspended in RPMI 1640 supplemented
with 10% heat-inactivated FCS, stimulated with phytohe-
moagglutinin (PHA) (5 Ag/ml) for 2 days, and cultured in
IL-2 (5 U/ml) containing medium.
Plasmids
Mutants of HIV-1 vpr were generated using overlap PCR
and/or by Quick-change mutagenesis (Singh et al., 2001)
and were cloned under the control of the CMV promoter in
pCDNA3.1 (Invitrogen, CA) with a flag epitope. All the
Vpr mutant constructs were sequenced to verify the integrity
of the mutations. Proviral constructs pNL43 R+E�, pNL43
R�E�, pIIIB Env and HIV-1 LTR-luciferase reporter
constructs were obtained from NIH ARRRP, contributed
by Dr. Landau and Drs. Jeeninga and Berkhort, respectively.
pGRE (5X)-luciferase reporter plasmid was constructed by
PCR amplifying the 5XGRE consensus sequence from
pGRE5/EBV vector (USB, CA) using specific forward
(5VATACGCGGATCCTCTAGA AGATCCGCT3V) and
reverse (5VATCATACTCGAGGGCCCTCGCAGACA3V)primers. Amplified PCR product (290 bp) was cloned in
the upstream of a firefly luciferase gene reporter construct.
The human glucocorticoid receptor gene alpha (hGRa) was
PCR amplified using specific forward (5VATCGGGGATCCGATGGACTCCAAAGAATCA3 V) and reverse (5 VGTGGTCCTCGAGCTTTTGATGAAACAGAAG3V) pri-
mers and cloned into pcDNA 3.1 (Invitrogen) and pET41b
(+) (Novagen, CA) vectors for further eukaryotic and
prokaryotic expression studies, respectively. The integrity
of the plasmid DNA was tested by DNA sequence analysis
(ABI 7700, CA).
In vitro pull-down assay
HIV-1 GST-Vprwt and GST-hGRa were expressed
following 1 mM IPTG induction in BL21 cells. Protein
was purified using the BugBuster system (Novagen)
according to the manufacturer’s instruction. The integrity
D. Thotala et al. / Virology 328 (2004) 89–10098
and purity of the proteins were analyzed by SDS-poly-
acrylamide gel electrophoresis followed by Coomassie blue
staining. For in vitro pull-down assays, GST beads
containing GR or Vpr were used. Vpr mutants were in
vitro translated using T7 in vitro transcription/translation
system (Promega, WI) as per the manufacturer’s instruction.
Equal amounts of in vitro translated products were added to
50 Al of GST-GR beads in 500 Al of GST bind/wash buffer
(Novagen) and rocked at 4 8C for 90 min. GST beads were
then washed with wash buffer three times and resuspended
in protein sample buffer. Proteins bound to GST beads were
resolved on a 12% SDS-PAGE and autoradiographed.
Transfection and luciferase assay
HeLa and CV-1 cells were transfected with HIV-1 LTR
luciferase or GRE5X-luciferase reporter plasmid (1 Ag) inthe presence and absence of Vpr mutants (0.5 Ag), pGR and
pCMVh-Gal using lipofectamine (Invitrogen). Forty-eight
hours posttransfection, cells were lysed in 500 Al of 1�reporter lysis buffer and luciferase activity was measured
following the manufacturer’s protocol (Promega). Trans-
fection efficiency was normalized by transfecting with
CMV h-gal plasmid and analyzing the h-gal activity
(Promega).
In vivo interaction studies
HeLa cells were transfected with Vpr mutant plasmids
using lipofectamine. Forty-eight hours posttransfection, the
cells were washed with PBS and lysed in RIPA buffer (50
mM Tris–HCl, [pH 7.6], 150 mM NaCl, 0.5% Triton X-100,
0.5% deoxycholic acid, 0.1% sodium dodecyl sulfate (SDS),
1 mM phenylmethylsulfonyl fluoride (PMSF) and PI
cocktail). Cell lysate was centrifuged to remove the cell
debris. Protein estimation of the cell lysate was carried out
using Bradford reagent (Bio-Rad, Richmond, CA). Equiv-
alent amount (150 Ag) of cellular protein was subjected to
immunoprecipitation using anti-GR antibodies. Immunopre-
cipitated complex was analyzed on SDS-PAGE and
immunoblotted for Vpr using M2 anti-FLAG antibody
(1:200) from Sigma, MO.
