Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M407863200 on November 19, 2004

J. Biol. Chem., Vol. 280, Issue 9, 8553-8563, March 4, 2005
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
280/9/8553    most recent
M407863200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Rajendra Kumar, P.
Right arrow Articles by Mahalingam, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Rajendra Kumar, P.
Right arrow Articles by Mahalingam, S.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Phosphorylation by MAPK Regulates Simian Immunodeficiency Virus Vpx Protein Nuclear Import and Virus Infectivity*

Palakurthy Rajendra Kumar, Prabhat K. Singhal, Malireddi R. K. Subba Rao, and Sundarasamy Mahalingam{ddagger}

From the Laboratory of Molecular Virology, Centre for DNA Fingerprinting and Diagnostics, ECIL Road, Hyderabad 500 076, India

Received for publication, July 13, 2004 , and in revised form, November 18, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Transport of the viral genome into the nucleus required phosphorylation of components in the preintegration complex by virion-associated host cellular kinases. In this study, we showed that ERK-2/MAPK is associated with simian immunodeficiency virus (SIV) virions and regulated the nuclear transport of Vpx and virus replication in non-proliferating target cells by phosphorylating Vpx. Suppression of the virion-associated ERK-2 activity by MAPK pathway inhibitors impaired both Vpx nuclear import and viral infectivity without affecting virus particle maturation and release. In addition, mutation analysis indicated that the inactivation of Vpx phosphorylation precluded nuclear import and reduced virus replication in macrophage cultures, even when functional integrase and Gag matrix proteins implicated in viral preintegration complex nuclear import are present. In this study, we also showed that co-localization of Vpx with Gag precursor in the cytoplasm is a prerequisite for Vpx incorporation into virus particles. Substitution of hydrophobic Leu-74 and Ile-75 with serines in the helical domain abrogated Vpx nuclear import, and its incorporation into virus particles, despite its localization in the cytoplasm, suggested that the structural integrity of helical domains is critical for Vpx functions. Taken together, these studies demonstrated that the host cell MAPK signal transduction pathway regulated an early step in SIV infection.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
A critical step in the process of retrovirus infection is the transfer of viral DNA into the nucleus of the infected cells (1-3). Lentiviruses like HIV1/SIV are capable of infecting non-dividing cells such as terminally differentiated macrophages and memory T-cells, which are important for viral dissemination and persistence (4). In contrast, prototypic retroviruses do not replicate efficiently in non-dividing cells (5). Although the mechanisms that underlie this restriction are not fully understood, inefficient nuclear transport of viral DNA appears to be one of them. HIV/SIV possess determinants that ensure effective nuclear import of viral DNA to the nucleus in non-dividing cells by exploiting cellular pathways (6-11). After entry of the virus into the cell, the genomic HIV/SIV RNA is reverse-transcribed into linear double-stranded DNA, which remains associated with a nucleoprotein complex called the preintegration complex (PIC) (12-15). The viral PICs are then imported into the nucleus through the nuclear envelope via an active mechanism within 4-6 h after infection (3). One cis-acting element, central DNA flap (15), and at least three different proteins, namely Integrase (16), Gag matrix (MA) (13, 16), and Vpr (14), have been identified as possible mediators of the nuclear import of the HIV-1 PIC; however, the roles of GagMA protein and central DNA flap in this process are still controversial (17-19). Based on its karyophilic properties, Vpr protein has been implicated in this process (20-26).

Viruses in the HIV-2/SIVsm/SIVmac lineage contain a vpr gene as well as an evolutionarily related vpx gene (27). A recent report (10) has demonstrated that SIVsm Vpr and Vpx proteins have distinct and non-complementary functions. Vpr induces cell cycle arrest at the G2 stage (13-28), whereas Vpx is mainly involved in the nuclear import of the viral PIC (9, 10). Vpx is an 18-kDa, 112-amino acid protein, which is highly conserved among all the divergent isolates of HIV-2 and SIVsm (7, 8). Vpx mutant SIVsm is significantly reduced in its ability to replicate in non-dividing target cells such as macaque macrophages (6, 10, 29). Vpx is also essential for efficient in vivo dissemination and spread of SIVsm following mucosal and intravenous infection of macaques (4). Within viral particles, Vpx seems to be localized within the viral core (30). Vpx is packaged efficiently in the progeny virions formed in the absence of the pol and env gene products and is independent of viral RNA encapsidation (6, 8, 14). These results indicate that expression of the HIV-2/SIV Gag precursor (Pr55Gag) is sufficient to mediate the incorporation of Vpx into virions. It is likely that the mechanism by which Vpx enters the assembling virion also dictates its association with the viral PICs. Recent studies (7, 9, 11, 29, 31, 32) have shown that the packaging of Vpx into viral particles depends on the C-terminal p6 domain of the Gag polyprotein. As the Vpx protein is incorporated into the virion it becomes available during early replication events, immediately following entry of the new virion into a target cell even before de novo viral protein synthesis could start. Based on such late expression during virus production and early availability during initial infection, it has been proposed that Vpx is involved in the early stages of viral life cycle, particularly in the efficient import of viral genome into the nuclear compartment of non-proliferating target cells. The domain(s) and/or the amino acid(s) required for various functions of Vpx and its relevance to the optimal virus replication in non-dividing target cells have not been reported so far. Also, the mechanism by which Vpx incorporates into the virus particles and mediates the nuclear import of HIV-2/SIVsm PICs remains unknown. Mutations within the nuclear targeting domain abrogate nuclear import function of Vpx and attenuate replication of HIV-2 and SIV in macrophages (6, 29, 33).

Phosphorylation plays a critical role in the nuclear localization signal (NLS)-mediated nuclear transport, cell cycle progression, and gene expression (34-37). Phosphorylation-regulated NLSs were found to control nuclear transport in eukaryotic cells from yeast and plants to higher mammals (35). For example, the archetypal NLS-containing simian virus 40 large T-antigen is regulated by the CcN motif. This motif comprises the T-antigen NLS (N), the phosphorylation site (C) of casein kinase II, 13 amino acids N-terminal to the NLS modulating the rate of nuclear import, and a cyclin-dependent kinase site (c) adjacent to the NLS regulating the maximal level of nuclear accumulation. Phosphorylation has been shown to regulate the progression of cell cycle and gene expression by changing the nuclear localization of various proteins, as well as their association with transcriptional activation factors (36, 37). Recent studies demonstrated that serine-threonine kinases of the host cell are incorporated within HIV-1 particles (38-42) and regulate early steps in the viral life cycle (38-41). Phosphorylation of HIV-1 Gagp17MA and Gagp6 by the virion-associated ERK-2/MAPK is shown to be essential for its association with the viral PIC and also for the release of the virus particle from the infected cells (42, 43). By having demonstrated that Vpx is the critical determinant for HIV-2/SIV replication in non-dividing cells, the present study was designed to understand the contribution of the Vpx nuclear transport and phosphorylation in the HIV-2/SIV life cycle.

In this study, we show for the first time that ERK-2/MAPK is packaged into the SIVsm(PBj1.9) virions and that it phosphorylates Vpx and regulates SIV replication. Furthermore, we demonstrate that the phosphorylation was directly implicated in the Vpx nuclear import and subsequent virus infection but not on viral maturation, assembly, and Vpx packaging into virus particles. These results reveal a new level of regulation of HIV-2 and SIV infection.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Construction of Vpx Expression Vectors—Mutational analyses were performed by using the infectious molecular clone SIVsm(PBj1.9) (44). The quick-change site-directed mutagenesis kit (Stratagene) was used to introduce mutations into the PBj1.9 vpx gene (subcloned as an internal SpeI-ClaI DNA fragment). Mutagenized vpx genes were PCR-amplified and inserted into the mammalian expression vector pCDNA3 (Invitrogen) and also were reinserted into the PBj1.9 proviral vector using a series of subcloning steps. None of the introduced nucleotide substitutions resulted in amino acid changes in overlapping the virion infectivity factor (Vif) open reading frame. All introduced mutations were confirmed by DNA sequence analysis.

Cell Culture, Transfection, and Infection—293T, COS-7, Vero, and HeLa cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with penicillin (100 units/ml), streptomycin (100 µg/ml), and 10% fetal bovine serum (FBS). CEMx174 and Jurkat cells were maintained in RPMI 1640 supplemented with 2 mM L-glutamine, penicillin (100 units/ml), streptomycin (100 µg/ml), and 10% FBS. Macaque PBMCs were obtained from heparin-treated whole blood using lymphocyte separation medium (Organon Teknika). Macrophages were purified from unstimulated macaque PBMCs as described elsewhere (32). Briefly, 3 x 106 macaque PBMCs were placed in 12-well tissue culture plates in macrophage medium containing 10% autologous macaque serum and conditioned medium to supply growth factors. Non-adherent cells were removed after 30-60 min of incubation at 37 °C, followed by extensive washing with phosphate-buffered saline. Cells were allowed to differentiate in macrophage medium for 10-12 days prior to virus infection. To examine the effects of MAPK inhibitor on virus infectivity, T-cells were stimulated with PMA, and the infectious virus particles were produced in the presence or absence of the MAPK inhibitor hypericin and were used for infection in macaque macrophages.

