Human Immunodeficiency Virus Type 1 Vpr Is a Positive Regulator of  Viral Transcription and Infectivity in Primary Human Macrophages

Isolation and Culture of Peripheral Blood–derived Macrophages.

Blood units from HIV seronegative volunteers were obtained from the Hôpital Maisonneuve Rosemont (Montreal, Canada). PBMCs were isolated by Ficoll-Paque centrifugation as recommended by the manufacturer (Pharmacia Biotech AB, Uppsala, Sweden), washed thoroughly to remove contaminating platelets, and cryopreserved. An enriched population of monocyte-derived macrophages were first isolated by adherence to plastic as previously described (5). Further purification was achieved by negative selection where the enriched macrophage population was incubated with a cocktail of monoclonal antibodies against T and B cells followed by subsequent depletion using a secondary antibody conjugated to magnetic beads (Dynal Inc., Lake Success, NY) as previously described (20). Purified macrophages were plated in 6-well plates at a concentration of 2.5 × 10⁶ cells per well and maintained in endotoxin-free RPMI 1640 media (GIBCO BRL, Gaithersburg, MD) containing 10% FCS, l-glutamine, penicillin (100 U/μl), streptomycin (100 U/μl), and gentamycin (10 μg/ml). The resulting adherent macrophages were >93% positive by nonspecific esterase test (Sigma Chemical Co., St. Louis, MO).

Proviral Constructs, Infections, and Replication Kinetics.

The previously well-characterized isogenic HxBRU based clones (21) were rendered macrophage-tropic by transferring the SalI– BamHI fragment from another well-characterized macrophage-tropic construct ADA (NLHXADA-SM), provided by Dr. Lee Ratner (University of Washington School of Medicine, St. Louis, MO) (9). The construction of the HxBRU parental clones that code for the wild-type Vpr or the Vpr-negative ATG⁻ mutant (where the Vpr ATG initiation codon has been mutated to prevent protein translation) have been previously described (21). The R62P and R80A were subcloned in this background using a two-step PCR-based method as previously described (21).

Cos-7 cells were plated at 8 × 10⁵ cells per 100-mm plate, cultured overnight, and transfected with proviral constructs by calcium phosphate method. To generate viral stocks, supernatants were collected 72 h after transfection, concentrated by ultracentrifugation at 35,000 rpm for 2.5 h, filtered through 0.45-μm filters, treated with DNase (25 U/ml) to remove contaminating plasmid DNA, and stored at −80°C. In vitro differentiated macrophages that had been in culture for 7 d were adsorbed with virus for 8 h at 37°C and were subsequently washed thoroughly to remove free virus and were maintained in RPMI 1640 complete medium containing 100 nM Palinavir (BioMega-Boehringer Ingelheim, Laval, Quebec, Canada), a previously well characterized protease inhibitor (22). Half of the medium was changed every 3–4 d with fresh media supplemented with 100 nM Palinavir. Virus production in cultures was determined by measuring supernatant p24 levels by standard ELISA techniques.

In Situ Hybridization.

For in situ hybridization purposes, purified macrophages were maintained after purification for 7 d in Lab-Tech slides (Nunc™, Naperville, IL) and subsequently infected at varying viral inputs under conditions similar to the ones used for one-step replication studies in the presence of 100 nM Palinavir. Hybridization procedures were performed using standard protocols (23, 24) using an antisense probe previously described in detail (25). This probe contains a 92-bp sequence complementary to the Sty-1 (nucleotide 7591) to HindIII (nucleotide 7683) fragment overlapping the HxBc2 envelope sequences found in the ADA chimeric clones (9). Thus, the hybridization procedure identified both full-length and singly spliced mRNAs, both of which contain the target envelope sequences. Based on earlier optimization procedures, RNA pattern was determined at 14 d after infection as all donors tested clearly showed the Vpr-mediated phenotype of augmented replication by this time.

PCR Analysis and Southern Blotting.

Total DNA lysates were prepared to monitor the efficiency of reverse transcription at different time points early in the infection as previously described (26). Alternatively, proviral levels found in the nuclear compartment were monitored as previously described (26). PCR analysis to detect HIV-1 proviral products was performed using the following primers in the Gag sequence: sense oligonucleotide 5′-ATA ATC CAC CTA TCC CAG TAG GAG AAA T-3′, and antisense oligonucleotide 5′-TTT GGT CCT TGT CTT ATG TCC AGA ATG C-3′. These primers amplified a fragment of 114 bp corresponding to the sequence 1090 to 1204 in the HxBRU strain. Alternatively, the cellular β2-adrenergic receptor (β2-AR) gene sequences were detected using the following primers: sense 5′-TAG GCC TTC AAA GAA GAC CTC C-3′, and antisense 5′-CGT CTA CTC CAG GGT CTT TCA G-3′. PCR products from this amplification generated a 399-bp fragment that corresponded to the sequence 1465 to 1821 of the human β2-AR gene. Amplification was performed using Taq DNA polymerase (Perkin Elmer, Norwalk, CT) for 30 cycles of 1 min at 94°C, 2 min at 50°C, and 3 min at 72°C. α-[³²P]dATP-labeled probes capable of recognizing the PCR products were generated as follows: HIV-1 Gag probe was generated by nick translating with α-[³²P]dATP (3,000 Ci/mmol; ICN Radiochemicals, Costa Mesa, CA), the 591-bp ApaI to PstI digestion fragment corresponding to positions 964 to 1555 in the HxBc2 provirus sequence. β2-AR probe was generated by similarly nick translating a fragment corresponding to the sequence 1465 to 1821 from a well characterized β2-AR eukaryotic expressor construct (27).