Immunofluorescence and confocal microscopy
HeLa cells were seeded in four-well chamber slides and
transfected with Vpr or GR expression plasmid DNA using
Lipofectamine. Forty-eight hours posttransfection, cells
were washed with PBS and fixed in methanol at room
temperature for 10 min. After washing three times with
PBS, cells were incubated with anti-GR antibody (Affinity
BioReagents, CO) at 37 8C in a humidified incubator for 90
min. Primary antibody was washed three times with PBS
and then incubated with anti-Flag M2 monoclonal antibody-
FITC (Sigma) to detect Vpr, and anti rabbit-TRITC to detect
GR. Following several washes with PBS, cells were
mounted with Vectashield containing DAPI (Vector Labo-
ratories, CA). Immunofluorescence was detected using
Nikon inverted fluorescence microscope with appropriate
filters. Confocal microscopy was performed using a Leica
TCS NT confocal tri-laser scanning inverted microscope
(Wetzlar, Germany).
Electrophoretic mobility shift assay
Oligonucleotide containing the GRE consensus sequence
(5VGACCCTAGAGGATCTGTACAGGATGTTCTAGAT
3V) was either used as a probe or competitor. One picomolar
of GRE oligonucleotide was end-labeled with 2 Al of p32ATP using T4-polynucleotide kinase (Promega) in a 50-Alreaction. The labeled oligonucleotide was purified to
remove unincorporated labels using a Qiaquick nucleotide
removal kit (Qiagen, Valencia, CA). HeLa cells were
transfected with Vpr wild type or Vpr mutants as described
above. Forty-eight hours posttransfection, cells were washed
with PBS and nuclear extracts were prepared using the NE-
PER nuclear and cytoplasmic extraction kit following the
manufacturer’s instructions (Pierce, IL). DNA binding
reactions were carried out with approximately 1 pM of
p32 labeled GR consensus double-strand oligonucleotide
(50,000 cpm) and 5.0 Ag nuclear extract (Tseng et al., 2001).Briefly, nuclear extract was pre-incubated in a binding
buffer [50 mM Tris–HCl (pH 7.6), 0.5 mM EDTA (pH 8.0),
0.5 mM DTT, 50% Glycerol, 2 Ag of poly: dIdC] in a final
volume of 30 Al at room temperature for 10 min. The
labeled oligo was then added and incubated at room
temperature for 30 min. For competition experiments, a
cold probe was added to the nuclear extract 20 min before
the addition of labeled probe. The samples were analyzed on
a 5% non-denaturing polyacrylamide gel (5% polyacryla-
mide gel, 50 mM Tris–Glycine) and autoradiographed.
Single round infection assay
To assess the effect of Vpr as a virion-associated
molecule, we have used pseudotype viruses containing
Vprwt or Vpr mutant molecules. A single round infection
assay was used to study the effect of different Vpr mutants
on HIV-1 replication by transfecting 293T cells with
proviral DNA pNL43 R�E� with pEnv and Vpr expression
plasmid produced pseudotype viruses. Seventy-two hours
posttransfection culture supernatant was collected and
assayed for virus production by quantitating the p24 by
ELISA. PBMCs (5 � 106) were infected with 10 ng of p24
antigen-equivalent viruses for 4 h, washed thoroughly with
RPMI medium, and resuspended in appropriate growth
medium. Supernatant was collected at 24-h intervals to
measure virus replication by analyzing p24 by ELISA
(AIDS Reagent program, NIH). Additionally, to measure
the effect of virion-associated Vpr molecules during early
infection, CEM-GFP cells were infected with the leucine
mutant complemented pNL43 R-E-/VSV-G-Env viruses at
D. Thotala et al. / Virology 328 (2004) 89–100 99
0.01 multiplicity of infection (MOI) for 6 h at 37 8C as
described (Gervaix et al., 1997). The cells then were washed
three times with PBS, resuspended in culture medium, and
incubated at 378C in 5% CO2. Aliquots of cells were taken
at days 2 and 4 and fixed in 2% paraformaldehyde. The
cells were analyzed by flow cytometry followed by cell
quest software to measure the MFI and percent positive
cells.
Acknowledgments
HIV-1 LTR-luciferase construct, pNL43 R+E� and
pNL43 R�E�, pHXB2-Env, pHIV-1 LTR-luciferase plas-
mids and CEM-GFP were obtained from the AIDS Research
and reference reagent Program, Division of AIDS, NIAID.
We thank Dr. Pavlakis for his generous gift of RU486 for
our initial experiments. We thank Dr. Frank Jenkins at the
University of Pittsburgh for CV-1 cells and Dr. Michelle
Calos, at Stanford University, for 293T cells. We thank Dr.