For the generation of virus stocks, 293T cells were transfected with wild type and vpx mutant SIVsm(PBj1.9) proviral DNAs (10 µg) using the Effectene transfection kit (Qiagen). Cell culture supernatants were collected 48 h after transfection and analyzed for core antigen (p27Gag) content using an SIV core antigen assay (Coulter). CEMx174 cells were then infected with supernatants containing 10 ng of p27Gag and were incubated overnight at 37 °C in 5% CO2. Terminally differentiated macaque macrophages were infected with virion preparations containing 10 ng of p27Gag in 12-well plates overnight at 37 °C in 5% CO2. Infected cells were washed extensively to remove residual virus and incubated at 37 °C. Culture supernatants were collected after every 3 days and were frozen at -70 °C for p27Gag determination at the conclusion of the experiments.

Metabolic Labeling and Immunoprecipitation—The infection-transfection protocol for the vaccinia virus expression system was as described elsewhere (29). Briefly, Vero cells were grown to 90% confluence on 60-mm-diameter plates, infected for 1 h at 37 °C with vTF7-3, a vaccinia virus expressing T7 RNA polymerase (45), at a multiplicity of infection of 10, and then transfected with wild type or relevant vpx mutant constructs. Ten to 12 h after transfection, the cells were labeled for 5 h with 1.5 ml of phosphate-free DMEM containing 1.0 mCi of 32Pi (Bhabha Atomic Research Center), 1% dialyzed FBS, or Met/Cys-free DMEM containing 150 µCi of [35S]methionine (Bhabha Atomic Research Center). The labeled cells were lysed with radioimmunoprecipitation buffer without SDS (1% (v/v) Triton X-100, 0.5% (w/v) deoxycholate, and 0.2 mM phenylmethylsulfonyl fluoride in phosphate-buffered saline, with 0.2 mM Na2VO4 added to 32Pi-labeled samples). Then 3 µl of anti-Vpx antiserum was added, and the mixture was incubated for 2 h at 4 °C. Twenty microliters of immobilized protein-A beads was added, and the mixture was incubated for 90 min at 4 °C with gentle rotation. Immunoprecipitates were analyzed by SDS-12% PAGE after extensive washing in buffer containing high salt and bovine serum albumin. The gels were dried and exposed to a XAR-5 film (Eastman Kodak Co.) at -80 °C.

Western Blot Analysis—COS-7 cells in 60-mm-diameter dishes were infected with vTF7-3 at a multiplicity of infection of 10 for 1 h in 5% CO2 at 37 °C and transfected with 5 µg of various Vpx expression plasmids using Lipofectin (Invitrogen). Transfected cells were lysed with lysis buffer (20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM {beta}-glycerophosphate, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin), solubilized in loading buffer (62.5 mM Tris-HCl (pH 6.8), 0.2% SDS, 5% 2-mercaptoethanol, 10% glycerol), and separated on a SDS-12% PAGE. Following electrophoresis, proteins were transferred to a Hybond-P membrane (Amersham Biosciences) and probed with the monoclonal Vpx antibody (1:1000). Protein-bound antibodies were probed with horseradish peroxidase-conjugated specific secondary antibodies (at a 1:2000 dilution) and developed using the enhanced chemiluminescence-plus detection system (Amersham Biosciences). For determining the ability of Vpx packaging into virus particles, virions from the culture supernatants (293T cells were transfected with Vpx wild type and mutant proviral clones) were concentrated by ultracentrifugation (125,000 x g, 2 h) through a 20% sucrose cushion, and viral pellets were separated on a SDS-12% PAGE. Following electrophoresis, Western blot was performed as described above using monoclonal anti-Vpx and anti-Gag antibodies.

Isolation of Nuclear and Cytoplasmic Fractions—Vpx expression plasmids were transfected into Vero cells using Lipofectin as described above. Transfected cells were labeled with 32Pi for 5 h, and the cytoplasmic and nuclear extracts were prepared by resuspending the cell pellets with hypotonic buffer (10 mM HEPES (pH 7.9), 10 mM KCl, 1 mM EDTA, 1 mM EGTA, and 1.5 mM dithiothreitol) and incubated on ice for 45 min. Nuclear and cytoplasmic fractions were isolated by performing centrifugation of cell lysates at 12,500 rpm for 1 min after adding 0.1% Nonidet P-40 and were followed by immunoprecipitation using monoclonal Vpx antibody as described above.

Fluorescence Microscopy—Vero cells in Chamber culture slides (Nunc) were infected with vTF7-3 and transfected with Vpx expression plasmids using Lipofectin as described above. Ten to 12 h after transfection, the cells were fixed with 3% paraformaldehyde and probed with the monoclonal anti-Vpx antibody (1:250) for 90 min at 37 °C. Alexa Fluor 488-conjugated affinity-purified goat anti-mouse IgG (Molecular Probes) was used as a secondary antibody to visualize the subcellular localization of Vpx proteins. Vpx and Gag co-localization in the Vpx and Gag expression vectors co-transfected cells were determined by probing the cells with monoclonal anti-Vpx antibody (1:250) and polyclonal FLAG antibody (1:500), which recognizes the FLAG-Gag fusion protein. Alexa Fluor 488-conjugated affinity-purified goat anti-mouse IgG and Alexa Fluor 594-conjugated affinity-purified goat anti-rabbit IgG were used as secondary antibodies to visualize the subcellular localization of Vpx and Gag proteins, respectively. The cells were mounted in mounting medium (Vector Laboratories) containing 4,6-diamidino-2-phenylindole to stain the nuclei. Samples were viewed with an upright Nikon E800 microscope and photographed with a DXM1200 camera using Image Pro-plus software (Media Cybernetics Inc.). Confocal laser microscopy was performed on a Zeiss LSM510 META microscope.

Construction, Expression, and Purification of His-Vpx, GST-Gag p17MA, and GST-c-Jun Fusion Proteins—Vpx from SIVsm(PBj1.9) was amplified by PCR using the forward (NdeI) primer 5'-GGTCGTCATATGATGTCAGATCCCAGGGAGAG-3' and the reverse (BamHI) primer 5'-GATTAGGGATCCTTATGCTAGTCCTGGAGGGGG-3'. The PCR fragments were then cloned into pET16b vector at the NdeI and BamHI sites. JNK phosphorylation domain of c-Jun (forward primer BamHI, 5'-ATCGGGGATCCGATGACTGCAAAGATGGAA-3', and reverse primer XhoI, 5'-CTAGGGCTCGAGTCAGGGGCACAGGAACTGGGT-3') and SIVsm(PBj1.9) Gagp17MA (forward primer EcoRI, 5'-GATCCGAATTCTATGGGCGCGAGAAAC-3', and reverse primer XhoI, 5'-GATCAGCTCGAGTTAGTAATTTCCTCCTTTGCCACT-3') were amplified and cloned into pET41b vector as GST fusion. All the plasmids were transformed into the BL-21 DE3 strain of Escherichia coli and induced with 1 mM isopropyl-{beta}-D-thiogalactopyranoside to produce fusion proteins. The bacteria were collected by centrifugation and resuspended in E. coli lysis buffer containing 40 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride, and 1% Triton X-100. Vigorous sonication was performed before centrifugation at 10,000 x g for 30 min. The resulting supernatants containing fusion proteins were purified by affinity chromatography with nickel-nitrilotriacetic acid (Vpx) and glutathione-Sepharose beads (c-Jun and Gagp17MA) according to the manufacturer's (Qiagen) instructions. Elution buffer containing 20 mM Tris-HCl (pH 8.5), 200 mM NaCl, and 150 mM imidazole was used for Vpx purification. Purified proteins were stored at -70 °C.

In Vitro Kinase Assay—Purified recombinant Vpx protein and c-Jun (3 µg each) were incubated separately with ERK-2/MAPK and JNK immunoprecipitates from T-cells activated with PMA (100 nm/ml) for 1 h in the presence or absence of the MAPK inhibitor hypericin in 20 µl of kinase reaction buffer (20 mM HEPES (pH 7.4), 10 mM MgCl2, 1 mM dithiothreitol, 20 mM {beta}-glycerol phosphate, and 0.1 mM vanadate containing 10 µCi of [{gamma}-32P]ATP). Samples were incubated for 30 min at 30 °C, and reactions were terminated by adding 7 µl of SDS sample buffer and boiled for 5 min. Identical reactions were also carried out by incubating recombinant Vpx or GST-Gagp17MA with ERK-2/MAPK (Upstate Biotechnology, Inc.). A portion of the sample (5 µl) was separated on a SDS-12% PAGE and autoradiographed.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Vpx Is Localized in the Nucleus and Phosphorylated by Host Cellular Kinases—To understand the mechanism of Vpx nuclear transport and its effects on virus replication, Vpx from SIVsm(PBj1.9) proviral clone was selected as a representative of the HIV-2/SIVsm group of viruses, and the subcellular localization of Vpx in intact cells was assessed using vaccinia virus T7-RNA polymerase system (vTF7-3) (45). In the present study, vTF7-3-infected Vero cells were transfected with Vpx expression plasmid, and expression was driven by the bacteriophage T7 promoter. Cells were probed with anti-Vpx antiserum and mAb414 (a monoclonal antibody that specifically react with FXFG containing nucleoporins at the nuclear envelope) 18 h after transfection. Results in Fig. 1A indicate that Vpx was localized predominantly in the nucleus of transfected cells. Therefore, it appears that Vpx does not depend on any other viral factors for its nuclear localization and appears to possess a specific signal for its nuclear import.