Correlation of the G2-arrest and Nuclear Localization Phenotypes of Vpr with its Ability to Establish a Productive Infection in Macrophages.

To explore what role, if any, Vpr-mediated G2-arrest and nuclear localization played during viral replication in nondividing target cells such as macrophages, we compared the replication of a virus that coded for a wild-type Vpr or no Vpr at all (ATG⁻ mutant), to that of viruses coding for mutations in the gene that selectively affected either the G2-arrest or the nuclear localization activity of the protein. For this purpose, the macrophage-tropic envelope region from the well-characterized NLHX-ADA-SM construct (9) was transferred into HIV-1 constructs that coded either for the wild-type Vpr or for a mutated version of the protein (21). The two mutations used in this study to gauge how the G2-arrest and nuclear localization phenotypes of Vpr contribute to HIV-1 replication in macrophages were chosen based on previously published reports that clearly separate these functions at the structural level (28, 29).The R62P and R80A proviruses expressed, respectively, a Vpr-mutant that substituted the Arginine residue at position 62 with a Proline and one that substituted the Arginine residue at position 80 with an Alanine. The Proline substitution mutant, R62P, retained its ability to G2-arrest cells but lost its ability to localize to the nucleus as it disrupted a helical domain shown by earlier studies to affect the nuclear localization phenotype specifically (29–32). The R80A mutant was capable of localizing to the nucleus but was impaired in its ability to growth-arrest T cells, also as previously shown (28). These observations are in agreement with a number of earlier functional studies that have characterized central helical and COOH-terminal domains of Vpr (21, 28, 29), and which are summarized in Table 1.

Whether or not Vpr is dispensable for viral replication in macrophages has been controversial as there is limited consensus on the extent to which the virus is impaired in the absence of the protein. Reports from independent groups have documented anywhere from 2-fold to close to 100-fold impairment (5, 7, 9). As some investigators have suggested that the viral input may contribute to the in vitro replication kinetics of viruses mutated in another nucleophilic determinant, matrix p17 (33), as well as viruses mutated in Nef, the accessory protein (34, 35), we tested if the input multiplicity can compensate for the Vpr-mediated augmentation of viral replication. To address this issue, we infected purified macrophages kept in culture for 7 d with the wild-type or Vpr-mutant viruses at either a high input titer (50 ng) or one third serial dilutions thereof (16.7 ng, 5.6 ng, 1.85 ng, 619 pg). One-step replication was ensured by maintaining the infected cultures in the presence of the potent protease inhibitor Palinavir (BioMega-Boehringer Ingelheim), which effectively prevented multiple rounds of infection from newly generated viruses in this system (data not shown). We have previously shown that treatment with this inhibitor results in the release of noninfectious particles into the supernatant due to a block at the virion maturation step (22). This culture system allowed us to look at a one-step viral replication without the complication of the progression of viral infection through the culture.

Our data suggests that at low viral inputs both the ability of the protein to localize to the nucleus and its ability to G2-arrest dividing cells appeared to be essential for increased viral replication in macrophages. The wild-type protein capable of both activities established a readily apparent infection even at the lowest multiplicity tested (Fig. 1). However, the R62P mutant, which lacks the nuclear localization activity, and the R80A mutant, which lacks the G2-arrest phenotype (Table 1) are both impaired at low viral inputs (Fig. 1). The ATG⁻ mutant, which lacks both these biological activities, is clearly impaired at the low multiplicities tested (Fig. 1).

However, at high viral inputs, the requirement for the nuclear localization is no longer essential for augmented replication, whereas the G2-arrest ability is still required. Note that the R80A mutant that lacks the G2-arrest activity shows impairment of replication at the high viral inputs even though it is capable of localizing to the nucleus, whereas the R62P mutant possessing the G2-arrest activity is rescued even though it fails to localize to the nucleus (Fig. 1).

Ability of the Wild-type and Mutant Vpr Proteins to Target Proviral DNA to the Nuclear Compartment.

We sought to understand if either the nuclear localization or the G2-arrest activity associated with Vpr affected the protein's ability to target the proviral DNA to the nuclear compartment. To formally address this issue, we used a semiquantitative PCR approach to monitor the total levels of HIV-1 proviral generation and the levels of provirus found in the nuclear compartment at various time points after addition of the initial viral inoculum as previously described (26). Single-step viral replication conditions identical to the ones used to monitor productive infection by p24 were used for this purpose. The total proviral synthesis was approximately the same both in the presence and absence of Vpr, indicating that neither viral entry nor reverse transcription was radically affected by the protein (data not shown), an observation that is in agreement with previous reports (11, 12). However, absence of Vpr drastically reduced the targeting of the proviral DNA to the nuclear compartment and this effect, interestingly, only became apparent at low viral inputs (Fig. 2, A and B, 1.85- and 0.619-ng lanes). It is also clear that the drastic impairment in nuclear targeting is not due to a temporal delay in transport, as nuclear proviral levels remain constant after 72 h (Fig. 2 A). However, at high viral inputs the PCR data clearly demonstrates that the proviral import function proceeds efficiently in spite of the absence of Vpr (ATG⁻), or its inability to localize to the nucleus (R62P) (Fig. 2 B). This is probably due to complementation of the nuclear import function at high viral inputs by the viral integrase protein (13) and the Gag matrix p17 product (11, 12, 36). Note, however, that at lower viral inputs Vpr was essential for efficient nuclear targeting of the provirus regardless of the presence of these complementary viral nucleophilic factors (Fig. 2 B, compare the 1.85-ng lanes). Also, note that at lower viral inputs, inability of Vpr to localize to the nucleus impairs its ability to target the proviral DNA to the nuclear compartment (compare the 1.85-ng lanes of R62P to that of wild-type).

However, an additional contribution to the augmented replication is provided by the protein's G2-arrest ability, as similar nuclear levels of proviral DNA present at higher viral inputs (Fig. 2, compare the 50-ng lanes), still lead to a nonproductive infection in the R80A and ATG⁻ cultures (Fig. 1). This clearly suggests that access to the nucleus by itself is not sufficient to ensure productive infection, and that G2-arrest ability is essential to induce productive infection after achieving efficient proviral nuclear targeting. This conclusion is underscored by the productive infection of R62P mutation once efficient targeting of its proviral DNA is achieved by the use of high viral titers (Fig. 1).

RNA Expression Pattern Among Cultures Infected with Wild-Type and Vpr-Mutant Viruses.

Though analysis of replication kinetics by monitoring supernatant p24 levels allowed us to gauge productive infection, this did not clarify if the highly reduced viral production apparent in some cultures (Fig. 1, ATG⁻ and R80A) is due to a reduction in the number of infected cells, or, alternatively, due to the same number of infected cells producing lower amounts of viruses per cell. For example, it is conceivable that a subpopulation of macrophages are susceptible to HIV-1 infection in the absence of Vpr, whereas others are not. This is a possibility that needs to be considered as it is clear that blood-derived macrophages are a heterogeneous population of cells. Also, it is conceivable that infection itself is established in a small fraction of proliferating cells as previously suggested (37). Hence, in theory, the saturation seen in the p24 assay (Fig. 1) may result from saturation of this subpopulation, after which increasing the viral input results in no further infection as there are no further susceptible cells.

To explore the issue we performed in situ hybridization in lab-tech slides under conditions identical to the ones used to monitor viral replication by p24 and proviral DNA targeting to the nucleus (Fig. 3). Hybridization procedures were performed according to standard protocols (23, 24) using an antisense probe previously described in detail (25). This probe identified both full length and singly spliced mRNAs in HIV-1 infected primary cells (20). We chose to look at two input titers in this experiment, one at the higher saturating level (50 ng) and another at the lower end (1.85 ng).

Note that the in situ evidence clearly suggests that Vpr upregulates transcription in these nondividing cells, an effect linked to its ability to induce G2-arrest: mutants lacking the Vpr-G2-arrest capability (ATG⁻ and R80A) clearly show drastic reduction in productively infected cells, even though the total number of infected cells were similar (in the range of 90%) at high viral inputs (Fig. 3 c and e, and Table 2). Interestingly, as exemplified by the R62P mutation at low viral inputs, the lack of nuclear targeting takes precedence over the ability to increase transcription as lack of proviral nuclear targeting impedes the establishment of infection (Fig. 2 B, and Table 2). However, the importance of this late transcriptional role is underscored by the lack of productive infection in the R80A mutant where close to wild-type levels of nuclear import occurs (Table 2). Also, the Vpr-negative ATG mutant still fails to show productive infection (Fig. 1 and Table 2) even after effective targeting of its proviral DNA to the nuclear compartment at high viral inputs (Fig. 2 B, 50-ng lane).

Also, both the wild-type and R62P cultures (both of which growth-arrest proliferating cells in the G2 phase) failed to change the DNA profile of these terminally differentiated macrophages, as expected. Both the uninfected and infected cultures showed <1% of the cells in the G2 phase of the cell-cycle (data not shown) even though in the range of 90% of the cells were shown to be infected (Table 2). Hence, the observed correlation between G2-arrest activity and the increased replication in macrophages is likely due to biochemical events rather than actual DNA duplication that characterize the G2-arrest status in proliferating T cells.