Indranil Mukhopadhyay, University of Pittsburgh for his
help with the statistical analysis. This work was supported
by AI50463 from NIAID, NIH to VA.
Appendix A. Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.virol.2004.07.
013.
References
Agostini, I., Navarro, J.M., Rey, F., Bouhamdan, M., Spire, B., Vigne, R.,
Sire, J., 1996. The human immunodeficiency virus type 1 Vpr
transactivator: cooperation with promoter-bound activator domains
and binding to TFIIB. J. Mol. Biol. 261, 599–606.
Ayyavoo, V., Mahboubi, A., Mahalingam, S., Ramalingam, R.,
Kudchodkar, S., Williams, W.V., Green, D.R., Weiner, D.B., 1997.
HIV-1 Vpr suppresses immune activation and apoptosis through
regulation of nuclear factor kB. Nat. Med. 3, 1117–1123.
Balliet, J.W., Kolson, D.L., Eiger, G., Kim, F.M., McGann, K.A.,
Srinivasan, A., Collman, R., 1994. Distinct effects in primary macro-
phages and lymphocytes of the human immunodeficiency virus type 1
accessory genes vpr, vpu, and nef: mutational analysis of a primary
HIV-1 isolate. Virology 200, 623–631.
Cadepond, F., Gasc, J.M., Delahaye, F., Jibard, N., Schweizer-Groyer, G.,
Segard-Maurel, I., Evans, R., Baulieu, E.E., 1992. Hormonal regulation
of the nuclear localization signals of the human glucocorticosteroid
receptor. Exp. Cell Res. 201, 99–108.
Cohen, E.A., Dehni, G., Sodroski, J.G., Haseltine, W.A., 1990. Human
immunodeficiency virus vpr product is a virion-associated regulatory
protein. J. Virol. 64, 3097–3099.
Connor, R.I., Chen, B.K., Choe, S., Landau, N.R., 1995. Vpr is required for
efficient replication of human immunodeficiency virus type-1 in
mononuclear phagocytes. Virology 206, 935–944.
DeFranco, D.B., 2002. Navigating steroid homone receptors through the
nuclear compartment. Mol. Endocrinol. 16, 1449–1455.
Di Marzio, P., Choe, S., Ebright, M., Knoblauch, R., Landau, N.R., 1995.
Mutational analysis of cell cycle arrest, nuclear localization, and virion
packaging of human immunodeficiency virus type 1 Vpr. J. Virol. 69,
7909–7916.
Felzien, L.K., Woffendin, C., Hottiger, M.O., Subbramanian, R.A., Cohen,
E.A., Nabel, G.J., 1998. HIV transcriptional activation by the accessory
protein, VPR is mediated by the p300 co-activator. Proc. Natl. Acad.
Sci. U.S.A. 95, 5281–5286.
Feng, W., Ribeiro, R.C., Wagner, R.L., Nguyen, H., Apriletti, J.W.,
Fletterick, R.J., Baxter, J.D., Kushner, P.J., West, B.L., 1998. Hormone
dependent coactivator binding to a hydrophobic cleft on nuclear
receptors. Science 280, 1747–1749.
Gervaix, A., West, D., Leoni, L.M., Richman, D.D., Wong-Staal, F.,
Corbeil, J.A., 1997. A new reporter cell line to monitor HIV infection
and drug susceptibility in vitro. Proc. Natl. Acad. Sci. U.S.A. 94,
4653–4658.
Goh, W.C., Rogel, M.E., Kinsey, C.M., Michael, S.F., Fultz, P.N., Nowak,
M.A., Hahn, B.H., Emerman, M., 1998. HIV-1 Vpr increases viral
expression by manipulation of the cell cycle: a mechanism for selection
of Vpr in vivo. Nat. Med. 4, 65–71.
Gummuluru, S., Emerman, M., 1999. Cell cycle- and Vpr-mediated
regulation of human immunodeficiency virus type 1 expression in
primary and transformed T-cell lines. J. Virol. 73, 5422–5430.
Heery, D.M., Kalkhoven, E., Hoare, S., Parker, M.G., 1997. A signature
motif in transcriptional co-activators mediates binding to nuclear
receptors. Nature 387, 733–736.
Heinzinger, N.K., Bukinsky, M.I., Haggerty, S.A., Kewalramani, V., Lee,
M.A., Gendelman, H.E., Ratner, L., Stevenson, M., Emerman, M.,
1994. The Vpr protein of human immunodeficiency virus type 1
influences nuclear localization of viral nucleic acids in non-dividing
host cells. Proc. Natl. Acad. Sci. U.S.A. 91, 7311–7315.