View larger version (12K):
[in this window]
[in a new window]
  		FIG. 1.
Vpx localized to the nucleus and phosphorylated by host cell kinases. A, vTF7-3-infected Vero cells were transfected with the Vpx expression plasmid, and the localization of Vpx was detected by indirect immunofluorescence using anti-Vpx monoclonal antibody (mAb) followed by an anti-mouse Alexa Fluor 488-conjugated secondary antibody. Green indicates localization of Vpx; red indicates staining of nuclear membrane with mAb414. B, Vero cells were transfected with Vpx expression plasmids and labeled with 1 mCi of 32Pi per ml in phosphate-free medium containing 1% dialyzed fetal bovine serum or Met/Cys-free DMEM containing 0.15 mCi of [35S]methionine. The cells were lysed and immunoprecipitated with anti-Vpx antiserum. Labeled Vpx proteins were visualized by electrophoresis on SDS-12% PAGE and autoradiography. {Delta}Vpx, a control construct lacking a functional vpx open reading frame.

 
Several studies indicated that phosphorylation regulates the nuclear transport of both viral and cellular proteins (34-42). The exchange of potential phosphorylation residues in Vpx has disturbed its nuclear import, and this has suggested that phosphorylation plays an important role in modulating Vpx nuclear import (29). In order to define whether phosphorylation regulates Vpx functions, we first tested the phosphorylation status of Vpx by expressing Vpx using the vaccinia T7-RNA polymerase system in Vero cells and labeled with 32Pi (1 mCi/ml) for 5 h. The labeled cells were lysed and immunoprecipitated with anti-Vpx antibodies and separated by SDS-12% PAGE, and the phosphorylated Vpx was detected by exposure of the dried gel to x-ray film. A 32P-labeled band was evident in the cells expressing Vpx (Fig. 1B) but not {Delta}Vpx, suggesting that Vpx is phosphorylated by cellular kinases.

Vpx Is a Virion-associated Protein, and Mutations in the Helical Domains Affect Its Incorporation into Virus Particles—In order to define the underlying mechanism(s) that regulate Vpx incorporation into the virus particle, we first determined the presence of Vpx in the virus particles. SIVsm(PBj1.9) proviral DNAs were transfected into 293T cells, and the culture supernatants were collected 48 h after transfection and centrifuged over a 20% sucrose cushion, and the viral pellets (normalized by p27Gag content) were examined by Western blot analysis. Probing with an anti-Gag monoclonal antibody showed that the expression, processing, and assembly of Gag were not affected by the Vpx mutation as evidenced by an equal amount of Gag-matured product capsid p27 incorporated into the virions of both wild type and Vpx mutant viruses (Fig. 2, lower panel). Probing with a Vpx-specific antiserum revealed the presence of Vpx-specific signal in the wild type SIVsm(PBj1.9)-transfected samples (Fig. 2, upper panel), whereas the absence of Vpx from SIVsm(PBj1.9) {Delta}Vpx samples suggests that Vpx is selectively packaged into virus particles. {Delta}Vpx, a mutant in which the initiating and internal methionine codons were replaced with threonine and leucine residues and contained a stop codon at amino acid position 80, was used as a negative control.



View larger version (31K):
[in this window]
[in a new window]
  		FIG. 2.
Vpx is packaged into the SIVsm(PBj1.9) virus particles. Western blot analysis of SIVsm(PBj1.9) viral particles containing mutant Vpx proteins is shown. 293T cells were transfected with vpx mutant PBj1.9 proviral clones. Virus particles were concentrated from culture supernatants by ultracentrifugation through a 20% sucrose cushion, solubilized in gel loading buffer, and analyzed for protein content by Western blot analysis using monoclonal Gag and Vpx antibodies. wt, wild type; {Delta}Vpx, a control construct lacking a functional vpx open reading frame.

 
We next identified the determinants required for Vpx virion incorporation by introducing mutations into the vpx gene of the infectious molecular clone SIVsm(PBj1.9) (44). The conserved serine, threonine, and tyrosine residues, the targets for phosphorylation, were selected, and such phosphorylation in HIV-1 Gag plays a critical role in the nuclear import of viral genome and virus replication (13). In addition, we also targeted all the conserved residues of Vpx (Table I). The substitutions were with serines and alanines except in the N-terminal half of Vpx in most instances. The conserved residues at the N terminus of Vpx (amino acids 1-56) overlapping the Vif were exchanged with either Ile or Phe in order to maintain the wild type Vif open reading frame. The Vif protein is essential for HIV/SIV replication and pathogenesis in vivo and in restricted cell types in vitro (2). Proline-rich motifs and tryptophan residues have been shown to mediate protein-protein interactions and have been implicated in nuclear translocation of viral and cellular proteins, viral budding, and assembly processes (6, 46, 47). To understand the role of the conserved C-terminal proline-rich motif and tryptophans in Vpx functions, tryptophan and proline residues were substituted with serines.


View this table:
[in this window]
[in a new window]
  		TABLE I
Site-directed mutagenesis of the SIVsm(Pbj1.9) Vpx protein

The positions and identity of the amino acid substitutions that were introduced into the Vpx protein of SIVsm(Pbj1.9) are listed together with their subcellular distribution in the presence or absence of structural protein Pr55Gag expression. Subcellular localization was determined by co-transfection of Vero cells with Gag and variants of Vpx expression plasmids and indirect immunofluorescence assay. In most cases, the data reflect at least three independent experiments. Nuc, nuclear; Cyt, cytoplasmic; NA, not applicable.

 
SIVsm(PBj1.9) proviruses containing the variants of vpx were transfected into 293T cells, and the ability of the incorporation into virions by the Vpx mutant proteins was assessed. Cell culture supernatants were collected 48 h after transfection, and the viral pellets (normalized by p27Gag content) were examined by Western blot analysis (Fig. 2). Probing with anti-Vpx antibody revealed the absence of the Vpx signal from six mutants (W49S, Y66A/Y71A, Y69A/Y71A, Y66A/Y69A/Y71A, Y66S/Y69S/Y71S, and L74S/I75S) (Table II and Fig. 2). Most interestingly, introduction of the helical destabilizing proline residue in the helical domain I (E30P) did not abrogate virion incorporation, suggesting that the N-terminal {alpha}-helix I may be dispensable for Vpx virion incorporation. This was also true for mutations in the C-terminal region (amino acids 82-112) (Fig. 2, upper panel; Table II). Most interestingly, when we analyzed the individual tyrosine mutants such as Tyr-66 and Tyr-69, they retained wild type virion incorporation function, whereas exchange of Tyr-71 in combination with either Tyr-66 or Tyr-69 completely abrogated Vpx incorporation, suggesting the Tyr-71 may be critical for Vpx incorporation into virus particles (Fig. 2). Taken together, these data suggest that the amino acid residues Trp-49, Tyr-71, Leu-74, and Ile-75 may be involved in interactions with Gag, thereby facilitating Vpx incorporation into virus particles.


View this table:
[in this window]
[in a new window]
  		TABLE II
Effects of mutagenesis on Vpx expression, subcellular localization, virion incorporation, phosphorylation, and SIV replication

 
Vpx Interacts with Pr55Gag in the Cytoplasm and Incorporates into Virus Particles—Vpx incorporates into the virus particles through interaction with the C-terminal p6 region of the Gag precursor polyprotein. Gag was shown to be a nucleocytoplasmic shuttling protein and is sufficient to mediate Vpx incorporation into virions, but the underlying mechanism(s) of Vpx and Gag interaction inside the cells and its incorporation into virions remain elusive. We therefore analyzed the in vivo interaction between Vpx and Pr55Gag by using indirect immunofluorescence assay. Results in Fig. 3 indicate that Vpx colocalization with SIVGag precursor in the cytoplasm provides evidence that these two viral proteins interact in vivo. This interaction was specific, because there was no co-localization observed either between Vpx and HIV-1 Gag (data not shown) or green fluorescence protein and SIV Gag (Fig. 3B) in the cytoplasm, indicating that the SIV Pr55Gag interacts with Vpx and translocates Vpx to the site of viral assembly for incorporation into virus particles. These results suggest that interaction of Vpx with Pr55Gag involved specific determinants in the Vpx protein.



