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The human immunodeficiency virus type 1 (HIV-1) Vpr accessory gene product is a small virion-associated protein (15 kDa) that localizes in the nucleus of infected cells (6, 21). Several functions have been attributed to the Vpr protein, including cell cycle (11) and apoptosis control (2, 8, 13, 18, 23), nuclear import of preintegration complexes (16), and transactivation of cellular and viral promoters (12, 24). The importance of Vpr for viral persistence, replication, and pathogenesis is suggested by a number of studies. Although this protein does not confer a significant viral growth advantage in primary T cells (10), its function is strictly required for viral replication in nondividing cells (3, 7, 15). However, positive effects of Vpr on HIV replication have been observed in a number of cell types as a result of its ability to delay infected cells at the G₂/M phase of the cell cycle, in which the HIV long terminal repeat (LTR) is transcriptionally more active (14, 17, 25).

We recently described the isolation and the biological properties of genetically modified Jurkat T-cell clones expressing low levels of Vpr (8). In particular, we reported that Vpr expression was responsible for a decreased response of these cells to apoptotic stimuli (8). In the present study, we investigated the kinetics of HIV-1 replication in this cell model.

Mock- and Vpr-transfected clones, collected during logarithmic growth, were infected with the HIV-1_NL432 strain at a multiplicity of infection (MOI) of 0.1 for 1 h at 37°C. Cells were then extensively washed and seeded in fresh medium at a concentration of 2 × 10⁵ cells/ml. The extent of viral replication was monitored at different time points after infection by measuring the levels of p24 antigen released in culture supernatants by enzyme-linked immunosorbent assay (DuPont-NEN, Boston, Mass.). As shown in Fig. 1A, high levels of virus production were observed in the control clone as early as 11 days after infection, with peak production at day 14 (300 ng/ml). As regards Vpr clones, significant levels of p24 release were detected in only one out of three clones (Vpr7) only at day 25 and day 28 (4.7 and 12.5 ng/ml, respectively). In this clone, viral release was more than 20-fold lower than that observed in the control clone at the peak of replication. Only barely detectable levels of p24 were found in Vpr10 cells at late times of infection (30 pg/ml), whereas the Vpr4 clone was completely protected at all times (<12.5 pg/ml). This strong inhibition of HIV replication was still observed 2 months after viral challenge (data not shown). To investigate whether the restriction of virus replication in Vpr-expressing cells was associated with a decreased proviral DNA synthesis, the amount of fully reverse-transcribed HIV DNA was assessed by PCR at 24 h postinfection by using the primer pair LTR/gag, which detects only full-length or nearly completely synthesized viral DNA (26). As shown in Fig. 1B, a marked reduction in the number of proviral DNA copies was found in Vpr-expressing clones with respect to control cells. Likewise, the levels of HIV proteins started to be detected only at late times postinfection (day 21) and remained significantly low (day 28) in Vpr-expressing cells (Fig. 1C). Notably, a strong inhibition of proviral DNA and viral protein synthesis as well as of p24 release was still observed at an MOI of 1 (data not shown). To establish whether the restriction to HIV infection exhibited by Vpr transfectants was related to some changes in the expression of molecules involved in HIV entry, surface expression of CD4 was analyzed by flow cytometry after incubation of cells with fluorescein isothiocyanate-conjugated CD4 monoclonal antibody (125 ng/sample; Becton Dickinson, Mountain View, Calif.). As shown in Fig. 2A, there was a marked reduction in the expression of surface CD4 in all Vpr-expressing clones as compared to that of control cells. In contrast, comparable levels of CXCR4 were detected in all the clones analyzed independently of Vpr (data not shown). In spite of the significant reduction in surface expression of CD4, the total CD4 content was not modified in the presence of Vpr (Fig. 2B). The reduction of surface CD4 was directly related to Vpr expression, since culture of Vpr-expressing clones in the presence of antisense oligodeoxynucleotides (3, 8) completely restored the surface CD4 content to levels comparable to those of control clones (Fig. 2C).

View larger version (33K): [in this window] [in a new window]   FIG. 1.   Analysis of HIV-1 replication in Vpr-expressing clones. (A) HIV-1 p24 release in the supernatants of infected cultures at the indicated time points after infection. (B) Analysis of full-length proviral DNA (LTR/gag) at 24 h postinfection. As a control, DNA from the CMV2 clone infected with heat-inactivated virus () was used. The standard curve was obtained by amplifying DNA from the indicated number of 8E5 cells. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (C) Immunostaining of whole-cell extracts from infected clones subjected to sodium dodecyl sulfate-14% polyacrylamide gel electrophoresis by human sera containing antibodies to HIV-1 (DuPont-NEN) at 7, 11, 14, 21, 25, and 28 days postinfection (lanes 1 through 6, respectively). Results shown in panels A through C were obtained in three separate experiments.

View larger version (20K): [in this window] [in a new window]   FIG. 2.   Analysis of CD4 expression in Vpr-expressing cells. Surface (A) and intracellular (B) CD4 expression was evaluated in mock- and Vpr-transfected clones. Intact (A) or permeated (B) cells were stained with a fluorescein isothiocyanate-conjugated anti-CD4 monoclonal antibody and analyzed by FACS. (C) Mock- and Vpr-transfected clones were cultured in the presence or in the absence of antisense or control sense phosphorothioate oligodeoxynucleotides targeted to Vpr (15 µM). After 72 h, surface CD4 expression was evaluated. Results from two representative clones are shown in panels B and C.

