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International Immunology, Vol. 14, No. 1, 13-22, January 2002
© 2002 Japanese Society for Immunology

HIV-1 viral protein R compromises cellular immune function in vivo

Velpandi Ayyavoo1, Karuppiah Muthumani, Sagar Kudchodkar, Donghui Zhang, P. Ramanathan, Nathanael S. Dayes, J. J. Kim, Jeong-Im Sin, Luis J. Montaner2 and David B. Weiner

Department of Pathology and Laboratory Medicine, University of Pennsylvania, 505 Stellar Chance Laboratories, 422 Curie Boulevard, Philadelphia, PA 19104, USA
1 Department of Infectious Diseases & Microbiology, University of Pittsburgh, PA 15261, USA
2 The Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104, USA

Correspondence to: D. B. Weiner


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
HIV-1 viral protein R (Vpr) is a virion-associated gene product that profoundly affects T cell proliferation, induces apoptosis and can affect cytokine production in part through interfering with NF-{kappa}B-mediated transcription from host cells. Collectively, these effects support that Vpr could influence immune activation in vivo. However, this effect of Vpr has not been explored previously. Here we examined the effect of Vpr expression in an in vivo model system on the induction of antigen-specific immune responses using a DNA vaccine model. Vpr co-vaccination significantly altered the immune response to co-delivered antigen. Specifically, in the presence of Vpr, inflammation was markedly reduced compared to antigen alone. Vpr reduced antigen-specific CD8-mediated cytotoxic T lymphocyte activity and suppressed Th1 immune responses in vivo as evidenced by lower levels of IFN-{gamma}. In the presence of Vpr, there is a profound shift in isotype towards a Th2 response as determined by the IgG2a:IgG1 ratio. The data support that Vpr compromises antigen-specific immune responses and ultimately effector cell function, thus confirming a strong selective advantage to the virus at the expense of the host.

Keywords: antigen-presenting cell, antigen-specific immune response, HIV-1 viral protein R, T cell activation, Th1/Th2 cytokine, vaccination


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Viral protein R (Vpr) is the only HIV-1 accessory protein present in significant quantity in virions, suggesting that this protein has an important role early in the viral life cycle. For example, vpr plays an important role in facilitating infection of non-dividing cells by transporting the pre-integration complex into the nucleus (1–3). Additionally, Vpr regulates a number of host cellular events including cell cycle arrest, transcription, apoptosis and cytokine production, in part through affecting NF-{kappa}B functions (4–6). Interestingly, endogenous Vpr mediates these cellular effects in the absence of other HIV-1 viral proteins (7,8) as well as by virion-associated Vpr in the absence of de novo protein synthesis (9–11). Vpr is packaged in HIV-1 virions, through its interaction with group-specific antigen (Gag) (p6) protein and is present in equimolar concentrations to Gag (12,13). Although CD4+ cells do not require Vpr for HIV-1 infection, presence of full-length Vpr is critical for infection in macrophages. Upon infection, these target cells (both infected and uninfected) are exposed to HIV-1 antigens and components of virus particles (14,15).

Antigen-presenting cells (APC) (macrophages/dendritic cells), in addition to being a major target/reservoir for viral infection, play an important role in activating the immune system (16,17). HIV-1 Vpr increases viral production in infected cells through arrest of the cell cycle in the G2/M phase (7,18). This effect of Vpr is late, requiring 24–48 h to benefit the virus. The anti-HIV immune response, which includes large pools of antigen-specific cytotoxic T lymphocytes (CTL), can recognize and kill HIV-infected targets in <6 h, a period more rapid than cell cycle arrest (19,20). We suspected that Vpr could tip the host immune response to take advantage of its reported properties of aiding viral replication. Therefore we sought to further investigate the effect of Vpr on immune regulation.