View larger version (37K):
[in this window]
[in a new window]
  		FIG. 3.
The Vpx and Gag proteins of SIVsm(PBj1.9) co-localize in the cytoplasm of mammalian cells. A, variants of Vpx expression plasmids were co-transfected with the Gag expression plasmid in vTF7-3-infected Vero cells. Localization of Vpx and Gag proteins was detected by indirect immunofluorescence with an anti-Vpx monoclonal antibody and the anti-FLAG polyclonal antibody for FLAG-Gag, followed by an anti-mouse Alexa Fluor 488-conjugated secondary antibody (for Vpx) and anti-rabbit Alexa Fluor 594-conjugated secondary antibody (for FLAG-Gag). Green indicates localization of Vpx; red indicates Gag; blue indicates staining of nuclear compartment with 4,6-diamidino-2-phenylindole. B, Vpx specifically interacts and co-localizes with SIV Gag precursor in the cytoplasm for subsequent incorporation into virus particles. C, L74, I75S Vpx mutant proteins were localized in the cytoplasm but not co-localized with Gag precursor, whereas Vpxwt colocalized with Gag precursor in the cytoplasm of mammalian cells. Green indicates localization of Vpx; red indicates Gag.

 
We next analyzed the relationship between the co-localization of Vpx with the Gag precursor and Vpx virion incorporation. A library of Vpx mutants was generated and expressed in combination with Pr55Gag in Vero cells. We isolated six Vpx mutants (W49S, W49S/W53S/W56S, Y71A, Y66A/Y71A, Y69A/Y71A, and Y66A/Y69A/Y71A) that are unable to co-localize with Pr55Gag in the cytoplasm (Fig. 3, A and B; Table I) despite retaining the wild type nuclear localization as detected by immunofluorescence assay (Table I). The Trp-49, Tyr-71, Leu-74, and Ile-75 residues are located within the predicted helical domains II and III of Vpx, suggesting that the helical domains are critical for Vpx interaction with Gag precursor. As expected, the Vpx mutants that are co-localized with Gag precursor in the cytoplasm are incorporated into virus particles (Figs. 2 and 3; Table II). Most interestingly, the Vpx mutants (H82S, G86S/C87S, and P103S/P106S) that are defective for nuclear import but retained the ability to incorporate into virus particles like wild type Vpx protein, suggest that Vpx interacts with Gag in the cytoplasm for its virion incorporation (Table II). On the other hand, substitution of Leu-74 and Ile-75 resulted in cytoplasmic localization of mutant protein but failed to co-localize with Gag (Fig. 3, B and C) and subsequently did not incorporate into virus particles, suggesting that these residues are critical for Vpx and Gag interaction (Figs. 2 and 3). Furthermore, W49S and Y66A/Y69A/Y71A mutant Vpx proteins were localized to the nucleus in the presence or absence of Gag expression and not incorporated into virus particles (Fig. 2 and Fig. 3, A and B; Table II). Most interestingly, exchange of Tyr-71 with alanine reduced Vpx virion incorporation, but a combination with a mutation at either Tyr-66 or Tyr-69 completely prevented Vpx incorporation without altering wild type nuclear localization (Tables I and II). This striking correlation between the ability of Pr55Gag and Vpx co-localization inside the cells and the packaging of Vpx into virions indicates that Vpx is incorporated into virions through direct interaction with the Gag precursor in the cytoplasm (Figs. 2 and 3; Table II). Taken together, these results suggest that the co-localization of Vpx with the Gag precursor in the cytoplasm is essential for its incorporation into virus particles. Furthermore, these results suggest that residues in the helical domain II and III are critical for Vpx interaction with Pr55Gag and subsequent packaging into the virions to support virus replication.

ERK-2/MAPK Associates with SIVsm(PBj1.9) Virions and Phosphorylates Vpx—Because Vpx is incorporated into the budding virions in association with Gag and targets the HIV-2/SIV genome to the nucleus of non-dividing cells, we next investigated the mechanism by which these two opposing functions of Vpx are regulated. Previous studies (42, 48) suggested that cellular kinases like MAPK are selectively incorporated into HIV-1 particles and regulate virus infectivity. Having demonstrated that Vpx is a major determinant for HIV-2/SIV infection in non-dividing cells, here we investigated whether MAPK is present in the purified SIVsm(PBj1.9) virions and phosphorylates Vpx. Viral supernatants were clarified from productively infected CEMx174 cell lines and resolved on a sucrose density gradient, and the purified virions were examined by Western blotting with antibodies to ERK-2/MAPK. ERK-2/MAPK was selectively incorporated into the virus particles irrespective of Vpx virion incorporation (Fig. 4A). ERK-2/MAPK was not detectable in supernatants obtained from uninfected cells (data not shown), suggesting that ERK-2/MAPK was selectively associated with SIV virions.



View larger version (41K):
[in this window]
[in a new window]
  		FIG. 4.
ERK-2/MAPK is associated with SIVsm(PBj1.9) virions and phosphorylates Vpx. A, virus particles were obtained from productively infected CEMx174 cells and analyzed by Western blot analysis with anti-ERK-2, anti-Gag, and anti-Vpx antibodies. Twenty five ng of equivalent p27Gag was loaded in each well. Comparison of the ERK-2/MAPK proteins with SIVsm Gag in each sample indicates packaging of ERK-2/MAPK with virus particles in the presence or absence of Vpx virion incorporation. B, Vpx is a substrate for ERK-2/MAPK. Various cellular kinases were immunoaffinity-purified from uninfected PMA-activated Jurkat cells, and the activity was examined by in vitro kinase assay using purified recombinant SIVsm Vpx. Phosphorylated Vpx protein was visualized by electrophoresis on SDS-12% PAGE and autoradiography. C, MAPK inhibitors prevent Vpx phosphorylation. ERK-2/MAPK was immunoaffinity-purified from uninfected Jurkat cells treated with the MAPK inhibitor, hypericin, and the kinase activity was determined by using a recombinant Vpx as substrate. D, Vpx, GST-Gagp17MA, and GST-c-Jun proteins were expressed in E. coli, BL21-DE3, and purified by nickel-nitrilotriacetic acid affinity chromatography (for Vpx) and glutathione-Sepharose (for GST). E, recombinant Vpx and Gagp17MA were specifically phosphorylated by recombinant ERK-2. F, hypericin specifically inhibits ERK-2-mediated phosphorylation of Vpx but not the JNK activity. An arrow indicates Vpx, GST, c-Jun, and Gagp17MA.

 
We next examined whether Vpx is a substrate for ERK-2/MAPK and stimulation or inhibition of the MAPK pathway modulates the phosphorylation of Vpx. The mitogen phorbol myristate acetate (PMA) was used to activate the MAPK pathway, and hypericin was used to inhibit the activity of ERK-2/MAPK. Direct evidence of Vpx phosphorylation was obtained following immunoprecipitation of various cellular kinases (ERK-2/MAPK, ERK-1/MAPK, NIK, and LCK) from cell lysates by specific antibodies and was used for in vitro kinase assays. Immunoaffinity-purified ERK-2/MAPK was able to selectively phosphorylate recombinant Vpx and not by other kinases (Fig. 4B). This provides evidence that Vpx is a substrate for ERK-2/MAPK. SIVsm Vpx protein was expressed in E. coli BL-21DE3, purified by nickel-nitrilotriacetic acid chromatography (Fig. 4D), and used for in vitro kinase assays. Recent reports (42) suggested that hypericin at a 100 nM concentration efficiently inhibited MAPK activity. Most interestingly, we observed the inhibition of Vpx phosphorylation when ERK-2/MAPK immunoaffinity-purified from the cells was treated with the MAPK inhibitor hypericin (Fig. 4C, upper panel), suggesting that Vpx is the substrate for ERK-2/MAPK and further suggesting that hypericin inhibits only the activity of ERK-2 but not its expression level (Fig. 4C, lower panel). Because ERK-2-dependent phosphorylation was reported for Gagp17MA (42), GST and GST-Gagp17MA were used as negative and positive controls, respectively (Fig. 4, D and E). Recombinant ERK-2/MAPK was also able to phosphorylate recombinant Vpx and Gag p17MA in vitro (Fig. 4E). An equal amount of recombinant proteins loaded on each lane was ascertained by Coomassie Brilliant Blue staining (Fig. 4D). To prove further whether Vpx is the substrate for ERK-2 and that hypericin selectively inhibits ERK-2 activity, we purified ERK-2 and JNK (a serine-threonine kinase) from the cells treated with or without hypericin and subjected to in vitro kinase reaction using Vpx and c-Jun as substrates. The results in Fig. 4F indicate that Vpx is selectively phosphorylated by ERK-2 (Fig. 4F, upper panel, lanes 3 and 6). However, we noticed complete inhibition ERK-2-mediated Vpx phosphorylation (Fig. 4F, lower panel, lane 6) in the presence of hypericin. Most interestingly, we observed the equal level of c-Jun phosphorylation by JNK when the cells were treated with or without hypericin. Collectively, these results suggest that Vpx is phosphorylated by ERK-2/MAPK both in vitro and in vivo and that hypericin selectively inhibits ERK-2 activity.