Hogan, T.H., Nonnemacher, M.R., Krebs, F.C., Henderson, A., Wigdahl,
B., 2003. HIV-1 Vpr binding to HIV-1 LTR C/EBP cis-acting elements
and adjacent regions is sequence-specific. Biomed. Pharmacother. 57,
41–48.
Hrimech, M., Yao, X.J., Bachand, F., Rougeau, N., Cohen, E.A., 1999.
Human immunodeficiency virus type 1 (HIV-1) Vpr functions as
an immediate early protein during HIV-1 infection. J. Virol. 73,
4101–4109.
Htun, H., Barsony, J., Reni, I., Gould, D.L., Hager, G.L., 1996.
Visualization of glucocorticoid receptor translocation and intranuclear
organization in living cells with a green fluorescent protein chimers.
Proc. Natl. Acad. Sci. U.S.A. 93, 4845–4850.
Jowett, J.B., Planelles, V., Poon, B., Shah, N.P., Chen, M.L., Chen, I.S.,
1995. The human immunodeficiency virus type 1 Vpr gene arrests
infected T cells in the G2+M phase of the cell cycle. J. Virol. 69,
6304–6313.
Kamata, M., Aida, Y., 2000. Two putative a-helical domains of human
immunodeficiency virus type 1 Vpr mediate nuclear localization by at
least two mechanisms. J. Virol. 74, 7179–7186.
Kino, T., Gragerov, A., Kopp, J.B., Stauber, H., Pavalakis, G.N., Chrousos,
G.P., 1999. The HIV-1 virion-associated protein vpr is a coactivator of
the human glucocorticoid receptor. J. Exp. Med. 189, 51–62.
Kino, T., Gragerov, A., Slobodskaya, O., Tsopananomichalou, M.,
Chrouusos, G.P., Paylakis, G.N., 2002a. Human immunodeficiency
virus type 1 (HIV-1) accessory protein Vpr induces transcription of the
HIV-1 and glucocorticoid-responsive promoters by binding directly to
p300/CBP coactivators. J. Virol. 76, 9724–9734.
Kino, T., Tsukamoto, M., Chrousos, G.P., 2002b. Transcription factor
TFIIH components enhance the GR coactivator activity but not the cell
cycle-arresting activity of the human immunodeficiency virus type-1
protein Vpr. Biochem. Biophys. Res. Commun. 298, 17–23.
Levy, D.N., Fernandes, L.S., Williams, W.V., Weiner, D.B., 1993. Induction
of cell differentiation by human immunodeficiency virus 1 vpr. Cell 72,
541–550.
Levy, D.N., Rafaeli, Y., Weiner, D.B., 1995. Extracellular vpr protein
increases cellular permissiveness of human immunodeficiency virus
replication and reactivates virus from latency. J. Virol. 69, 1243–1252.
D. Thotala et al. / Virology 328 (2004) 89–100100
Li, D.P., Periyasamy, S., Jones, T.J., Sanchez, R., 2002. Heat and chemical
shock potential of glucocorticoid receptor transactivation requires heat
shock factor (HSF) activity. J. Biol. Chem. 275, 26058–26065.
Mahalingam, S., Khan, S.A., Jabbar, M.A., Monken, C.E., Collman, R.,
Srinivasan, A., 1995. Mutagenesis of the putative alpha helical domain
of the Vpr protein of human immunodeficiency virus type 1: effect on
stability and virion incorporation. Proc. Natl. Acad. Sci. U.S.A. 92,
3794–3798.
Mahalingam, S., Ayyavoo, V., Patel, M., Kieber-Emmons, T., Weiner, D.B.,
1997. Nuclear import, virion incorporation, and cell cycle arrest/
differentiation are mediated by distinct functional domains of human
immunodeficiency virus type 1 Vpr. J. Virol. 71, 6339–6347.
Morellet, N., Bouaziz, S., Petitjean, P., Roques, B.P., 2003. NMR structure
of the HIV-1 regulatory protein VPR. J. Mol. Biol. 327, 215–227.
Nakamura, T., Suzuki, H., Okamoto, T., Kotani, S., Atsuji, Y., Tanaka, T.,
Ito, Y., 2002. Recombinant Vpr (rVpr) causes augmentation of HIV-1
p24 Ag level in U1 cells through its ability to induce the secretion of
TNF. Virus Res. 90, 263–268.
Northrop, J.P., Crabtree, G.R., Mattila, P.S., 1992. Negative regulation of
interleukin 2 transcription by the glucocorticoid receptor. J. Exp. Med.
175 (5), 1235–1245.