To investigate the effect of Vpr on immune activation in vivo, we used a DNA vaccine model system. DNA immunization has been used to induce immune responses to foreign antigens of interest in vivo through inoculation of the host with plasmids encoding pathogen or tumor antigens (21–24). In vivo injection of plasmid results in protein production in local transfected cells as well as in directly transfected APC, which migrate to regional lymph nodes (25–29). This technique elicits both humoral and cellular responses to the specific immunizing antigens in animal models and humans (29–31). To investigate Vpr modulation of immune activation in vivo, we co-immunized mice with different HIV-1 plasmid-encoded antigens in the presence and absence of Vpr plasmid, and examined the immune responses induced by the immunizing antigen. Vpr specifically inhibited the development of strong CD8 CTL responses as well as the synthesis of protypic Th1-type cytokines and shifted the antibody response towards a Th2-type bias. These data support that in vivo Vpr can interfere with the development of antigen-specific immunity. Such an effect is likely to have consequences for immune control of HIV infection and viral pathogenesis.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Cells
HeLa, RD and NIH3T3 cells, obtained from the ATCC (Rockville, MD), were grown in a monolayer at 37°C in 5% CO2 in DMEM, 10% FBS, 1% penicillin, 1% streptomycin and 1% -glutamine. P815 cells obtained from ATCC were maintained as suspension cultures in RPMI 1640, 10% FBS, 1% penicillin, 1% streptomycin and 1% -glutamine at 37°C with 5% CO2.

Cloning and expression of DNA vaccine constructs
Plasmids expressing HIV-1 antigens Vpr, HIV-1 regulator factor (Nef), Gag-Pol and Vif were constructed using appropriate PCR primers as described (28,33,34). The pCDNA 3 vector was used which takes advantage of a cytomegalovirus promoter and a BGH poly(A) signal. The Gag-Pol insert was driven by the MPV CTE, which allows for Rev independent expression in murine and human cell lines. All the plasmids were sequenced to verify the coding region and were further analyzed for protein expression by immunoprecipitaion using specific antibodies (21,34,35). To further examine the expression and trafficking of Vpr antigen in vivo, we fused Vpr in frame with green fluorescent protein (GFP) and cloned Vpr-GFP into a eukaryotic expression vector as described (35).

Mice
BALB/c female mice aged 6–8 weeks were purchased from Harlan Sprague-Dawley (Indianapolis, IN). The mice were housed in a temperature-controlled, light-cycled room as per the guidelines of National Institute of Health and University of Pennsylvania.

DNA inoculation
We have utilized a facilitated DNA inoculation protocol, which results in increased protein expression levels from plasmid-delivered genes in vivo. Specifically, the quadriceps muscles of BALB/c mice were injected with 100 µl of 0.25% bupivacaine–HCl (Sigma, St Louis, MO) using a 27-gauge needle. Forty-eight hours later, 100 µg of DNA construct of interest in PBS was injected into the same region of the muscle as the bupivacaine injection. Mice were given one injection followed by a boost 2 weeks later. Two weeks after the second injection, half of the mice in each group were sacrificed for their spleens and the remaining mice were given a second boost with the appropriate DNA construct. To ensure against non-specific plasmid effects when comparing plasmid mixtures, pNef or pNef and pVpr or other vectors mixed with antigen-negative plasmids to adjuvant DNA concentrations as a specific control as required.

In vivo expression of Vpr by immunostaining
Eight-week-old BALB/c mice were immunized with 100 µg of Vpr-GFP expression plasmid (pcVpr-GFP), pEGF expression plasmid, control vector (pcDNA3) and saline as described above. Three days post-injection mice were sacrificed and the quadriceps muscle was removed. Muscle was cryopreserved and 0.2-µm sections were prepared for viewing. Slides were washed with PBS and stained with DAPI (0.1% in PBS; Sigma), and again washed and mounted using a fade-resistant mounting medium (Ted Pella, Redding, CA). Hematoxylin & eosin staining was performed as described (36).