Phosphorylation by ERK-2 Regulates Vpx Nuclear Transport—Recently, we reported (29) that the exchange of potential phosphorylation residues impaired Vpx nuclear transport and virus replication, and in the present study we found that Vpx is phosphorylated by host cell ERK-2/MAPK (Fig. 4). Because the underlying mechanism of Vpx nuclear transport remains unknown and having demonstrated that phosphorylation regulates the nuclear transport of various cellular and viral proteins, we analyzed the presence of the phosphorylated form of Vpx protein in different cellular compartments as described under "Materials and Methods." The results in Fig. 5A indicate that the phosphorylated form of Vpx transported and accumulated more in the nuclear fractions compared with cytoplasm, as compared with the nuclear localization of Vpx (Fig. 1A; Table I). This was further supported by the specific inhibition of Vpx nuclear transport when we analyzed how the subcellular fractions from the cells expressing Vpx were treated with hypericin followed by immunoprecipitation (Fig. 5A) and subcellular localization analysis (Fig. 5B). Most interestingly, we observed nearly equal levels of Vpx phosphorylation in the presence or absence of hypericin, but the phosphorylated form of Vpx accumulated more in the cytoplasm (block of nuclear transport) in the hypericin-treated cells suggesting that Vpx may be phosphorylated at multiple residues and the ERK-2-mediated phosphorylation may be required for Vpx nuclear import. Furthermore, the results in Fig. 5B show that the inhibitor (PD98059) of the upstream kinase MEK in the MAPK pathway also inhibits the nuclear import of Vpx. This inhibition was specific, because the tyrosine kinase inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo [3,4-d]pyrimidine did not alter the nuclear import of both wild type and mutant Vpx proteins (Fig. 5C). Altogether, these data suggest that phosphorylation plays an important role in regulating the transport of Vpx into the nucleus.



View larger version (39K):
[in this window]
[in a new window]
  		FIG. 5.
MAPK-mediated phosphorylation regulates SIVsm(PBj1.9) Vpx protein nuclear transport. A, vTF7-3-infected Vero cells were transfected with Vpx expression plasmids and labeled with 1 mCi of 32Pi per ml in phosphate-free medium. The presence of Vpx protein in the total cell lysate (t) cytoplasmic (C) and nuclear (N) fractions was performed by immunoprecipitation using monoclonal Vpx antibody. The phosphorylated form of Vpx transported and accumulated more in the nuclear compartment compared with cytoplasmic fractions. B, vTF7-3-infected Vero cells were transfected with Vpx expression plasmid and treated with the indicated MAPK pathway inhibitors. The localization of Vpx was detected by indirect immunofluorescence using anti-Vpx monoclonal antibody followed by an anti-mouse Alexa Fluor 488-conjugated secondary antibody 18 h after transfection. C, MAPK pathway inhibitors block the nuclear transport of Vpx. Vero cells were transfected with Vpx expression plasmids (wild type (wt) and Y66A/Y69A/Y71A (Y66,69,71A)) and treated with both MAPK inhibitor (hypericin, 100 nM) and tyrosine kinase inhibitor (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3-4-d]pyrimidine, 5 nM). The localization of Vpx proteins was determined by immunofluorescence assay by using monoclonal Vpx antibody. Nuclear import of Vpx was specifically inhibited by hypericin but not by the tyrosine kinase inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3-4-d]pyrimidine. a, no treatment; b, treated with MAPK inhibitor; c, treated with tyrosine kinase inhibitor. D, phosphorylation of Vpx mutant proteins. Vpx was labeled following transfection of COS-7 cells with variants of Vpx expression plasmids in the presence of 1 mCi of 32Pi per ml in phosphate-free medium containing 1% dialyzed fetal bovine serum. Labeled cells were lysed and immunoprecipitated with anti-Vpx antibodies. Labeled Vpx proteins (upper panel) were visualized by electrophoresis on SDS-12% PAGE and autoradiography. Western blot analysis of cell lysates using anti-Vpx antibody showed that an equal amount of Vpx proteins was expressed in all samples (lower panel). An arrow indicates the labeled Vpx. Wt, wild type.

 
To determine the amino acid residue(s) responsible for ERK-2-mediated Vpx phosphorylation, we transfected various vpx mutant plasmids into the vTF7-3-infected Vero cells and labeled with 32Pi (1 mCi/ml) as described above. As shown in Fig. 5D, S13N, E30P, and W49S mutant proteins retain wild type phosphorylation; in contrast, replacement of serine 63 and 65 and threonine 67 severely impaired Vpx phosphorylation, indicating that these residues account for host cell kinase-dependent Vpx phosphorylation (Fig. 5D, upper panel). Nevertheless, an equal amount of expression was observed for all mutant proteins (Fig. 5D, lower panel). To determine the interrelationship between various Vpx functions, we first compared the results of subcellular localization and virion incorporation experiments with those of phosphorylation for various Vpx mutants. As shown in Table II, phosphorylation-defective Vpx mutants failed to localize to the nucleus but showed wild type virion incorporation. For example, S63A/S65A and H82S Vpx mutants were defective for phosphorylation and nuclear transport but retained wild type virion incorporation (Table II), suggesting that phosphorylation plays an important role in regulating the transport of Vpx into the nucleus but not for its incorporation into virus particles. This was further supported by inhibition of Vpx (wild type and Y66A/Y69A/Y71A) nuclear import by MAPK pathway inhibitors (Fig. 5, B and C). Collectively, these results strongly suggest that the phosphorylation and structural integrity of helical domain III (amino acids 64-82) play a critical role for the efficient transport of Vpx into the nucleus.

Phosphorylation of Vpx by ERK-2 Is Not Essential for Its Incorporation into Virus Particles—In order to further define whether phosphorylation modulates Vpx virion incorporation, we tested the virion incorporation ability of non-phosphorylated Vpx proteins. Because Gag expression was shown to be sufficient to mediate Vpx incorporation into virions, we cotransfected SIVsm(PBj1.9) Vpx and Gag expression plasmids into COS-7 cells. Transfected cells were treated with inhibitors of MAPK pathways and tyrosine kinases alone or in combination and labeled with 32Pi or [35S]methionine. Results in Fig. 6 indicate that Vpx was efficiently incorporated into virus-like particles generated by the Pr55Gag precursor and was not altered by either inhibitors of MAPK pathways or tyrosine kinases (Fig. 6A). Most interestingly, inhibitors of the MAPK pathway blocked Vpx phosphorylation (Fig. 6B) without altering its incorporation into virus-like particles as well as virus particles (Fig. 6A and 8A). Furthermore, we observed an equal amount of virus-like particle (Fig. 6C) as well as virus particle (Fig. 8A) release in the presence or absence of kinase inhibitors. Altogether, these data provide evidence that ERK-2-mediated phosphorylation is not required for incorporation of Vpx into virus particles.



View larger version (40K):
[in this window]
[in a new window]
  		FIG. 6.
Inhibition of Vpx phosphorylation by kinase inhibitors did not alter Vpx virion incorporation ability. A, Vpx and Gag expression plasmids were co-transfected in COS-7 cells and treated with inhibitors of MAPK pathways (100 nM hypericin and 100 µM PD98059) and tyrosine kinase (2.6 µM genistein and 5 nM 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3-4-d]pyrimidine). Cells were labeled with [35S]methionine, and the Vpx incorporation was monitored in the Pr55Gag precursor-generated virus-like particles by immunoprecipitation using anti-Vpx antibody. B, similarly, the transfected cells were labeled with 32Pi, and the incorporation of the phosphorylated and non-phosphorylated forms of Vpx proteins was monitored. C, the results suggested that phosphorylation did not modulate Vpx virion incorporation. Neither the MAPK pathway nor tyrosine kinase inhibitors altered the release of virus particles into the culture medium. Culture media from [35S]methionine-labeled cells were collected and concentrated by Centricon-30 and subjected to immunoprecipitation using monoclonal anti-Gag antibody.

 



View larger version (25K):
[in this window]
[in a new window]
  		FIG. 8.
ERK-2/MAPK modulates SIV infectivity. A, ERK-2/MAPK associated with SIVsm(PBj1.9) virions and the MAPK inhibitor does not influence the packaging of ERK-2/MAPK into virus particles. Virus particles were obtained from the productively infected CEMx174 cells in the presence or absence of MAPK inhibitor and purified as described under "Materials and Methods." Virus particles were pelleted and analyzed by Western blot analysis using anti-ERK-2, anti-Gag, and anti-Vpx antibodies. Twenty five ng of equivalent p27Gag was loaded in each well. Comparison of the ERK-2/MAPK proteins with SIVsm Gag in each sample indicates efficient packaging of ERK-2/MAPK with virus particles in the presence or absence of Vpx virion incorporation. B, terminally differentiated macaque macrophages were infected with the SIVsm(PBj1.9) virus produced in the presence or absence of MAPK inhibitor and equilibrated by p27Gag content (10 ng of p27Gag). Virus replication was assessed by quantifying the amounts of p27Gag antigen in culture supernatants at 3-day post-infection intervals. wt, wild type; {Delta}Vpx, negative control; wt post-treatment, presence of MAPK inhibitor during and throughout the virus infection period; wt pre-treatment, viruses were produced in the presence of MAPK inhibitor.