Paliogianni, F., Boumpas, D.T., 1995. Glucocorticoids regulate calcineurin-
dependent trans-activating pathways for interleukin-2 gene transcription
in human T lymphocytes. Transplantation 59 (9), 1333–1339.
Sawaya, B.E., Khalili, K., Gordon, J., Taube, R., Amini, S., 2000.
Cooperative interaction between HIV-1 regulatory proteins Tat and
Vpr modulates transcription of the viral genome. J. Biol. Chem. 275,
35209–35214.
Schmidt, J., Fleissner, S., Heimann-Weitschat, I., Lindstaedt, R., Pomberg,
B., Werner, U., Szelenyi, I., 1994. Effect of corticosteroids, cyclosporin
A and methotrexate on cytokine release from monocytes and T-cell
subsets. Immunopharmacology 27 (3), 173–179.
Sherman, M.P., de Noronha, C.M., Pearce, D., Greene, W.C., 2000. Human
immunodeficiency virus type 1 vpr contains two leucine rich helices
that mediate glucocorticoid receptor co-activation independently of its
effects on cell cycle arrest. J. Virol. 74, 8159–8165.
Singh, S.P., Tungaturthi, P., Cartas, M., Tomkowicz, B., Rizyi, T.A., Khan,
S.A., Kalyanaraman, V.S., Srinivasan, A., 2001. Virion-associated HIV-
1 Vpr: variable amount in virus particles derived from cells upon virus
infection or proviral DNA transfection. Virology 283, 78–83.
Subbramanian, R.A., Kessous-Elbaz, A., Lodge, R., Forget, J., Yao,
X.J., Bergeron, D., Cohen, E.A., 1998. Human immunodeficiency
virus type 1 Vpr is a positive regulator of viral transcription and
infectivity in primary human macrophages. J. Exp. Med. 187,
1103–1111.
Tacke, E., Schmitz, J., Prufer, D., Rohde, W., 1993. Mutational analysis of
the nucleic acid-binding 17-kDa phosphoprotein of potato leafroll
luteovirus identifies an amphipathic alpha helix as the domain for
protein/protein interactions. Virology 197, 274–282.
Tseng, Y.T., Stabila, J.P., Nguyen, T.T., McGonnigal, B.G., Waschek, J.A.,
Padbury, J.F., 2001. A novel glucocorticoid regulatory unit mediates the
hormone responsiveness of the beta1-adrenergic receptor gene. Mol.
Cell. Endocrinol. 181, 165–178.
Tungaturthi, P.K., Sawaya, B.E., Singh, S.P., Tomkowicz, B., Ayyavoo, V.,
Khalili, K., Collman, R.G., Amini, S., Srinivasan, A., 2003. Role of
HIV-1 Vpr in AIDS pathogenesis: relevance and implications of
intravirion, intracellular and free Vpr. Biomed. Pharmacother. 57,
20–24.
Vanitharani, R., Mahalingam, S., Rafaeli, Y., Singh, S.P., Srinivasan, A.,
Weiner, D.B., Ayyavoo, V., 2001. HIV-1 vpr regulates viral tran-
scription in part through the GRE and upstream region (�176 to �670)
of HIV-1 LTR. Virology 289, 334–342.
Wang, L., Mukherjee, S., Jia, F., Narayan, O., Zhao, L.J., 1995. Interaction
of virion protein Vpr of human immunodeficiency virus type 1 with
cellular transcription factor Sp1 and trans-activation of viral long
terminal repeat. J. Biol. Chem. 270, 25564–25569.
Wecker, K., Morellet, N., Bouaziz, S., Roques, B.P., 2002. NMR structure
of the HIV-1 regulatory protein Vpr in H20/trifluoroethanol. Compar-
ison with the Vpr N-terminal (1–51) and C-terminal (52–96) domains.
Eur. J. Biochem. 269, 3779–3788.
Yao, X.J., Mouland, A.J., Subbramanian, R.A., Forget, J., Rougeau, N.,
Bergeron, D., Cohen, E.A., 1998. Vpr stimulates viral expression and
induces cell killing in human immunodeficiency virus type-1 infected
dividing Jurkat T cells. J. Virol. 72, 4686–4693.
Zhang, S., Pointer, D., Singer, G., Feng, Y., Park, K., Zhao, L.J., 1998.
Direct binding to nucleic acids by Vpr of human immunodeficiency
virus type 1. Gene 212, 157–166.
Zhao, L.J., Mukherjee, S., Narayan, O., 1994. Biochemical mechanism of
HIV-1 Vpr function: specific interaction with a cellular protein. J. Biol.
Chem. 269, 15577–15582.