CTL assay
Recombinant vaccinia viruses (vMN462, vVK1, VV:gag, vTFnef, vSC8) were obtained from the NIH AIDS Research and Reference Reagent Program, and P815 cell line was obtained from ATCC (Rockville, MD). A 5-h 51Cr-release assay was performed using vaccinia-infected targets. The effectors were stimulated for 24 h with concanavalin A (Sigma) at 2 µg/ml concentration followed by specific stimulation with vaccinia infected P815 cells, which were fixed with 0.1% gluteraldehyde for 2–3 days. A standard 51Cr-release assay was performed in which the target cells were labeled with 100 µCi/ml Na251CrO4 for 2 h and incubated with the stimulated effector splenocytes for 6 h at 37°C. CTL activity was determined at E:T ratios ranging from 50:1 to 12.5:1. Percent specific lysis was determined from the formula: 100x (experimental release - spontaneous release/maximum release - spontaneous release). Maximum release was determined by lysis of target cells in 1% Triton X-100-containing medium.

ELISA
Fifty microliters of recombinant Nef (Intracel, Cambridge, MA) or purified prostate-specific antigen (PSA) protein (Fitzgerald Industries, Concord, MA) diluted in 0.1 M carbonate– bicarbonate buffer (pH 9.5) to 2 µg/ml concentrations was adsorbed onto microtiter wells overnight at 4°C as described (3,22). Mouse sera (pre-immune and post-immune) were diluted and incubated for 1 h at 37°C, then incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (Sigma). The plates were washed and developed with 3'3'5'5' TMB buffer and the plates were read at OD450.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Vpr function in mouse cells
We examined the effect of Vpr on immune activation in vivo using a mouse model. Although Vpr is known to alter cell cycle events in human cells of different lineages, it is important to demonstrate that HIV-1 Vpr can exert similar effects in murine cells. We transfected both HeLa (human) and NIH3T3 (murine) cells with Vpr expression plasmids, and compared Vpr subcellular localization and its ability to inhibit cell proliferation. HeLa and NIH3T3 cells, maintained in DMEM containing 10% FBS, were transfected with a CMV Vpr expression plasmid or control vector plasmid. Localization of Vpr was detected by indirect immunofluorescence as described in methods. Results indicate a similar Vpr localization pattern in both HeLa and NIH3T3 cells (Fig. 1Go). In an effort to further confirm whether Vpr exhibits a similar localization pattern in mouse primary cells, we infected mouse peritoneal macrophages with HIV-1 complemented with vesicular stomatitis virus (VSV)-G-Env and stained for Vpr localization. Results demonstrate that in mouse primary cells Vpr showed a nuclear localization pattern similar to its localization in human primary cells.



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  		Fig. 1. Subcellular localization of Vpr in human and murine cells by indirect immunofluorescence assay: HeLa (Human) and NIH3T3 (mouse) cells were transfected with HIV-1 Vpr as described (35), and cells were stained with an anti-Vpr antibody followed by phycoerythrin-conjugated goat anti-rabbit secondary antibody to detect Vpr.

 
In addition to the cellular localization of Vpr in HeLa (human) and NIH3T3 (murine) cells, we have also tested the effect of Vpr on cell cycle arrest. We examined these effects (Fig. 2Go) using native or a mutant Vprs to further confirm specificity. We compared the wild-type as well as a lost of function mutant Vpr mutated at amino acid 30. The A30S substitute does not exhibit cell cycle arrest in human cells. This mutation has been previously described (34). No differences were observed in the HeLa (human) or NIH3T3 (mouse) cell lines as Vpr or mutated Vpr activities were consistent in both. The results indicate that Vpr modulates cell proliferation in both of these human and murine cell lines in a similar manner, suggesting that Vpr exerts similar effects on the basic cellular machinery of each cell type. Taken together, the localization and cell proliferation analysis in these two cell lines support the appropriateness of a murine model for the in vivo immune studies.



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  		Fig. 2. Comparison of Vpr-mediated cell cycle arrest in human and mouse cells. HeLa and NIH 3T3 cells were transfected with 10 µg of either Vpr expression construct (wild-type or A30S mutant) using the DOTAP method. Transfected cells were selected and analyzed for cell growth and cell cycle arrest by FACS analysis as described (35). Gate M1 indicates the G1 phase and M2 indicates the G2/M phase.