 
Consequences of MAPK-mediated Phosphorylation on Vpx Nuclear Transport and SIVsm(PBj1.9) Replication in Differentiated Macaque Macrophages—Our results were compatible with a role for Vpx in the nuclear transport of HIV-2/SIV PIC and hence for efficient infection in non-dividing target cells. To probe this issue in detail, we examined the ability of wild type and nuclear transport/phosphorylation-defective vpx mutant SIVsm(PBj1.9) proviruses to elicit a spreading infection in monocyte-derived macaque macrophage cultures. The proviral clones with (a) wild type Vpx virion incorporation with defective nuclear transport and phosphorylation and (b) wild type Vpx localization pattern with defective virion incorporation function were selected to examine whether the phosphorylation and nuclear transport property of Vpx are required for optimal virus replication in non-dividing cells. All vpx mutant viruses replicated efficiently and to high titers in proliferating CEMx174 cells like wild type (Fig. 7B; Table II). However, this was not the case in terminally differentiated macaque macrophage cultures (Fig. 7A; Table II). As expected, mutant Vpx proteins that failed to phosphorylate and localize to the nucleus were severely impaired in their ability to support virus replication in macrophages. For example, impairment of virus replication in macrophages was observed for Vpx H82S and P103S/P106S mutants that are packaged into virus particles similar to wild type levels but failed to phosphorylate and localize to the nucleus (Table II; Fig. 7). Furthermore, failure to replicate in macrophages was also observed for the Y66A/Y69A/Y71A mutant that localized to the nucleus but was not packaged into virus particles (Table II; Figs. 2 and 7). In three independent experiments, PBj1.9 mutants with impairment of Vpx phosphorylation and nuclear import replicated poorly in macrophages, suggesting that phosphorylation plays a critical role in Vpx-mediated nuclear transport of the HIV-2/SIV genome for the establishment of virus infection in macrophages. In addition, our results also suggest that the packaging of Vpx into virus particles with wild type nuclear transport function is critical for optimal virus replication in non-dividing cells.



View larger version (16K):
[in this window]
[in a new window]
  		FIG. 7.
Replication potential of wild type and vpx mutant SIVsm(PBj1.9) proviruses. A, terminally differentiated macaque macrophages; B, CEMx174 cells were infected with the indicated SIVsm(PBj1.9) virus constructs equilibrated by p27Gag (10 ng of p27Gag). The isolation and infection of primary macaque macrophages are described under "Materials and Methods." Virus replication was assessed by quantifying the amounts of p27Gag antigen in culture supernatants at 3-day intervals post-infection. wt, wild type; {Delta}Vpx, negative control.

 
ERK-2/MAPK Regulates SIVsm(PBj1.9) Infectivity—We next determined whether modulation of virion-associated ERK-2/MAPK activity influences the early steps in viral infectivity. Virus particles were produced in the presence or absence of hypericin (ERK-2/MAPK inhibitor), and infectivity was examined in macaque macrophages. Virus particles produced in the presence of hypericin have significantly impaired infectivity in macrophages (Fig. 8) but had no apparent effect on virus maturation, production, or release as indicated by the similar levels of structural Gag proteins incorporated into the virus particles in the presence or absence of MAPK inhibitor (Figs. 6C and 8A). Addition of hypericin to target cells resulted in inhibition of virus replication 12 days after infection (Fig. 8B), suggesting that ERK-2/MAPK regulates an early step in the virus life cycle, which is critical for the establishment of virus replication in macrophages. To characterize further the impairment of virus replication by MAPK inhibitors, we examined whether the MAPK pathway inhibitors (hypericin or PD98059) alter the nuclear transport activity of Vpx. Results in Figs. 4 and 5 indicate that the MAPK pathway inhibitors impair Vpx nuclear transport possibly by preventing Vpx phosphorylation (Fig. 4C). This was further supported by the low levels of nuclear accumulation and transport of Vpx when the cells were treated with MAPK inhibitors (Fig. 5, A and B). Taken together, these results suggest that the phosphorylation of Vpx by ERK-2/MAPK ultimately influences its subcellular distribution in the target cell. As a consequence, the inhibition of ERK-2/MAPK activity in the virion impairs the relative ability of Vpx to translocate to the nucleus in virus-infected cells. Taken together, these data suggest that the activity of the virion-associated ERK-2/MAPK is critical for Vpx-mediated nuclear transport of the functional HIV-2/SIV preintegration complex for optimal virus replication in non-proliferating target cells.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
HIV-2/SIV Vpx has two distinct localization properties that direct it either to the nucleus or, in association with Gag, to budding virus particles at the plasma membrane (12, 29, 32, 33, 49). These distinct Vpx localizations may be mediated through different protein-protein interactions. This study undertakes extensive mapping of the determinants for Vpx virion incorporation, subcellular localization, phosphorylation, and characterization of the inter-relationship between various properties of Vpx as relevant to the virus replication. Our data indicate that the ERK-2/MAPK is selectively packaged into the SIV virions and regulates Vpx nuclear transport and virus replication in macaque macrophages. This is supported by the following findings. (i) Vpx was phosphorylated by ERK-2/MAPK both in vivo and in vitro. (ii) Mutations that are capable of affecting the ability of Vpx phosphorylation impair its nuclear import and did not support the virus replication in macrophages. (iii) Inhibitors of ERK-2/MAPK pathway block Vpx phosphorylation as well as impair Vpx nuclear import and virus replication in primary macaque macrophages. (iv) Mutations that prevent Vpx co-localization with Gag in the cytoplasm did not support its incorporation into virus particles. (v) Helical domains are critical for both Vpx virion incorporation and nuclear transport.

Virion incorporation occurs late in the infection when de novo synthesized Vpx is re-routed to the plasma membrane for packaging into budding virions through its interaction with viral Gag precursor polyproteins. Hence, the nuclear localization of Vpx is likely to be relevant early in the infection when Vpx found in the virion helps to translocate the viral preintegration complex to the nuclear compartment. In fact, this ability of the virion-associated Vpx to act as a nuclear targeting signal for the viral PICs is the major function that current consensus attributes to Vpx protein. However, very little is known about the mechanism(s) by which Vpx incorporates into virus particles and how exactly Vpx nuclear transport is regulated to support virus replication in macrophages. Vpx is one of the regulatory proteins of primate lentiviruses packaged in large amounts into virus particles. We have shown that Vpx protein of SIVsm(PBj1.9) interacts and co-localizes with homologous Gag precursor in the cytoplasm for its packaging into virus particles. Most interestingly, Vpx mutants that are co-localized with Pr55Gag in the cytoplasm are efficiently incorporated into virions. On the other hand, the L74S/I75S mutant Vpx localized in the cytoplasm failed to co-localize with Gag and incorporated into virus particles (see Fig. 3C), strongly suggesting that Vpx and Pr55Gag interaction/co-localization in the cytoplasm is critical for Vpx packaging into virus particles. Prediction of the amphipathic profile of Vpx helical structure indicated that the Tyr-71, Leu-74, and Ile-75 are on the hydrophobic faces of the helices (Fig. 9, A and B), and replacement of these residues disturbed the amphipathic character of the helix and impaired Vpx incorporation into virions possibly by preventing its interaction with Gag (Figs. 2 and 3; Table II). Similarly, helical domains are implicated in HIV-1 Vpr nuclear import and virion incorporation (50). Helical domains are known to be involved in protein-protein, protein-nucleic acid, and protein-lipid interactions (51, 52). Taken together, these results suggest that the structural integrity of helical domains in Vpx is essential for its interaction with Gag in the cytoplasm for subsequent incorporation into virus particles. The precise mechanism of Vpx and Gag interaction (either direct or indirect) remains to be explored.



View larger version (18K):
[in this window]
[in a new window]
  		FIG. 9.
Structure prediction of SIV Vpx. A, linear sequence of Vpx from SIV isolate PBj1.9. Three helical domains were identified in 112 amino acids of Vpx sequence such as helix I (amino acids 21-39); helix II (amino acids 42-55), and helix III (amino acids 64-82) and a prolinerich C-terminal domain. B, helical wheel representation of the predicted structure denoting the {alpha}-helical organization of the motifs. Hydrophobic residues are shown in boldface type.

 
Our subcellular localization analysis demonstrates that wild type Vpx protein is efficiently translocated to the nuclear membrane and to the nuclear interior. Given the nuclear accumulation of wild type protein, we sought to characterize the underlying mechanism(s) that regulate Vpx nuclear transport. Nuclear import of proteins in general is a multistep process where proteins are first targeted to nuclear pore complexes and subsequently translocated to the nuclear interior (34, 35). This appears to be the case for the Vpx, which is targeted to the nuclear membrane and subsequently imported into the nuclear interior as observed by its localization patterns in this study. Because the nuclear targeting domain identified in Vpx is distinct from other known nuclear localization signals, it is reasonable to hypothesize that Vpx is transported to the nucleus by interacting with novel nuclear transport receptor(s) or by a piggyback mechanism. The region responsible for Vpx nuclear import (Table I) contains conserved serines, threonines, and tyrosines. These residues are frequently involved in phosphorylation and protein-protein interactions in signal transduction pathways (13, 53, 54). The results outlined here suggest that Vpx is phosphorylated by the host cell kinase, ERK-2/MAPK, and the exchange of potential phosphorylation residues blocked Vpx nuclear transport. Our results also demonstrate that the nuclear transport property of Vpx is not required for its incorporation into virus particles. Most interestingly, W49S and Y66A/Y69A/Y71A mutants failed to package into the virus particle but retained wild type nuclear localization, suggesting that Vpx may be exported to the cytoplasm. The exchange of Trp-49 and Tyr-66, -69, and -71 residues block cytoplasmic localization and subsequent Vpx incorporation into virus particles. A recent report (33) suggests that Vpx is not exported to the cytoplasm, unlike the Vpr of HIV-1. However, data from our present study suggest that Vpx may be a nucleocytoplasmic shuttling protein, and it is reasonable to hypothesize that the nuclear export of Vpx may be required for its incorporation into the virus particle like HIV-1 Vpr (55-57). Because phosphorylation regulates the nucleo-cytoplasmic transport of the proteins (13, 53, 54), it will be interesting to define whether phosphorylation regulates Vpx nuclear export activity, which may be required for its interaction with the Gag precursor for virion incorporation in order to support virus replication.