To verify whether the down-modulation of CD4 could be reproduced during the course of an acute HIV-1 infection, we infected Jurkat parental cells with either a wild-type (wt) or a Vpr-mutant (Af2) HIV-1_NL432 carrying a highly unstable truncated Vpr protein (20), and the expression of surface CD4 was monitored by fluorescence-activated cell sorter (FACS) analysis in both infected and uninfected control cultures at different time points. As shown in Fig. 3 (upper panel), a significant reduction in the levels of surface CD4 was observed 48 h postinfection in cultures infected with the wt HIV-1 compared to the uninfected cultures kept under the same experimental conditions. Interestingly, CD4 expression was not down-modulated in cultures infected with Af2 HIV-1 (Fig. 3, upper panel). At this time point, the percentage of infected cells, assessed by staining cells with anti-HIV-1 p24 monoclonal antibody (1:20; Coulter Corporation, Miami, Fla.) was identical in cultures infected with either wt or Af2 viruses (Fig. 3, middle panel), indicating that the reduction of CD4 expression observed in wt-virus-infected cultures was not due to a higher efficiency of infection. Notably, single-cell analysis aimed at simultaneously identifying productively infected cells (p24 positive) and CD4 expression clearly indicated that the down-modulation of CD4 predominantly occurred in infected cells (Fig. 3, middle panel). Furthermore, when the analysis was restricted to p24-positive cells, a more marked Vpr-dependent down-modulation of CD4 was detected (Fig. 3, lower panel). At later times postinfection (day 7), a pronounced reduction in surface CD4 expression was observed independently of Vpr expression (Fig. 3, upper panel).

View larger version (48K): [in this window] [in a new window]   FIG. 3.   Analysis of surface CD4 expression during HIV-1 infection with wt and Vpr-mutant HIV-1. Parental Jurkat cells were infected with wt or Af2 HIV-1NL432 at an MOI of 1. (Upper panels) The expression of surface CD4 was monitored at different time points after infection by FACS analysis. (Middle panels) Double fluorescence analysis of surface CD4 molecule and intracellular p24 was performed at 48 h postinfection. Region b shows the percentage of p24-positive cells detected in wt and Af2 HIV-1NL432-infected cultures. Controls were uninfected cultures. (Lower panels) Surface CD4 expression was specifically analyzed in p24-positive cells (region b shown in middle panels). In the left panel, the median values of flow cytometric CD4 analysis obtained from three different experiments plus standard errors are reported. A highly significant difference (P < 0.001 in Student t test analysis) was found between CD4 expression in control and wt-infected cultures at 48 h postinfection.

To specifically address the question of whether the restriction to HIV-1 infection observed in Vpr-expressing clones was due to a CD4-mediated block of virus entry, experiments were carried out by using HIV-1 virions pseudotyped with the G protein of vesicular stomatitis virus (VSV). As shown in Fig. 4, a comparable number of p24-positive cells was detected at 48 h postinfection in control and Vpr-expressing cells infected with VSV G protein-pseudotyped HIV-1 virions. As expected, productive infection with wt HIV-1 was observed in control clones but not in Vpr-expressing clones (Fig. 4).

View larger version (26K): [in this window] [in a new window]   FIG. 4.   Detection of VSV G protein-pseudotyped HIV-1 replication by p24 expression. Mock- and Vpr-transfected Jurkat cells were infected with wt and VSV G protein-pseudotyped HIV-1 at an MOI of 1. The extent of viral replication was monitored by FACS analysis of intracellular p24 expression at 48 h postinfection. The results of one representative experiment out of three are shown.

Our results clearly indicate that Vpr expression in Jurkat cells before HIV infection results in a strong restriction to viral replication which occurs at the level of viral entry and involves the down-modulation of surface CD4 receptors. The CD4 steady-state levels are not affected by Vpr expression, suggesting that its maturation and/or intracellular trafficking is somehow impaired. Interestingly, a Vpr-mediated down-modulation of CD4 was also observed during the course of HIV infection. To the best of our knowledge, our study is the first to describe a Vpr-induced reduction in the expression of cell surface CD4. Although different HIV proteins (i.e., gp160-Env, Vpu, and Nef) have been involved in the down-regulation of CD4 (1, 4, 5, 22), the redundant function of four HIV genes (including vpr) is probably required for ensuring the complete suppression of CD4 receptors at all stages of viral infection.

A number of studies have shown that Vpr, imported by the virion (17), newly synthesized (14, 25), or exogenously added (19), can positively regulate HIV replication in different cell types. In this report, we studied the effect of Vpr, constitutively expressed before HIV infection, on the susceptibility of Jurkat T cells to viral replication. Our results, as well as data from the literature, suggest that Vpr can differently affect HIV replication, either by acting directly on specific steps of the virus life cycle or by modifying cellular processes involved in the control of infection. We have previously reported that Vpr-expressing cells are less susceptible to apoptosis induced by different stimuli (8). Moreover, we have recently shown that Vpr can exert an antiapoptotic function at early stages of infection (9). On the basis of these results, we speculate that in HIV-infected cells Vpr acts as an early factor in determining an impairment in apoptosis susceptibility as well as in the CD4-mediated immune recognition. Similar effects could also result from the interaction of Vpr, released in the extracellular milieu at sites of infection, with bystander uninfected cells. This could lead to an altered response of activated T cells to antigens, thus contributing to the pathogenesis of AIDS.