 
Vpr is nuclear localized in vivo in mouse tissues
Six-week-old mice were injected i.m. with 50 µg of Vpr-GFP expression plasmid, GFP expression plasmid or the control vector plasmid in quadriceps muscle. Mice were sacrificed after 72 hrs and the quadriceps muscle was removed and frozen in OCT (Sakura, Tokyo, Japan). The frozen sections were cut into 0.2-µm sections and expression of Vpr-GFP was visualized directly by fluorescence microscopy under a FITC filter. Results demonstrate that expression of Vpr is detected in the muscle. In muscle, sections Vpr-GFP showed a dramatic and distinct nuclear staining (Fig. 3AGo, b) in contrast to GFP expression, which was distributed in the cytoplasm and not similarly localized (data not shown). As expected, staining for the vector plasmid immunized mice did not show any specific staining (Fig 3AGo, d). These are the first results to indicate that in vivo expression of Vpr does result in nuclear localization in the muscle fiber of a living animal. Plasmid injection into muscle also results in transfection of a small number of dendritic cells and macrophages that migrate to the regional lymph node and activate the T cells (25). To identify the cells expressing Vpr antigen in lymph nodes further in vivo studies were performed. Direct visualization of Vpr-GFP was performed in lymph node sections obtained from pVpr-GFP inoculated mice 3 days after plasmid injection, as previously described (25,35). Vpr-GFP-expressing cells were detectable in the proximal lymph nodes. The frequency of green cells was counted and calculated (Fig. 3BGo). These data support the transport of plasmid delivered Vpr to the regional lymph nodes—a property of HIV-1 virally delivered Vpr where it is possible that Vpr could affect the host immune response.




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  		Fig. 3. Expression and localization of Vpr in vivo. (A) Mice were immunized into the right quadriceps muscle with pVpr-GFP, pGFP or pcDNA3 vector plasmid. Three days post-immunization, mice were sacrificed, and the right quadriceps muscle was frozen in OTC and sectioned. Sections were viewed directly and photographed for Vpr-GFP expression using a FITC filter. Slides from (a) and (b) represent the pVpr-GFP immunized mice muscle sections, and (c) and (d) from control vector (pCDNA 3) immunized mice muscle sections. Panels (a) and (c) represent the nuclear staining (DAPI), and panel (b) represents the pVpr-GFP immunized mice muscle sections. Note how Vpr-GFP mirrors DAPI staining. The panels are shown at x40 magnification. (B) GFP localization in the lymph nodes following DNA inoculation. Lymph nodes (popliteal and inguinal femoral) were removed 3 days post-injection from mice inoculated with saline control, pcDNA control plasmid, pEGFP plasmid and pVpr-GFP plasmid. Lymph nodes were cryopreserved and 0.2-µm sections were prepared for viewing and counting. Data shown indicate the results of four randomly chosen section planes.

 
Effect of Vpr on inflammation in vivo
Injection of antigen expression cassettes frequently results in lymphocyte infiltration at the site of injection. Ten-week-old BALB/c mice were co-immunized with a Vpr expression plasmid (pVpr) or a control plasmid along with one of two different HIV-1 antigen immunization constructs (pGag or pNef). As an example of responses observed with these antigens, pNef immunization in muscle resulted in significant infiltration at the immunization site (Fig. 4Go). In order to determine whether Vpr interferes with the trafficking and recruitment of immune cells to the inflammatory site, we analyzed muscle sections of mice immunized with pNef in the presence or absence of pVpr on day 7 as described in Methods. Previous studies have established that co-delivery of plasmids results in co-transfection of mixed plasmids into target cells in vivo (28,37). Figure 5Go shows that the number of infiltrating cells is much higher in mice immunized with pNef and control plasmid, whereas pVpr co-immunization reduced infiltration and inflammation dramatically. Similar results were observed with pGag and pVpr co-immunization (data not shown). Overall, it is evident that antigen induced inflammation is suppressed by pVpr co-immunization.



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  		Fig. 4. Immunohistochemical analysis of lymphocyte infiltration at the site of antigen expression. Frozen muscle sections from naive, pNef and control plasmid, and pNef- and pVpr-immunized mice (n = 4) were prepared 7 days post-immunization. Panel H&E represents the sections stained with hematoxylin & eosin stain for visualization of infiltrating lymphocytes. The nuclei are shown in blue and the cytoplasm is shown in red stain. Five panels were examined for each staining for each experiment. Similar results were obtained in multiple experiments.

 


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  		Fig. 5. CTL response induced by pNef or pGag-Pol in the presence or absence of pVpr co-immunization. BALB/c mice were immunized with 100 µg of pNef and control vector or 100 µg of pNef and pVpr. Splenocytes were obtained from the mice (n = 4) 2 weeks after the first and second boost, and an antigen-specific CTL assay was performed using the 6-h 51Cr-release assay (22). (A and B) Specific lysis (%) induced by pNef vaccine in the presence or absence of pVpr co-immunization, 2 weeks after first and second boost respectively. (C and D) Same as described above, except here the antigen expression plasmid is pGag-Pol instead of pNef. These studies were repeated 4 times with similar results.

 
Vpr modulates the antigen-driven CD8+-mediated CTL response
To investigate whether a correlation exists between Vpr-mediated effects on inflammation and cellular immunity, splenocytes were collected from mice co-immunized with HIV-1 antigen plasmids and Vpr plasmid, and assayed for antigen-specific CTL activity. Nef-specific CTL activity measured in pNef and pVpr co-immunized mice was suppressed significantly in comparison to mice immunized with pNef alone (Fig. 5Go). Mice immunized with pNef and control vector exhibited 37 and 53% specific lysis at a 50:1 E:T ratio after the second and third injections respectively. In contrast, mice receiving equal amounts of pNef and pVpr resulted in <17 and 19% specific lysis at a 50:1 E:T ratio after second and third immunizations respectively. Similar results were observed after co-immunization of pGag-Pol with pVpr (Fig. 5Go). This finding supports that the CD8 suppressive effect of Vpr is not dependent on any particular antigen. To further examine the specificity of the Vpr effect, we co-immunized mice with pNef and pGag-Pol and pNef and pEnv, and assayed again for Nef-specific CTL activity. Results indicate that neither pGag-Pol nor pEnv had any effect on Nef-specific CTL activity (data not shown) indicating that decreased CTL activity was mediated specifically and solely by Vpr.

The pattern of cytokine expression influences the nature and persistence of the inflammatory response. For instance, production of IFN-{gamma} and tumor necrosis factor (TNF)-{alpha} are well suited to enhance cellular immunity, whereas IL-4 and IL-10 are important for humoral immunity. We suspected an effect on cytokine production in vivo, and therefore we examined in vivo effects of Vpr on the release of the cytokines IL-4 and IFN-{gamma} from antigen-stimulated splenocytes collected from immunized mice. Figure 6Go shows that splenocytes of mice co-immunized with pVpr and pNef and stimulated with specific antigen produced significantly less IFN-{gamma} compared to mice immunized with pNef and control vector. In contrast, no change was observed in IL-4 production in either group. Mice immunized with pNef in the presence of pVpr produced 5-fold less IFN-{gamma} (19.9 pg/ml), whereas mice immunized with pNef + control vector produced 95.3 pg/ml of IFN-{gamma}. In parallel with recent in vitro studies (4) that treatment of peripheral blood mononuclear cells with Vpr suppressed production of certain cytokines (IL-2, IL-12 and TNF-{alpha}), this study provides in vivo evidence that the Vpr-mediated immunosuppressive effect is targeted in particular at Th1-mediated cellular immunity.



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  		Fig. 6. Cytokine production in splenocytes obtained from mice co-immunized with pNef in the presence and absence of pVpr. Splenocytes harvested from mice immunized with pNef with or with out pVpr were stimulated with P815 cells infected with vaccinia expressing Nef (vTFnef) for 2 days. Cell-free supernatants were collected and assayed for the production of IL-4 and IFN-{gamma} by capture ELISA following the manufacturer's instructions (Intracel).

 
Effect of Vpr on humoral responses
Since co-immunization of pVpr resulted in down-regulation of CTL responses, we also tested the effect of Vpr on humoral responses by measuring Nef-specific antibodies elicited by pNef immunization in the presence or absence of pVpr by ELISA. Interestingly, co-immunization of pVpr with pNef did not alter the Nef-specific antibody titers (Fig. 7AGo). Since pNef by itself does not induce a very high titered antibody response, we selected a plasmid encoding PSA (pPSA), which generates a significantly higher humoral response (22). We analyzed the sera from the immunized animals for the presence of PSA-specific antibodies by ELISA. The results presented in Fig. 7(B)Go show that pPSA alone or in the presence of pVpr induces similar titered antibody responses. The OD value for pPSA with vector control or pPSA with pVpr is 0.707 and 0.69 respectively at a serum dilution of 1:128, which titers accordingly with higher sera dilutions. To reconcile this result with the effects of Vpr on cellular immune cell function, we next examined antibody subsets as an indicator of the Th1 versus Th2 phenotype. The relative ratios of IgG1 to IgG2a and IgG2a to IgG1 were determined in the presence or absence of pVpr co-injection and are shown in Table 1Go. The pPSA-immunized group had an IgG2a:IgG1 ratio of 0.8. On the other hand, co-injection of pVpr decreased the relative ratio to 0.2, indicating a shift towards a Th2 response. The 4-fold reduction seen in IgG2a:IgG1 ratio with pVpr co-injection indicates that Vpr significantly affects the Th1-type response, consistent with the results of cytokine release studies showing changes in IFN-{gamma} production.



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  		Fig. 7. Effect of pVpr on humoral responses generated by different antigens. Mice (n = 4) were co-immunized with 100 µg pNef or pPSA and 100 µg of control vector or pVpr intramuscularly at day 0, and boosted again on day 14 and 28 as described (54). The sera samples were collected at 0 and 28 days post-immunization and assayed for anti-Nef- (A) and anti-PSA- (B) specific antibodies at different dilutions as described (23). The OD value of the pre-immune sera was subtracted from the post-immune sera to account for non-specific binding. These experiments were repeated 3 times with similar results.

 

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  		Table 1. Effect of Vpr on the relative ratio of IgG1 to IgG2a
 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
In this study, we investigated the role of Vpr, a virion-associated HIV-1 protein, on immune activation. HIV-1 vpr exerts significant effects on cellular proliferation, differentiation, regulation of apoptosis, modulation of cytokine production and suppression of host cell-mediated NF-{kappa}B transcription (4–6). Many of the Vpr-mediated cellular events have been observed in a wide variety of cell lineages suggesting that Vpr targets basic eukaryotic cellular pathways (38,39). Additionally, Vpr being packaged in the virion, makes this molecule likely important as an early mediator of host pathogenesis in vivo. However, viral infection and viral spread in vivo is a dynamic process that is required to occur in the presence of an active targeted host cellular immune response. This response would easily be projected to have diverse consequences for viral production in vivo. Specifically the host contains high levels of functionally active anti-HIV CTL that should effectively destroy viral factories. One important property of Vpr is inhibition of cell cycle at the G2/M phase. This is associated with an increase in viral transcription after a 24-h lag period. This window of viral production may be too late to be of benefit to a virally infected cell. The host immune response would be expected to effectively kill this cell within 2–6 h. This period allows the immune system a 4- to 8-fold window to effectively eradicate viral reservoirs prior to a Vpr effect. How the virus lengthens this window has not been previously investigated in vivo. We hypothesized that Vpr could modulate local immune effects and tip this important balance in favor of the virus rather than the host. The effect of Vpr on APC suggested one important avenue for this effect. Interestingly, HIV-1 infection in macrophages (a critical APC) and other poorly dividing APC is Vpr dependent (1–3). In addition to being the target cells for HIV-1 infection, macrophages play an important role as APC in activation of the antigen-specific immune response.