We next assessed the role of Vpx phosphorylation and nuclear transport on the replication potential of vpx mutant SIVsm(PBj1.9) in non-dividing cells. Viruses that encoded phosphorylation and the nuclear import-defective Vpx protein failed to replicate or grew very poorly in macaque macrophage cultures (Table II). The reduced efficiency of vpx mutant virus replication in macrophages in the presence of functional GagMA and integrase NLS supports the notion that nuclear import of Vpx is critical for the optimal replication of HIV-2/SIV in macrophages. This is in agreement with the current consensus that suggests Vpx is the major nucleophilic determinant coded by HIV-2/SIV. Collectively, these results suggest that phosphorylation regulates Vpx-mediated nuclear transport of the viral genome. Although these results do not provide a quantitative measure for the contribution of Vpx phosphorylation to the overall defect in replication of Vpx mutant viruses in macrophages, results in the present study demonstrate a clear deficiency in nuclear import by the phosphorylation-defective Vpx mutants and suggest that differences in the replication potential of Vpx mutant viruses may be explained, at least in part, by the effect of these mutations in viral genome nuclear import. Thus, future studies will focus to what extent the phosphorylation/nuclear import properties of Vpx govern transport of the viral genome into the nucleus of non-dividing cells.

The exact mechanism of Vpx nuclear transport and its role in HIV-2/SIV replication remain unknown. The studies outlined here imply that the host cell ERK-2/MAPK is selectively incorporated into virus particles and regulates SIV infectivity, perhaps through phosphorylation of Vpx. Thus, it is likely that the MAPK inhibitor blocks SIV infection by interfering with the nuclear import of Vpx, thereby restricting subsequent virus replication in macrophages. ERK-2/MAPK is a proline-directed kinase (58), and there are no consensus ERK-2/MAPK recognition sites within Vpx. Despite this, immunoprecipitates of ERK-2/MAPK from cell lysates as well as the recombinant ERK-2/MAPK were able to phosphorylate Vpx, indicating that Vpx is an ERK-2/MAPK substrate. ERK-2/MAPK phosphorylation of a protein, which lacks MAPK consensus recognition sites, has been reported recently (59) for both viral and cellular proteins and has suggested that conformation of the substrate protein was sufficient to allow recognition by MAPK. For example, GagMA protein of HIV-1, which lacks MAPK consensus recognition motifs, was phosphorylated by ERK-2/MAPK and regulates the transport of viral DNA to the target cell nucleus (42). We found here that the impairment of host cell ERK-2/MAPK activity by a synthetic inhibitor resulted in the production of virions with reduced infectivity as assessed by infection assays performed in macaque macrophages. Western blot analysis of virions protein contents from cells exposed or not exposed to the MAPK inhibitor reveals that it had no apparent effect on virus production and/or particle release from the infected cells. These results suggest that the defects in viral infectivity might rely on the inhibition of virus-associated ERK-2/MAPK activity, which is critical for Vpx-mediated nuclear translocation of viral genome. Although the consequences of the ERK-2/MAPK-dependent phosphorylation of Vpx remain to be defined, a possible implication of Vpx phosphorylation in transporting the viral genome can be proposed. Indeed, evidence has been obtained from the present studies that ERK-2/MAPK-mediated phosphorylation regulates Vpx nuclear transport. The precise role of Vpx phosphorylation in modulating virus replication in non-dividing target cells remains to be explored.

In conclusion, host cell kinase associated with SIV particles, ERK-2/MAPK, might play an important role in the virus life cycle by modifying Vpx protein and regulating the transport of the viral genome to the nucleus for the establishment of viral infection. It is also important to consider that the presence of host cell ERK-2/MAPK in the virus particles might be of special interest in de-regulating the host cell activation level by triggering abnormal signaling. Identifying the precise relationship between the virus-associated protein kinases and the viral life cycle will hopefully reveal novel targets for the development of specific and new antiviral agents.


   FOOTNOTES
 
* This work was supported by a grant from Department of Biotechnology, Government of India (to S. M.), core support from the Department of Biotechnology and Centre for DNA Fingerprinting and Diagnostics, and by graduate fellowships from Council of Scientific and Industrial Research, Government of India (to P. R. K., P. K. S., and M. R. K. S. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed: Laboratory of Molecular Virology, Centre for DNA Fingerprinting and Diagnostics, ECIL Rd., Nacharam, Hyderabad 500 076, India. Tel.: 11-91-40-27171478; Fax: 11-91-40-27155610; E-mail: maha{at}cdfd.org.in.

1 The abbreviations used are: HIV, human immunodeficiency virus; MAPK, mitogen activated protein kinase; ERK, extra cellular signal-regulated kinase; SIV, simian immunodeficiency virus; HIV-1, HIV type 1; HIV-2, HIV type 2; PIC, preintegration complex; NE, nuclear envelope; NLS, nuclear localization signal; MA, matrix; PMA, phorbol myristate acetate; GST, glutathione S-transferase; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; PBMC, peripheral blood mononuclear cell; Vif, virion infectivity factor. Back