To address the effect of Vpr on host immunity in vivo, we used a DNA vaccination model system. In vivo co-immunization results in multiple plasmids being delivered and expressed together in cells in vivo (40,41). The expressed antigens are in part taken up by local professional APC including macrophages and dendritic cells through direct transfection mechanisms (25,26,28,42). APC process the antigen(s) and effectively induce specific immune response to foreign antigens (32). In this model the present studies demonstrate that expression of Vpr can effectively decrease CTL effector function of a co-expressed antigen in vivo. These data suggest that Vpr targets CTL effector function perhaps least in part by interfering with co-stimulatory molecule expression on APC. Combined with effects on cytokines, these Vpr-mediated events would compromise in particular local T cell activation, expansion and T cell survival.

Vpr effects in vivo are likely to contribute to an impairment of a localized targeted cellular immune response. Expansion of HIV-1 antigen-specific CD4+ T lymphocytes results in effective maintenance of the immune system and contributes to control of viremia (43–45). The presence of virus-specific CD8+ T cell response is essential for virus clearance in many viral infection models (44,46). Additionally, CD8+ T cells can inhibit HIV-1 replication in vitro (13,47,48). Recent evidence supports that CD8+ T cells can contribute significantly to controlling viral load in vivo (49). The reduction in the number of effector CD8+ T cells in HIV-1-infected patients has been correlated with reduced anti-viral effect and disease progression in parallel with the deterioration of the immune system (50,51). In this respect, data presented here provide in vivo evidence that CD8 effector function may be a target of HIV-1 Vpr.

Lessons learned from HIV-1+ long-term non-progressors and asymptomatic individuals suggest that the presence of a strong CTL response is correlated with slow disease progression (19,33,52,53). This report on Vpr modulating host cellular immunity and recent studies by Collins et al. (54) and Geleziunas et al. (55) on Nef support the relevance of HIV-1 accessory genes in viral pathogenesis and disease progression. Nef blocks the expression of MHC class 1 on the surface of infected cells and up-regulates Fas ligand, thereby sheltering virally infected cells from CTL-mediated destruction. However, Nef transcription is required for this effect. For this effect of Nef to be beneficial to the virus perhaps, functions must extend the window of viral replication and prevent immune destruction. Recently, Nef has been reported to be a strong chemoattractant for immune cells (31). However, such an attractant would be expected to result in immune enhancement against HIV-1. In this light it is interesting to speculate that, together, Nef by attracting targets of immune lineage and also blocking immune effector's activity and Vpr by blocking their natural ability to mount a significant anti-HIV immune response combined with a late effect of Env on CD4 Th cell function corrupt the host immune response (Fig. 4Go). This multi-pronged approach would deliver a coordinated attack on the immune system, which ultimately benefits the virus and allows viral persistence and expansion in the presence of the host immune response. This hypothesis has important implications for HIV pathogenesis. Furthermore, it will be important to investigate this effect in a relevant primate model system. Clarifying the molecular/cellular roles of the accessory genes in HIV-1 pathogenesis will likely generate novel approaches for therapy and enhance our strategies for HIV-1 vaccine development.


   Acknowledgments
 
We thank Michael Chattergoon for critically reviewing this manuscript and Tzvete Dentchev for her technical assistance in animal studies. We greatly appreciate the assistance of Dr James F. Sanzo, Biomedical Imaging core facility, University of Pennsylvania for the confocal photographs. This work was supported in parts by grants from National Institutes of Health to V. A. and L. J. M. This work was supported by grants from NIH to D. B. W. as well as the University of Pennsylvania CFAR core laboratories.


   Abbreviations
 
APC antigen-presenting cell
CTL cytotoxic T lymphocytes
Gag group-specific antigen
GFP green fluorescent protein
HRP horseradish peroxidase
Nef HIV-negative regulator factor
PSA prostate-specific antigen
TNF tumor necrosis factor
Vpr viral protein R
VSV vesicular stomatitis virus

   Notes
 
Transmitting editor: D. R. Green

Received 11 June 2001, accepted 21 September 2001.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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