   ACKNOWLEDGMENTS
 
We are indebted to Drs. S. E. Hasnain and Rama Sharma for critically reading the manuscript. We thank V. Vamsee Krishna, T. Giribabu, and A. Jagan for technical assistance. We thank Nandini Rangaraj, Centre for Cellular and Molecular Biology, for use of the Confocal Microscope Facility.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Cullen, B. R. (1998) Cell 93, 685-692[CrossRef][Medline] [Order article via Infotrieve]
  2. Trono, D. (1998) Nat. Med. 4, 1368-1369[CrossRef][Medline] [Order article via Infotrieve]
  3. Emerman, M. (1996) Curr. Biol. 6, 1096-1103[CrossRef][Medline] [Order article via Infotrieve]
  4. Hirsch, V. M., Sharkey, M. E., Byrum, R., Elkins, W. R., Hahn, B. H., Lifson, J. D. & Stevenson, M. (1998) Nat. Med. 4, 1401-1408[CrossRef][Medline] [Order article via Infotrieve]
  5. Lewis, P. F. & Emerman, M. (1994) J. Virol. 68, 510-516[Abstract/Free Full Text]
  6. Pancio, H. A., Vander Heyden, N. & Ratner, L. (2000) J. Virol. 74, 6162-6167[Abstract/Free Full Text]
  7. Henderson, L. E., Sowder, R. C., Copeland, T D., Benveniste, R. E. & Oroszlan, S. (1988) Science 241, 199-202[Abstract/Free Full Text]
  8. Kappes, J. C., Morrow, C. D., Lee, S. W., Jameson, B. A., Kent, S. B. H., Hood, L. E., Shaw, G. M. & Hahn, B. H. (1989) J. Virol. 62, 3501-3505
  9. Wu, X., Conway, J. A., Kim, J. & Kappes, J. C. (1994) J. Virol. 68, 6161-6169[Abstract/Free Full Text]
  10. Fletcher, T. M., Brichacek, B., Sharova, N., Newman, M. A., Stivahtis, G., Sharp, P. M., Emerman, M., Hahn, B. H. & Stevenson, M. (1996) EMBO J. 15, 6155-6165[Medline] [Order article via Infotrieve]
  11. Accola, M. A., Bukovsky, A. A., Jones, M. S. & Göttlinger, H. G. (1999) J. Virol. 73, 9992-9999[Abstract/Free Full Text]
  12. Bukrinsky, M. I., Haggerty, S., Dempsey, M. P., Sharova, N., Adzhubel, A., Spitz, L., Lewis, P., Goldfarb, D., Emerman, M. & Stevenson, M. (1993) Nature 365, 666-669[CrossRef][Medline] [Order article via Infotrieve]
  13. Gallay, P., Swingler, S., Aiken, C. & Trono, D. (1995) Cell 80, 379-381[CrossRef][Medline] [Order article via Infotrieve]
  14. Heinzinger, N. K., Bukinsky, M. I., Haggerty, S. A., Ragland, A. M., Kewalramani, V., Lee, M. A., Gendelman, H. E., Ratner, L., Stevenson, M. & Emerman, M. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 7311-7315[Abstract/Free Full Text]
  15. Zennou, V., Petit, C., Guetard, D., Nerhbass, U., Montagnier, L. & Charneau, P. (2000) Cell 14, 173-185
  16. Gallay, P., Hope, T., Chin, D. & Trono, D (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 9825-9830[Abstract/Free Full Text]
  17. Fouchier, R. A., Meyer, B. E., Simon, J. H., Fischer, U. & Malim, M. H. (1997) EMBO J. 16, 4531-4539[CrossRef][Medline] [Order article via Infotrieve]
  18. Dvorin, J. D., Bell, P., Maul, G. G., Yamashita, M., Emerman, M. & Malim, M. H. (2002) J. Virol. 76, 12087-12096[Abstract/Free Full Text]
  19. Limon, A., Nakajima, N., Lu, R., Ghory, H. Z. & Engelman, A. (2002) J. Virol. 76, 12078-12086[Abstract/Free Full Text]
  20. Lu, Y. L., Spearman, P. & Ratner, L. (1993) J. Virol. 67, 6542-6550[Abstract/Free Full Text]
  21. Yao, X. J., Subbramanian, R. A., Rougeau, N., Boisvert, F., Bergeron, D. & Cohen, E. A. (1995) J. Virol. 69, 7032-7044[Abstract]
  22. Mahalingam, S., Khan, S. A., Jabbar, M. A., Monken, C. E., Collman, R. G. & Srinivasan, A. (1995) Virology 207, 297-302[CrossRef][Medline] [Order article via Infotrieve]
  23. Vodicka, M. A., Koepp, D. M., Silver, P. A. & Emerman, M. (1998) Genes Dev. 12, 175-185[Abstract/Free Full Text]
  24. Jenkins, Y., McEntee, M., Weis, K. & Greene, W. C. (1998) J. Cell Biol. 143, 875-885[Abstract/Free Full Text]
  25. Fouchier, R. A., Meyer, B. E., Simon, J. H., Fischer, U., Albright, A. V., Gonzalez-Scarano, F. & Malim, M. H. (1998) J. Virol. 72, 6004-6013[Abstract/Free Full Text]
  26. de Noronha, C. M., Sherman, M. P., Lin, H. W., Cavrois, M. V., Moir, R. D., Goldman, R. D. & Greene, W. C. (2001) Science 294, 1105-1108[Abstract/Free Full Text]
  27. Hahn, B. H., Shaw, G. M., De Cock, K. M. & Sharp, P. M. (2000) Science 287, 607-614[Abstract/Free Full Text]
  28. Rogel, M. E., Wu, L. I. & Emerman, M. (1995) J. Virol. 69, 882-888[Abstract]
  29. Rajendra Kumar, P., Singhal, P. K., Vinod, S. S. & Mahalingam, S. (2003) J. Mol. Biol. 331, 1141-1156[CrossRef][Medline] [Order article via Infotrieve]
  30. Kewalramani, V. N. & Emerman, M. (1996) Virology 1, 159-168
  31. Kappes, J. C., Parkin, J. S., Conway, J. A., Kim, J., Brouillette, C. G., Shaw, G. M. & Hahn, B. H (1993) Virology 193, 222-233[CrossRef][Medline] [Order article via Infotrieve]
  32. Mahalingam, S., Van Tine, B., Santiago, M. L., Gao, F., Shaw, G. M. & Hahn, B. H. (2001) J. Virol. 75, 362-374[Abstract/Free Full Text]
  33. Belshan, M. & Ratner, L. (2003) Virology 311, 7-15[CrossRef][Medline] [Order article via Infotrieve]
  34. Fridell, R. A., Truant, R., Thorne, L., Benson, R. E. & Cullen, B. R. (1997) J. Cell Sci. 110, 1325-1331[Abstract]
  35. Jans, D. A. & Hubner, S. (1996) Physiol. Rev. 76, 651-685[Abstract/Free Full Text]
  36. Peterson, R. T. & Schreiber, S. L. (1999) Curr. Biol. 9, R521-R524[CrossRef][Medline] [Order article via Infotrieve]
  37. Schakney, S. E. & Shankey, T. V. (1999) Cytometry 35, 97-116[CrossRef][Medline] [Order article via Infotrieve]
  38. Camaur, D., Gallay, P., Swingler, S. & Trono, D. (1997) J. Virol. 71, 6834-6841[Abstract]
  39. Gallay, P., Swingler, S., Song, J., Bushman, F. & Trono, D. (1995) Cell 83, 569-576[CrossRef][Medline] [Order article via Infotrieve]
  40. Luo, T., Downing, J. R. & Garcia, J. V. (1997) J. Virol. 71, 2535-2539[Abstract]
  41. Paul, M. & Jabbar, M. A. (1997) Virology 232, 207-216[CrossRef][Medline] [Order article via Infotrieve]
  42. Jacque, J. M., Mann, A., Enslen, H., Sharova, N., Brichacek, B., Davis, R. J. & Stevenson, M. (1998) EMBO J. 17, 2607-2618[CrossRef][Medline] [Order article via Infotrieve]
  43. Hemonnot, B., Cartier, C., Gay, B., Rebuffat, S., Bardy, M., Devaux, C., Boyer, V. & Briant, L. (2004) J. Biol. Chem. 279, 32426-32434[Abstract/Free Full Text]
  44. Dewhurst, S., Embretson, J. E., Anderson, D. C., Mullins, J. I. & Fultz, P. N. (1990) Nature 345, 636-640[CrossRef][Medline] [Order article via Infotrieve]
  45. Fuerst, T. R., Earl, P. L. & Moss, B. (1987) Mol. Cell. Biol. 7, 2538-2544[Abstract/Free Full Text]
  46. Garnier, L., Wills, J. W., Verderame, M. F. & Sudol, M. (1996) Nature 381, 744-745[CrossRef][Medline] [Order article via Infotrieve]
  47. Harty, R. N., Paragas, J., Sudol, M. & Palese, P. (1999) J. Virol. 73, 2921-2929[Abstract/Free Full Text]
  48. Cartier, C., Hemonnot, B., Gay, B., Bardy, M., Sanchiz, C., Devaux, C. & Briant, L. (2003) J. Biol. Chem. 278, 35211-35219[Abstract/Free Full Text]
  49. Di Marzio, P., Choe, S., Ebright, M., Knoblauch, R. & Landau, N. R. (1995) J. Virol. 69, 7909-7916[Abstract]
  50. Mahalingam, S., Ayyavoo, V., Patel, M., Kieber-Emmons, T. & Weiner, D. B. (1997) J. Virol. 71, 6339-6347[Abstract]
  51. Saier, M. H. & McCaldon, P. (1988) J. Bacteriol. 170, 2296-2300[Abstract/Free Full Text]
  52. Tacke, E., Schmitz, J., Prufer, D. & Rohde, W. (1993) Virology 197, 274-282[CrossRef][Medline] [Order article via Infotrieve]
  53. Bresnahan, P. A., Yonemoto, W. & Greene, W. C. (1999) J. Immunol. 163, 2977-2981[Abstract/Free Full Text]
  54. Kang, H., Freund, C., Duke-Cohan, J. S., Musacchio, A., Wagner, G. & Rudd, C. E. (2000) EMBO J. 15, 2889-2899[CrossRef]
  55. Sherman, M. P., de Noronha, C. M., Heusch, M. I., Greene, S. & Greene, W. C. (2001) J. Virol. 75, 1522-1532[Abstract/Free Full Text]
  56. Jenkins, Y., Sanchez, P. V., Meyer, B. E. & Malim, M. H. (2001) J. Virol. 75, 8348-8352[Abstract/Free Full Text]
  57. Subbramanian, R. A., Yao, X. J., Dilhuydy, H., Rougeau, N., Bergeron, D., Robitaille, Y. & Cohen, E. A. (1998) J. Mol. Biol. 278, 13-30[CrossRef][Medline] [Order article via Infotrieve]
  58. Davis, R. J. (1993) J. Biol. Chem. 268, 14553-14556[Free Full Text]
  59. Corbalan-Garcia, S., Yang, S. S., Degenhardt, K. R. & Bar-Sagi, D. (1996) Mol. Cell. Biol. 16, 5674-5682[Abstract]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 J. Virol.Home page
C. Goujon, V. Arfi, T. Pertel, J. Luban, J. Lienard, D. Rigal, J.-L. Darlix, and A. Cimarelli
Characterization of Simian Immunodeficiency Virus SIVSM/Human Immunodeficiency Virus Type 2 Vpx Function in Human Myeloid Cells
J. Virol., December 15, 2008; 82(24): 12335 - 12345.
[Abstract] [Full Text] [PDF]

Home page
 J. Virol.Home page
P. K. Singhal, P. Rajendra Kumar, M. R. K. Subba Rao, and S. Mahalingam
Nuclear Export of Simian Immunodeficiency Virus Vpx Protein
J. Virol., December 15, 2006; 80(24): 12271 - 12282.
[Abstract] [Full Text] [PDF]

Home page
 J. Virol.Home page
P. K. Singhal, P. R. Kumar, M. R. K. S. Rao, M. Kyasani, and S. Mahalingam
Simian Immunodeficiency Virus Vpx Is Imported into the Nucleus via Importin Alpha-Dependent and -Independent Pathways
J. Virol., January 1, 2006; 80(1): 526 - 536.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
280/9/8553    most recent
M407863200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Rajendra Kumar, P.
Right arrow Articles by Mahalingam, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Rajendra Kumar, P.
Right arrow Articles by Mahalingam, S.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2005 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement