Human Immunodeficiency Virus Type 1 Modified To Package Simian Immunodeficiency Virus Vpx Efficiently Infects Macrophages and Dendritic Cells^,

Abstract

The lentiviral accessory protein Vpx is thought to facilitate the infection of macrophages and dendritic cells by counteracting an unidentified host restriction factor. Although human immunodeficiency virus type 1 (HIV-1) does not encode Vpx, the accessory protein can be provided to monocyte-derived macrophages (MDM) and monocyte-derived dendritic cells (MDDC) in virus-like particles, dramatically enhancing their susceptibility to HIV-1. Vpx and the related accessory protein Vpr are packaged into virions through a virus-specific interaction with the p6 carboxy-terminal domain of Gag. We localized the minimal Vpx packaging motif of simian immunodeficiency virus SIVmac₂₃₉ p6 to a 10-amino-acid motif and introduced this sequence into an infectious HIV-1 provirus. The chimeric virus packaged Vpx that was provided in trans and was substantially more infectious on MDDC and MDM than the wild-type virus. We further modified the virus by introducing the Vpx coding sequence in place of nef. The resulting virus produced Vpx and replicated efficiently in MDDC and MDM. The virus also induced a potent type I interferon response in MDDC. In a coculture system, the Vpx-containing HIV-1 was more efficiently transmitted from MDDC to T cells. These findings suggest that in vivo, Vpx may facilitate transmission of the virus from dendritic cells to T cells. In addition, the chimeric virus could be used to design dendritic cell vaccines that induce an enhanced innate immune response. This approach could also be useful in the design of lentiviral vectors that transduce these relatively resistant cells.

INTRODUCTION

Lentiviruses encode a set of accessory proteins (Nef, Vif, Vpr, Vpu, and Vpx) that play specific roles in virus replication and pathogenesis, several of which are directed at evasion of the host adaptive or innate immune response (32). Specifically, Nef downregulates major histocompatibility complex proteins to interfere with antigen presentation; Vif counteracts the APOBEC3 cytidine deaminases to protect the virus genome; and Vpu counteracts BST-2/tetherin to allow virus release. Although the roles of Vpx and Vpr are less well understood, they have been proposed to aid in evasion of an unidentified restriction to virus replication in macrophages and dendritic cells (17, 20, 40). Sharing significant sequence and structural homology, the two proteins appear to have arisen from the duplication of a common ancestral precursor gene (31, 42). Both are produced late in the viral life cycle and localize to the nucleus (3, 9, 14, 30). While all of the known lentiviruses encode Vpr, Vpx is restricted to viruses of the human immunodeficiency virus type 2 (HIV-2), simian immunodeficiency virus (SIV) of sooty mangabey (SIVsm), SIV of red-capped mangabey (SIVrcm), and SIV of macaque (SIVmac) lineages and is absent from HIV-1. Interestingly, SIV of African green monkey (SIVagm) encodes only Vpr, but this protein has some characteristics of Vpx, sharing the same virion packaging determinant and the same effect on virus replication in macrophages (1, 6).

Vpr and Vpx are unique among the lentivirus nonstructural proteins in that they are present in significant quantities in the virion (23, 25, 46). Both proteins are packaged into the assembling virion through a specific interaction with the carboxy-terminal Gag protein, p6. Deletion of the entire p6 region or of specific sequence motifs within p6 prevents packaging (30, 37, 44). The packaging of Vpr and Vpx is also virus specific (25, 29). HIV-1 will not package SIVmac Vpr or Vpx, nor will SIVmac package HIV-1 Vpr. By analyzing viruses with mutated or truncated p6, several groups have identified amino acid motifs in p6 that mediate Vpr and Vpx incorporation. In an analysis of the packaging determinants of HIV-1 Vpr, Kondo and Göttlinger identified the sequence ⁴¹LXXLF⁴⁵, near the carboxy terminus of p6 (28). Subsequently, Zhu et al. reported that removal of that motif did not prevent Vpr packaging (48). Instead, in a virus in which p6 residues 35 to 52 are deleted, the ¹⁵FRFG¹⁸ sequence near the amino terminus was required. In a study to identify the packaging determinant of SIVmac Vpx, Accola et al. mapped a conserved ¹⁷DXAXXLL²³ motif near the amino-terminal region of p6 (1). They further showed that the packaging of SIVagm Vpr was also dependent on this motif.

Vpr and Vpx are important for virus replication and pathogenicity in vivo, as demonstrated in the rhesus macaque model, where SIVmac₂₃₉ with Vpx deleted is attenuated and SIVmac₂₃₉ with both Vpx and Vpr deleted is further impaired (16). In vitro, neither Vpr nor Vpx is required for virus replication in activated CD4 T cells (2, 17, 18). However, in monocyte-derived macrophages (MDM) and monocyte-derived dendritic cells (MDDC), both proteins enhance virus infection, although the effect of Vpr is more modest (17, 20, 21, 24). The presence of Vpx and Vpr in the virion suggests that they play a role early in virus replication. Both proteins were initially proposed to facilitate nuclear import of the preintegration complex in the infection of nondividing cells (22). However, this view was challenged by the finding that HIV-1 with Vpr deleted maintained its ability to infect nondividing cells (45). More recently, Vpx and Vpr have been suggested to counteract an unidentified restriction factor in MDM and MDDC (17, 20, 40). Both proteins associate with an E3 ubiquitin ligase composed of damaged DNA binding protein 1 (DDB1), DDB1 cullin-associated factor 1 (DCAF1), and cullin 4A (Cul4A) (4, 20, 26, 39–41). This finding suggested, by analogy with Vif, that Vpx and Vpr might act as substrate receptors that induce the ubiquitination of a host restriction factor. Further evidence that Vpx plays a role in counteracting an MDM-specific host restriction was provided by somatic cell fusion experiments in which heterokaryons formed between MDM and COS cells were found to be nonpermissive for SIVmac with Vpx deleted (40). Moreover, domain mapping of Vpx localized an activation domain at the amino terminus that might serve as a binding site for the putative host restriction factor (20).

Goujon et al. found that Vpx, when introduced into the target cell in trans, could facilitate the infection of MDDC (17, 19). For this study, MDDC were first exposed to virus-like particles (VLP) that contained Vpx and were then infected with SIVmac from which Vpx had been deleted. The VLP treatment dramatically enhanced the infectibility of these cells. These findings were confirmed by Gramberg et al., who found that VLP generated with a codon-optimized Vpx expression vector boosted the infection of MDM by 100-fold, while Vpr had little effect (20). Measurement of the numbers of viral reverse transcription products generated in newly infected MDM and MDDC suggested that Vpx acts postentry, relieving a block to the reverse transcription or uncoating of SIV (12, 19, 20, 41). Although HIV-1 does not encode Vpx, the virus was found to be highly susceptible to its effects: pretreatment of MDM and MDDC with Vpx-containing VLP dramatically enhanced HIV-1 infection (18, 20). Recently, Manel et al. used Vpx-containing VLP to achieve high levels of HIV-1-infected MDDC (33). The infected MDDC strongly induced innate immune defenses, resulting in the production of type I interferon (IFN) and the upregulation of CD86. Induction of this response was not due to the incoming virus but rather to the newly produced Gag protein triggering an unidentified intracellular sensor.

Here we investigated the role of Vpx in virus replication using HIV-1 modified by the introduction of the Vpx packaging determinant of SIVmac p6. The resulting virus efficiently packaged SIVmac Vpx and, as a result, was highly infectious on MDM and MDDC, obviating the need for pretreatment of the target cells with Vpx-containing VLP. The virus induced high levels of type I IFN in infected MDDC. In an MDDC–T cell transfer assay, Vpx-containing HIV-1 was more efficiently transmitted from MDDC to activated T cells than was wild-type virus. These findings suggest a new role for Vpx in virus replication. In addition, the modified virus may be useful for the production of vaccines that stimulate innate immune responses and for the generation of lentiviral vectors that more efficiently infect MDM and MDDC.

MATERIALS AND METHODS

Cells and cell culture.

293T cells were cultured in Dulbecco's modified Eagle's medium (DMEM)–10% fetal bovine serum (FBS). MDM, MDDC, and T cells were cultured in RPMI 1640–5% human AB serum. Peripheral blood mononuclear cells (PBMC) were purified from normal human donor blood by use of a Ficoll density gradient. Monocytes were purified from healthy-donor PBMC by adherence to plastic or by positive selection on anti-CD14-coated magnetic beads (Miltenyi Biotec Inc.). Bead-purified monocytes were typically >98% CD14⁺. The monocytes were differentiated to MDM for 4 to 6 days in a medium containing 50 ng/ml granulocyte-macrophage colony-stimulating factor (GM-CSF; Invitrogen Inc.). MDDC were generated by culturing the monocytes for 5 to 6 days in a medium containing 50 ng/ml GM-CSF and 100 ng/ml interleukin 4 (IL-4) (R&D Systems). Autologous T cells were obtained from the CD14⁻ fraction by positive selection on anti-CD4-conjugated magnetic beads (Miltenyi Biotec Inc.) and were activated by the addition of anti-CD3/anti-CD28 beads (Invitrogen). The cells were cultured in a medium containing 10 ng/ml IL-2 (R&D Systems).

Plasmids.

Codon-optimized SIVmac₂₃₉ and HIV-2_rod Vpx and SIVagm Vpr expression vectors were generated by overlapping PCR (see Fig. S1 in the supplemental material). The amplicons were cloned into pcDNA6/myc-His (Invitrogen Inc.) at the EcoRI and XhoI sites, placing a Myc-6xHis epitope tag at the carboxy terminus. HIV-1 p6 chimeras and point mutants were generated in the pNL4-3-based luciferase reporter virus pNL.Luc3. An ApaI-to-EcoRI subclone containing the pNL4-3 p6 region was subcloned into pBS-KS+ (Fermentas Life Sciences) that had been modified to remove the PstI in the multiple cloning site. Mutations were introduced by overlapping PCR, and the mutated fragment was cloned back into the subcloned fragment in pBS-KS+ at the ApaI and PstI sites. The mutant fragment was then transferred into pNL4-3 at the ApaI and EcoRI sites. vpr and vpx were cloned into the nef position of NL.Luc3 by removing the luciferase gene with NotI and XhoI and replacing it with codon-optimized vpr and vpx amplicons that had been amplified with primers containing NotI and SalI sites.

Virus preparation and infections.

HIV-1 and SIVmac₂₃₉ luciferase reporter viruses were generated as described previously (7, 35). Briefly, to produce trans-complemented reporter viruses, 293T cells were cotransfected using Lipofectamine 2000 with pNL-Luc3-E⁻R⁻, pcVSV-G, and pcVpr.myc, pcVpx.myc, or pcDNA at a mass ratio of 2:1:1. Supernatants were harvested 48 h posttransfection, passed through a 0.45-μm-pore-size filter, aliquoted, and frozen at −80°C. The infectivity of the luciferase reporter virus was normalized for luciferase activity on 293T cells by infecting 2.0 × 10⁴ cells with 50 μl of virus. Three days later, luciferase activity was measured using Steadylite HTS reagents (Perkin-Elmer). The viruses typically yielded 1 × 10⁶ to 3 × 10⁶ cps per 50 μl. The p24 content of virus-containing supernatants was quantified by enzyme-linked immunosorbent assays (ELISA) using commercially available capture and sandwich antibodies (Aalto Bio Reagents, Ltd.). To determine the multiplicity of infection (MOI), 1.0 × 10⁵ ghost cells were infected with 10 μl, 25 μl, 50 μl, or 100 μl of virus-containing supernatant. After 72 h, the number of green fluorescent protein-positive (GFP⁺) cells was determined by flow cytometry.

To determine the infectivities of the chimeric luciferase reporter viruses, 1.0 × 10⁵ MDM and 1.25 × 10⁵ MDDC were seeded in a 96-well plate and were then infected in triplicate with a luciferase reporter virus (3.0 × 10⁵ cps). MDM were spin-infected with the reporter virus for 2 h at 500 × g. For intracellular p24 staining, 3.0 × 10⁵ MDM and 3.0 × 10⁵ MDDC were infected with 20 ng p24. For infections in which Vpx was produced in cis, 1.25 × 10⁵ MDDC were infected with 50 ng p24. To control for input virus, 25 μM zidovudine (AZT) (AIDS Research and Reference Reagent Program, NIH) was added to one well prior to infection with the p6 chimeric virus encoding Vpx. To limit replication to a single round, 3 μM nelfinavir (AIDS Research and Reference Reagent Program, NIH) was added to the samples 6 h postinfection. The cells were then fixed, permeabilized, and stained. For MDDC–T cell coculture infections, 2.5 × 10⁵ or 1.0 × 10⁵ MDDC were seeded in a 96-well culture plate. The next day, the cells were infected with 50 ng or 25 ng p24, respectively. After 6 h, the cells were washed three times with medium. After 48 h, autologous CD3/CD28-activated T cells (1.0 × 10⁶ or 4.0 × 10⁵) were added, resulting in an MDDC/T cell ratio of 1:4. To determine virus replication in T cells in the absence of MDDC, 4.0 × 10⁵ CD3/CD28-activated T cells were infected with 25 ng p24. After 6 h, the medium was changed. The supernatant was collected at 3, 5, 7, and 10 days or 3, 5, 7, 9, and 14 days postcoculture, and p24 was then quantified by ELISA. To detect intracellular p24, the cells were collected at 3 days postcoculture, fixed, permeabilized, and stained as described below.

qRT-PCR.

To quantify HIV-1 reverse transcripts, 3.0 × 10⁵ MDM were infected with 5 ng p24. To remove contaminating DNA, the viruses were treated for 1 h with 50 U/ml of Benzonase (Invitrogen, Inc.). To the sample infected with the p6 chimeric virus encoding Vpx, 25 μM AZT was added prior to infection in order to control for residual plasmid DNA. After 24 h and 48 h, DNA was isolated (Qiagen). Late reverse transcripts and two-long-terminal-repeat (2-LTR) circles were quantified by quantitative real-time PCR (qRT-PCR) using 250 ng of the DNA template as described previously (5, 20). The PCR products were detected with SYBR green (Applied Biosystems) with an ABI Prism 7300 system (Applied Biosystems). Absolute copy numbers were determined by normalization to standard curves generated from a serially diluted proviral plasmid and a 2-LTR plasmid.

IFN-β1 mRNA was quantified by qRT-PCR. For this purpose, 5 × 10⁵ MDDC were infected with 50 ng p24. To two samples, 25 μM AZT or 10 μM raltegravir (Merck) was added prior to infection. After 24 h and 48 h, the cultures were lysed, and RNA was isolated using Trizol (Invitrogen Inc.). cDNA was generated using Transcriptor reverse transcriptase (Roche) primed with oligo(dT). IFN-β1 and glucose-6-phosphate dehydrogenase (G6PDH) were then amplified by RT-PCR using primers previously described by Di Domizio et al. (8). The relative threshold cycle (C_T) value for IFN-β1 was normalized to that for G6PDH.

Immunoblot analysis.

Virions were harvested from culture supernatants 2 days posttransfection, filtered, pelleted through 20% sucrose at 100,000 × g for 20 min at 4°C, and then lysed in a buffer containing 0.5% NP-40. Cellular and virus proteins were analyzed by immunoblotting as described previously (20). The filters were probed with anti-myc monoclonal antibody (MAb) 9E10 (Covance), anti-p24 MAb 183-H12-SC (AIDS Research and Reference Reagent Program, NIH), and anti-α-tubulin (Sigma). The filters were then hybridized with biotinylated goat anti-mouse immunoglobulin and with streptavidin labeled with DyLight 680 or DyLight 800 (Pierce); they were then imaged on an Odyssey infrared imaging system (Li-Cor) at 700 or 800 nm, respectively.

Intracellular p24 staining.

Infected cells were removed from culture dishes by using phosphate-buffered saline (PBS) containing 5.0 mM EDTA, fixed, permeabilized with BD Cytofix/Cytoperm solution (BD Pharmingen), and washed with BD Perm/Wash buffer (BD Pharmingen) according to the manufacturer's instructions. The cells were then stained with a 1:200 dilution of either phycoerythrin (PE)- or fluorescein isothiocyanate (FITC)-conjugated anti-p24 MAb KC57 (Beckman Coulter) and were analyzed by flow cytometry using FlowJo software. The cells were gated for forward and side scatter and were analyzed for PE or FITC fluorescence with mock-infected cells as a negative control.

For analysis of the cells in the MDDC–T cell coculture infections, the cells in the cocultures were harvested and stained with a 1:25 dilution of allophycocyanin (APC)-conjugated CD11c (BD Pharmingen). The cells were then fixed, permeabilized, and stained with an FITC-conjugated anti-p24 MAb. T cells and MDDC were gated on CD11c, and intracellular p24 was detected on the FITC channel. Mock-infected cells were used as a negative control.

RESULTS

Mapping of the Vpx packaging determinant in Gag.

Vpx is packaged as the virus is assembled at the plasma membrane through an interaction with the carboxy-terminal domain of the Gag polyprotein precursor p6. The interaction is virus specific, such that Vpx is packaged by SIVmac₂₃₉ but not by HIV-1 (1, 25, 44). The p6 domain of Gag is structured as two α-helices in which the PTAPP late-domain motif that binds TSG101 is adjacent to helix 1 and the ALIX binding motif overlaps helix 2 (10, 47) (Fig. 1A). The Vpx packaging motif in p6 was mapped by Accola et al. to the amino acid sequence ¹⁷DPAVDLL²³, just carboxy-terminal to the PTAPP late domain (1). As a first step in engineering an HIV-1 variant that would package SIVmac₂₃₉ Vpx, we determined the minimal SIVmac₂₃₉ p6 amino acid sequence that could be introduced into HIV-1 to allow Vpx packaging. For this purpose, we generated chimeric HIV-1 genomes 17-23(a), 17-23(b), 17-28, 17-38, and 17-48, in which the indicated region of SIVmac₂₃₉ p6 was transferred to HIV-1 p6 (Fig. 1A). In chimera 17-23(a), the SIV sequence was displaced by six codons in order to preserve HIV-1 p6 amino acids 15 to 18, which contain the ¹⁵FRFG¹⁸ motif previously reported to play a role in Vpr packaging (48). To determine the ability of the chimeric viruses to package Vpx, we cotransfected 293T cells with the chimeric proviral DNA and two different amounts of the myc-tagged SIVmac₂₃₉ Vpx expression vector pcVpx.myc. After 2 days, the culture supernatant was harvested and the virions were pelleted by ultracentrifugation and were analyzed by immunoblotting (Fig. 1B). The results showed that chimeras 17-23(a) and 17-23(b) failed to package Vpx but that the addition of five more amino acids from SIVmac₂₃₉ p6 in chimera 17-28 enabled the virus to package Vpx. Extension of the chimeric region in chimeras 17-38 and 17-48 did not further increase Vpx packaging with either of the two amounts of cotransfected pcVpx.myc. The failure of the 17-23 chimera to package Vpx was unexpected, since this chimera contained the previously described ¹⁷DPAVDLL²³ motif, suggesting that the packaging motif extends further toward the carboxy terminus.

Fig. 1.

Identification of the minimal Vpx packaging motif in SIVmac p6 by transfer into HIV-1 p6. (A) Alignment of the NL4-3 and SIVmac₂₃₉ p6 amino acid sequences. The two proposed α-helices (α1 and α2) are shaded, and the PTAPP late domain and TSG101 binding site are boldface and underlined. Binding sites for ALIX and Vpx are also boldface and underlined. Below the alignment are diagrams of the HIV-1/SIVmac p6 chimeras. Open rectangle, HIV-1 sequence; filled rectangle, SIV sequence. The SIV sequence is inserted at position 14 of HIV-1 p6, except in chimera 17-23(a), where the Vpx binding motif is displaced to position 21 in order not to alter the ¹⁵FRFG¹⁸ Vpr packaging motif. (B) Immunoblot analysis shows packaging of SIVmac₂₃₉ Vpx in chimeras containing the Vpx packaging motif. 293T cells were cotransfected with pNL4-3 containing wild-type (WT) or chimeric p6 and either pcVpx.myc or an empty vector. Two days later, cell lysates and virions were pelleted by ultracentrifugation and were analyzed by immunoblotting. The immunoblot was probed with an antibody to myc-tagged Vpx, HIV-1 p24 CA, or tubulin. Transfection with pcVpx-myc alone was included in order to rule out nonspecific release of Vpx. (C) HIV-1 p6 chimeras that map amino acids required for Vpx packaging. Virions were prepared by transfection and were analyzed by immunoblotting as for panel B. (D) Effects of p6 mutations on packaging of HIV-1 Vpr. 293T cells were cotransfected with wild-type or chimeric pNL4-3 p6 and either pcVpr.myc or an empty vector. The cell lysate and virions were analyzed by immunoblotting as for panel B.

To more precisely define the Vpx packaging motif, we generated chimeras 17-24, 17-25, 17-26, and 17-27, in which the chimeric region was increased in single-amino-acid increments from positions 24 to 27 (Fig. 1C). Immunoblot analysis of virions derived from these chimeric genomes showed that the addition of SIVmac₂₃₉ p6 amino acids 24 and 25 did not allow for Vpx packaging. When the chimeric region was extended to position 26, packaging was restored. We concluded that the minimal Vpx packaging motif required for efficient packaging of SIVmac₂₃₉ Vpx into HIV-1 virions is ¹⁷DPAVDLLKNY²⁶.

Mapping of the Vpr packaging determinant in HIV-1 Gag.

Because p6 also contains the packaging determinant for Vpr, it was possible that alteration of this region in the chimeric virus would affect its packaging. Previous reports had mapped the Vpr packaging motif to two different locations. Kondo and Göttlinger mapped the Vpr packaging determinant in HIV-1 and SIVmac₂₃₉ to the ⁴¹LXXLF⁴⁵ motif near the carboxy terminus of p6, overlapping with the ALIX binding motif (Fig. 2A, motif 2) (28). Subsequently, Zhu et al. found that p6 with motif 2 deleted retained the ability to package Vpr but that the ¹⁵FRFG¹⁸ motif (Fig. 2A, motif 1), near the N terminus of p6, was required (48). To evaluate the roles of ¹⁵FRFG¹⁸ and ⁴¹LXXLF⁴⁵ (motifs 1 and 2, respectively) in Vpr packaging, we generated point mutants in both motifs of p6 and tested the virions for Vpr packaging. In the analysis of motif 1, we found that the F15A and F17A mutations decreased packaging by about 80%, while the R16A and G18A mutants were similar to the wild type (Fig. 2B). Mutation of the entire ¹⁵FRFG¹⁸ motif to alanine (M1A) did not further reduce the amount of Vpr packaged. In the case of motif 2, we found that single mutations to alanine had little effect (Fig. 2C). Although L38-mutated virions appeared to contain a reduced amount of Vpr, the mutant produced fewer virions, suggesting that the Vpr content was not affected. Mutation of amino acid S43 or F45 caused an apparent increase in the amount of Vpr packaged. These two mutant virions were defective in Gag processing, presumably because of the proximity of the amino acid residues to the proteolytic processing site. Such unprocessed virions may be more stable than wild-type virions and may therefore bind more Vpr (27). Although single amino acid mutations in motif 2 had little effect, mutation of the four conserved leucines (M2Aa) blocked Vpr packaging, confirming the importance of the motif in Vpr packaging. Combinations of motif 1 and motif 2 mutations (M1 and 2a; M1 and 2b) also prevented Vpr packaging. We concluded that both motifs play roles in Vpr packaging.

Fig. 2.

Relative contributions of the two proposed Vpr packaging motifs of HIV-1. (A) Sequences of the two reported Vpr packaging motifs of HIV-1 p6. The two motifs are underlined and boldface. (B) Immunoblot analysis of Vpr packaged by virions with mutations in motif 1. Virions were generated by transfection of 293T cells with pNL4-3 containing wild-type (WT) or mutant p6 and either a pcVpr.myc expression vector or an empty vector. The virions were pelleted from the culture supernatants by ultracentrifugation and were analyzed on an immunoblot probed with an antibody to myc-tagged Vpr, HIV-1 CA p24, or tubulin. (C) Immunoblot analysis of Vpr packaged by virions with mutations in motif 2. Mutant virions were prepared as for panel B. In the M1A mutant, F15, R16, F17, and G18 in motif 1 are mutated to alanine. In the M2Aa mutant, L35, L38, L41, and L44 in motif 2 are mutated to alanine. M2Ab is M2Aa with the additional mutations of S40, S43, and F45 to alanine. “M1 and 2a” is the combination of M1A and M2Aa. “M1 and 2b” is the combination of M1A and M2Ab.

Having identified both motifs as important, we tested the ability of the p6 chimeric virions to package Vpr. We found that chimera 17-23a, in which the ¹⁵FRFG¹⁸ motif is intact, maintained its ability to package Vpr, while the other chimeras, in which the ¹⁵FRFG¹⁸ motif was altered, packaged about one-third as much Vpr (Fig. 1D; see also Fig. S2 in the supplemental material). The 17-28 chimera reproducibly packaged Vpr somewhat better than the other chimeras (70% of wild-type packaging), perhaps due to a conformational effect on motif 2.

An HIV-1 p6 chimera that contains SIVmac Vpx efficiently infects MDDC and MDM.

The infectivity of HIV-1 on MDM and MDDC can be dramatically enhanced by pretreatment with Vpx-containing VLP (17, 20). This finding predicts that HIV-1 virions that package Vpx should have increased infectivity on these cells. To determine whether this is the case, we tested the ability of the 17-26 p6 chimera, which contains the minimal Vpx packaging sequence, to infect MDDC and MDM. For this purpose, we generated chimeric and wild-type luciferase reporter viruses as vesicular stomatitis virus glycoprotein (VSV-G) pseudotypes in 293T cells cotransfected with increasing amounts of pcVpx.myc. The viruses were normalized for infectivity on 293T cells and were then used to infect MDDC and MDM prepared from three donors. Immunoblot analysis of the virions showed that the wild-type virions contained only a small amount of Vpx over the Vpx expression vector titration. In contrast, the p6 chimeric virions packaged Vpx in proportion to the amount of pcVpx.myc transfected (Fig. 3A). On MDDC, the wild-type virus that lacked Vpx was poorly infectious. Complementation of this virus with Vpx slightly enhanced infection by this virus, suggesting that the small amount of packaged Vpx had been sufficient to boost its infectivity (Fig. 3B). The p6 chimeric virus complemented with increasing amounts of Vpx became even more infectious. The smallest amount of pcVpx.myc used, which produced virions with barely detectable Vpx, enhanced the infectivity of the virus an average of 100-fold. Increasing the amount of packaged Vpx boosted infectivity 600- to 800-fold, depending on the donor. Similar results were obtained using cell preparations from seven additional donors (not shown). Vpx also enhanced the infection of MDM, although the increase in infectivity was about 3-fold lower. In the titration of pcVpx.myc on MDM, infectivity reached the half-maximal level with the smallest amount of the cotransfected Vpx expression vector, suggesting that MDM are more sensitive than MDDC to packaged Vpx.

Fig. 3.

Chimeric HIV-1 containing the Vpx packaging motif has an enhanced ability to infect MDDC and MDM. (A) Expression and packaging of Vpx provided in trans. Luciferase reporter viruses were generated by cotransfection of 293T cells with a wild-type (WT) or p6 chimeric (SIVp6 17-26) proviral reporter virus plasmid (18 μg) and increasing amounts of pcVpx.myc (0.3 μg, 0.6 μg, 3.0 μg, and 6.0 μg), with the total mass of DNA held constant by the addition of the pcDNA plasmid. The cell lysate and the resulting virions were analyzed on an immunoblot probed with an antibody to myc-tagged Vpx, HIV-1 CA p24, or tubulin. (B) Effects of Vpx on infection of MDDC and MDM. MDDC (top) and MDM (bottom) were infected with VSV-G-pseudotyped luciferase reporter viruses at 300,000 cps based on normalization of infectivity on 293T cells, an amount corresponding to an average MOI of 0.3. After 4 days, the cultures were harvested, and the luciferase activity was determined. The data are displayed as fold enhancement of luciferase activity (calculated as the luciferase activity of the virus containing Vpx divided by that of the virus lacking Vpx). Error bars indicate the standard deviations of triplicate measurements. Results from three MDDC and two MDM donors are shown. (C) p24 analysis of MDDC and MDM infection by HIV-1 containing Vpx. MDDC (top) and MDM (bottom) were infected with a luciferase reporter virus at 20 ng p24, corresponding to an average MOI of 1.1. Three days later, the cells were collected, and intracellular p24-FITC levels were determined by flow cytometry.

The results obtained with the luciferase reporter viruses do not distinguish between effects on the number of cells infected and the transcriptional activity of the provirus per cell. To determine the effect of Vpx on the number of infected MDM and MDDC, we quantified intracellular p24 by flow cytometry (Fig. 3C). We found that the wild-type and p6 chimeric viruses that lacked Vpx were poorly infectious on MDDC, as was the wild-type virus complemented with Vpx. In contrast, the p6 chimeric virus complemented with Vpx infected 98-fold more cells. On MDM, the Vpx-complemented chimeric virus infected 84-fold more cells. On these cells, the wild-type virus complemented with Vpx was also more infectious, infecting nearly 40% as many cells as the complemented p6 chimeric virus. The increased sensitivity of MDM to small amounts of packaged Vpx suggests that these cells are less restrictive than MDDC. The mean fluorescence intensity of the cells was not affected, indicating that Vpx did not affect the transcription of the provirus or the translation of viral proteins.

Comparison with other lentivirus Vpx proteins.

Lentiviruses other than SIVmac encode Vpx. These include HIV-2_rod and possibly SIVagm, which encodes a single polypeptide that, although termed Vpr, is similar to Vpx, sharing the ability to enhance infection of MDM (6, 43). To determine whether HIV-2_rod Vpx and SIVagm Vpr function similarly to SIVmac Vpx and enhance HIV-1 infection, we generated wild-type and p6 chimeric reporter viruses complemented with each accessory protein. Immunoblot analysis showed that HIV-2_rod Vpx and SIVagm Vpr were packaged into the p6 chimeric virus but not into the wild-type virus (Fig. 4A). However, due to their low level of expression in the cell, SIVagm Vpr and HIV-2_rod Vpx were packaged in smaller amounts than SIVmac Vpx. The results showed that none of the accessory proteins affected the infectivity of the wild-type virus on MDDC. In contrast, both SIVmac₂₃₉ Vpx and HIV-2_rod Vpx enhanced the infectivity of the p6 chimeric virus (Fig. 4B, top). HIV-2_rod Vpx was only about 8% as active as SIVmac₂₃₉ Vpx, most likely because of its low expression level. SIVmac₂₃₉ Vpx and HIV-2_rod Vpx also enhanced the infectivity of the p6 chimeric virus on MDM (Fig. 4B, bottom). As in the preceding experiments, MDM were less restrictive than MDDC. SIVagm Vpr had no detectable effect on the infection of MDDC or MDM. The lack of an effect may indicate a species restriction of the AGM protein, although we could not rule out the possibility that the carboxy-terminal Myc tag interfered with the biological activity of the protein. Intracellular p24 staining of infected MDDC further supported these findings (not shown).

Fig. 4.

Comparison of SIVmac Vpx, SIVagm Vpr, and HIV-2_rod Vpx. Luciferase reporter viruses were generated by cotransfection of 293T cells with wild-type (WT) or p6 chimeric HIV-1 and an expression vector for SIVmac₂₃₉ Vpx, HIV-2_rod Vpx, or SIVagm Vpr. (A) Expression and packaging of codon-optimized SIVmac₂₃₉ Vpx, HIV-2_rod Vpx, and SIVagm Vpr. Cell lysates and pelleted virions were analyzed on an immunoblot probed with an antibody to myc-tagged Vpx, myc-tagged Vpr, HIV-1 p24 CA, or tubulin. (B) Effects of HIV-1 packaging of SIVmac₂₃₉ Vpx, HIV-2_rod Vpx, or SIVagm Vpr on MDDC and MDM infection. MDDC and MDM were infected with the indicated virus at 300,000 cps based on normalization of luciferase activity on 293T cells, corresponding to an average MOI of 0.3. Four days postinfection, luciferase activity was determined. The data are displayed as fold enhancement of luciferase activity (luciferase activity of the virus containing Vpx divided by that of the virus lacking Vpx). Error bars indicate the standard deviations of triplicate measurements. Results for three MDDC donors and three MDM donors are shown.

A p6 chimeric virus that encodes Vpx.

In the experiments described above, the Vpx-containing virions were produced by trans-complementation. Expression of Vpx in cis from the virus genome would allow for Vpx expression through multiple rounds of virus replication and would obviate the need to complement by cotransfection. To generate a virus that expressed Vpx in cis, we replaced HIV-1 nef with a codon-optimized vpx open reading frame. Insertions in this position have been used previously to express foreign genes without affecting virus replication in culture (7, 35). vpx could have been inserted in place of vpr, but this would have disrupted overlapping reading frames and splice signals. The virus we constructed is based on NL.Ba-L, an NL4-3 variant that contains the CCR5-tropic envelope glycoprotein of HIV-1 Ba-L. To allow for Vpx expression and packaging, we placed the SIVmac₂₃₉ p6 17-26 minimal Vpx packaging motif into Gag and a codon-optimized SIVmac₂₃₉ vpx, HIV-2_rod vpx, or SIVagm vpr into nef. Immunoblot analysis of transfected 293T cells showed that the cis viruses expressed the accessory proteins, although at levels lower than those of the trans viruses. To increase the sensitivity of detection, the cells were pretreated with MG132 (Fig. 5A). Immunoblot analysis of virions produced by the p6 chimeric virus with SIVmac Vpx in cis showed that they had packaged the accessory protein. Because of the lower level of Vpx produced, we used 10-fold more cis virus than in the trans virus experiments described above (Fig. 5B).

Fig. 5.

A CCR5-using chimeric virus with Vpx in cis replicates more efficiently in MDDC and MDM than wild-type virus. Wild-type (WT) and p6 chimeric NL.Ba-L viruses that express Vpx in cis were generated by transfection of 293T cells. (A) cis viruses encoding SIVmac₂₃₉ Vpx, HIV-2_rod Vpx, and SIVagm Vpr produce the accessory proteins. 293T cells were transfected with proviral plasmid DNA and, 5 h prior to harvest, were treated with 20 μM MG132. Cell lysates were prepared and analyzed on an immunoblot probed with an antibody to myc-tagged Vpx, myc-tagged Vpr, or tubulin. (B) The HIV-1 p6 chimeric virus with SIVmac₂₃₉ Vpx in cis packages Vpx. Wild-type and p6 chimeric pNL.Ba-L viruses encoding SIVmac₂₃₉ Vpx virions were pelleted from the culture supernatants of 10 culture dishes (10 times as many dishes than were used in the analysis of the trans virus). Pelleted virions and cell lysates were analyzed on an immunoblot probed with an antibody to myc-tagged Vpx, HIV-1 CA p24, or tubulin. For the p24 immunoblot analysis, 1/10 as much virus lysate was used as for the Vpx immunoblot analysis. (C) Vpx packaged by the cis p6 chimeric virus results in the generation of more reverse transcripts (RT). MDM were infected with 5 ng wild-type or cis p6 chimeric virus, corresponding to an average MOI of 0.04. After 24 and 48 h, DNA was prepared, and the reverse transcripts were quantified by qRT-PCR using primers specific for the late products and 2-LTR circles. Early products were not analyzed, because these were found to be present in the virions prior to infection. To control for plasmid contamination and intravirion reverse transcripts, AZT (25 μM) was added to the sample infected with the p6 chimeric virus encoding SIVmac Vpx (SIVp6: vpx⁺). (D) The cis p6 chimeric virus that encodes SIVmac₂₃₉ Vpx is more active than those encoding HIV-2_rod Vpx or SIVagm Vpr. MDDC were infected with 50 ng p24, corresponding to an average MOI of 0.5. After 3 days, the cells were collected, stained for intracellular p24, and analyzed by flow cytometry. AZT (25 μM) was used to control for contamination with the input virus. Nelfinavir (Nel) (3 μM) was used to limit replication to a single cycle. Results from two donors are shown.

Because of the low level of Vpx production by the cis virus, we measured the infectivities of the cis viruses by the sensitive method of qRT-PCR. For this purpose, we infected MDM with the cis virus encoding SIVmac₂₃₉ Vpx and, after 24 and 48 h, measured the late reverse transcription products and 2-LTR circles. The analysis showed that at both time points, the p6 chimeric virus encoding Vpx generated about 4-fold more late cDNA molecules than virus with wild-type p6 or virus that lacked Vpx. After 48 h, the p6 chimeric virus with Vpx in cis generated 6-fold more 2-LTR circles (Fig. 5C). To determine the relative numbers of infected cells, we infected MDDC with the cis viruses and analyzed them for intracellular p24 by flow cytometry. The analysis showed that the p6 chimeric virus encoding SIVmac₂₃₉ Vpx infected 8-fold more MDDC than viruses lacking Vpx (Fig. 5D). Infection with viruses encoding HIV-2_rod Vpx or SIVagm Vpr was not significantly enhanced over that of the controls. Addition of the protease inhibitor nelfinavir to the SIVmac₂₃₉ Vpx-encoding p6 chimeric virus decreased the number of infected cells by an average of 58%, suggesting that the virus had replicated beyond the first cycle. Taken together, the results suggest that the cis virus contained a small amount of Vpx but that this amount was sufficient to enhance its replication in MDM and MDDC.

HIV-1 containing Vpx induces type I IFN in MDDC.

Manel et al. recently showed that HIV-1 infection of MDDC pretreated with Vpx-containing VLP activates an innate immune response that induces CD86 and type I IFN (33). Induction of the response appeared to have been triggered by newly synthesized Gag protein through an unidentified intracellular sensor. These findings predict that Vpx-containing HIV-1 would similarly induce an innate immune response in MDDC. To test this, we infected MDDC with a p6 chimeric virus containing or lacking Vpx, and after 24 and 48 h, we quantified IFN-β mRNA. The results showed a robust, Vpx-dependent induction of IFN-β 48 h postinfection (Fig. 6). The IFN-β induction was blocked by AZT and raltegravir, inhibitors that block reverse transcription and integration, respectively. This result suggested that the response was triggered postintegration and not by the incoming virion, consistent with the findings of Manel et al. (33). The delay in IFN-β mRNA induction until 48 h postinfection is consistent with an event late in virus replication. These results suggest that the p6 chimeric virus induced an innate immune response in the MDDC.

Fig. 6.

Infection of MDDC with a Vpx-containing virus stimulates an innate immune response. MDDC from two healthy donors were infected with the p6 chimeric virus complemented in trans with Vpx. The virus was used at 50 ng p24, which corresponded to an average MOI of 2.7. AZT (25 μM) or raltegravir (Ral) (10 μM) was added to the indicated samples. The cultures were harvested 24 and 48 h postinfection, and IFN-β mRNA was quantified by qRT-PCR. The data are presented relative to the levels of G6PDH mRNA amplified in parallel.

Vpx facilitates the transfer of HIV-1 from MDDC to T cells.

In vitro, MDDC can be shown to transmit HIV-1 to CD4 T cells by trans-infection, a process in which the virus binds to lectin-like proteins, such as dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN), on the surfaces of MDDC and is then transferred to the T cell (15). In trans-infection, the MDDC does not become infected but binds the virus at the plasma membrane. Because Vpx enhances the infection of MDDC, we hypothesized that, in addition to transmission through trans-infection, HIV-1 virions containing Vpx could be efficiently transmitted from MDDC to CD4 T cells through a direct infection mechanism analogous to cell-to-cell transmission by T cells. To test whether Vpx would allow for enhanced transmission of HIV-1 from MDDC to CD4 T cells, we infected MDDC with the NL.Ba-L p6 chimeric virus containing Vpx (Vpx⁺) or lacking Vpx (Vpx⁻) in cis. Free virus was removed, and 2 days later, autologous activated CD4 T cells were added. The culture supernatants were sampled over 10 days for p24 quantification. The results showed that the chimeric virus containing Vpx replicated with more-rapid kinetics, producing >8-fold more p24 at earlier time points than the virus that lacked Vpx (Fig. 7A).

Fig. 7.

Exposure of MDDC to a Vpx-containing virus allows for efficient transfer of the virus to T cells. (A) MDDC were infected with NL.Ba-L wild-type or p6 chimeric virus containing vpx in cis, with or without additional Vpx complementation in trans. The virus was added at 50 ng p24, corresponding to an average MOI of 0.24. After 6 h, free virus was removed, and after 48 h, CD3/CD28-activated autologous CD4 T cells were added. Levels of the p24 supernatant were measured over 10 days. Results are representative of MDDC and CD4 T cells from two donors. (B) MDDC were infected as for panel A but with the virus at 25 ng p24, corresponding an average MOI of 0.3. After 6 h, free virus was removed. After 48 h, medium or CD3/CD28-activated autologous T cells were added. In parallel, T cells alone were infected as for panel A, and after 6 h, free virus was removed. Levels of the p24 supernatant were measured over 14 days. The results shown are representative of MDDC and CD4⁺ T cells from two donors. (C) MDDC and T cells were infected as for panel B, and cells were collected at day 3 postcoculture. The cells were incubated with APC-conjugated anti-CD11c and FITC-conjugated anti-p24 and were analyzed by flow cytometry. The cell populations were gated on CD11c and were then evaluated for intracellular p24. The results shown are representative of three donors tested.

In the coculture experiment, the increased virus replication could have been due simply to more-efficient replication in the MDDC. Alternatively, it was possible that Vpx enhanced HIV-1 replication in the activated CD4 T cells. To determine the relative contributions of the two cell types to virus replication in the coculture, we established three cultures: (i) MDDC alone, (ii) activated CD4 T cells alone, and (iii) a coculture of activated CD4 T cells and MDDC. The cultures were infected with NL.Ba-L p6 chimeric virus either containing Vpx (Vpx⁺) or lacking Vpx (Vpx⁻) in cis. For the coculture, the MDDC were infected for 6 h, after which the virus was removed; then, 2 days later, the activated CD4 T cells were added. Supernatants were collected over 14 days for p24 quantification. The results showed that in the MDDC-alone culture, no p24 produced by the Vpx⁻ was detectable, while the Vpx⁺ virus produced moderate levels of p24 after 10 days (Fig. 7B). In the coculture, the Vpx⁻ virus replicated to a similar moderate level, while the Vpx⁺ virus replicated faster and produced more p24. This result could have been due to more-efficient transfer of the Vpx⁺ virus from MDDC to CD4 T cells or, alternatively, to better replication of the Vpx⁺ virus in the T cells independently of the MDDC. In the culture with CD4 T cells alone, the Vpx⁺ and Vpx⁻ viruses replicated with similar kinetics. This result suggests that Vpx does not enhance virus replication directly in CD4 T cells, consistent with earlier findings (18). Thus, in the coculture, the increased virus replication was caused by transfer of the virus from infected MDDC to activated CD4 T cells.

To determine the number of infected CD4 T cells and MDDC in the cocultures, we stained the cells for intracellular p24 and gated on the CD11c⁺ (MDDC) and CD11c⁻ (T cell) populations. The analysis showed that both the CD11c⁺ and CD11c⁻ cells were infected more efficiently by the Vpx⁺ virus than by the Vpx⁻ virus (Fig. 7C). Since Vpx does not have a significant effect on HIV-1 replication in T cells, the result further suggests a role for Vpx in the transmission of virus from MDDC to CD4 T cells.

DISCUSSION

We report here on the development of an HIV-1 variant that has been modified to allow the packaging of Vpx, a lentiviral protein not encoded by the virus. With the addition of a 10-amino-acid Vpx packaging motif derived from SIVmac₂₃₉ p6, the engineered virus efficiently packaged SIVmac₂₃₉ Vpx, HIV-2_rod Vpx, or SIVagm Vpr expressed in trans in the producer cell or in cis from the viral genome. The ability of the virus containing SIVmac₂₃₉ Vpx to infect MDDC and MDM was dramatically enhanced, and it induced a strong innate immune response. Vpx had no effect on the replication of the virus in activated CD4 T cells but caused the virus to replicate more efficiently in MDDC–CD4 T cell cocultures. Replication-competent CCR5-tropic p6 chimeric viruses that expressed SIVmac Vpx, HIV-2_rod Vpx, or SIVagm Vpr in cis produced relatively small amounts of the accessory proteins, but in the case of SIVmac₂₃₉ Vpx, this was sufficient to provide a significant replicative advantage in MDDC. MDDC were more restrictive than MDM, an effect that was apparent in infections with viruses that contained a small amount of Vpx. In addition, cell preparations from different donors differed in the stringency of the restriction. Since Vpx has been proposed to counteract a host restriction factor, this result suggests that MDDC express more of the restriction factor than MDM and that donors differ with regard to the amount of the factor expressed by their cells.

To generate an HIV-1 variant that would package Vpx, we introduced the amino acid motif of SIVmac₂₃₉ p6 that mediates Vpx packaging. To construct the virus, we first identified the sequence ¹⁷DPAVDLLKNY²⁶ as the minimal motif of SIVmac₂₃₉ p6 needed to confer Vpx packaging on HIV-1. This motif includes the ¹⁷DXAXXLL²³ sequence that was identified by Accola et al. with the addition of three carboxy-terminal amino acids (1). Introduction of the motif adjacent to the PTAPP late domain resulted in a chimeric virus that efficiently packaged SIVmac₂₃₉ Vpx, HIV-2_rod Vpx, and SIVagm Vpr. When SIVmac₂₃₉ Vpx was provided in trans, the virus was 10- to 100-fold more infectious on MDM and MDDC. Due to a low level of expression, HIV-2_rod Vpx only slightly enhanced MDM and MDDC infection.

SIVagm Vpr was packaged into the p6 chimeric HIV-1 yet did not enhance the infection of MDM or MDDC. Whether its failure to promote infection is due to the lack of a role in MDM and MDDC infection or to species specificity in its interaction with the hypothetical Vpx-targeted restriction factor is unclear. SIVagm Vpr is similar to HIV-2 Vpx in sequence and length but is closer phylogenetically to HIV-2 Vpr (13, 42). Functionally, SIVagm Vpr bears properties of both Vpr and Vpx. It is packaged into virions, localizes to the nucleus, promotes infection of macaque MDM, and induces G₂ cell cycle arrest in AGM CV-1 cells (6, 38). To more definitively characterize SIVagm Vpr as Vpr or Vpx, it will be important to determine whether it facilitates the infection of AGM MDDC.

SIVagm Vpr, SIVmac Vpr, and SIVmac Vpx share the same packaging determinant, while HIV-1 Vpr packaging is mediated by a separate amino acid motif in p6 (1, 28, 48). Two groups have reported on p6 amino acid motifs required for HIV-1 Vpr packaging but with different results. Kondo and Göttlinger transferred HIV-1 p6 into murine leukemia virus (MLV) and showed that the leucine motif, termed motif 2 here, was required in order for it to package HIV-1 Vpr (28). In contrast, Zhu et al. found that a virus with p6 truncated at amino acid 35, and thus lacking motif 2, maintained Vpr packaging (48). In the truncated virus, Vpr packaging mapped to ¹⁵FRFG¹⁸, here termed motif 1. To investigate this apparent discrepancy, we analyzed p6 mutants of both motifs in the context of full-length Gag. We found that both motifs play roles in Vpr packaging, although our results differ somewhat from those of both groups. Kondo and Göttlinger found that single amino acid mutations in motif 2 at position 41, 44, or 45 prevented Vpr packaging. In contrast, we found that the individual mutations had no effect. Instead, mutation of all four hydrophobic residues was required to prevent Vpr packaging. This difference may be due to the fact that Kondo and Göttlinger tested the mutants in the context of MLV rather than HIV-1. For motif 1, we found, unlike Zhu et al., that single amino acid mutations only partially reduced Vpr packaging. This difference may have been due to our analysis in the context of full-length rather than truncated Gag. The roles of motifs 1 and 2 are not clear, but we speculate that one is the Vpr binding site and the other affects p6 conformation. Both motifs lie in predicted α-helices (10), but in motif 2, the hydrophobic leucines are predicted to be buried in the protein and thus to be less likely to serve as a binding site. The 17-26 p6 chimeric virus lacks the ¹⁵FRFG¹⁸ motif yet packages a significant amount of Vpr, presumably because it retains motif 2.

The SIVmac₂₃₉ Vpx cis virus, despite the use of a codon-optimized open reading frame, expressed less of the accessory protein than the transfected pcVpx.myc. Two factors may account for the reduced expression of Vpx. First, Vpx is rapidly degraded in the cell through a proteasomal pathway (data not shown). When Vpx is expressed by the high-copy-number expression vector, the amount of protein may overwhelm the capacity of the degradative pathway, increasing its half-life. Second, expression of Vpx in the nef position may not be optimal. Nef is expressed early in the virus replication cycle from a fully spliced mRNA (11). In contrast, virion assembly and Vpx packaging occur later, when there is a bias toward Rev-induced production of unspliced mRNAs that encode the structural proteins. Therefore, Vpx produced early in infection is likely to be degraded before it can be packaged.

In vivo, dendritic cells activate T cells by costimulation of the T cell receptor (TCR) and CD28 through a cell contact-dependent mechanism. This cell-cell interaction could provide an effective means by which the virus spreads throughout the body. Our findings with the replication of the chimeric virus in MDDC–T cell cocultures support such a mechanism. In trans-infection, virus binds to the MDDC surface through DC-SIGN and other type C lectins. The virus does not infect the cell but is transferred to the CD4 T cell through an infectious synapse (15, 34). We propose that Vpx provides a second mechanism in which MDDC are productively infected, releasing infectious virions that are then efficiently transmitted to CD4 T cells. Such a mechanism is supported by the finding that MDDC are productively infected at a low level and then transmit newly produced virus to T cells (36). Whether transmission occurs through a virological synapse or through cell-free virus could not be distinguished in the coculture system. A complication of this mechanism is that the infected MDDC induce IFN-β upon the production of Gag. IFN-β induces an antiviral state in bystander cells that might interfere with the transmission of virus to T cells, as suggested by Manel et al. (33). Whether the amount of IFN-β produced would be sufficient to dampen virus transmission would depend on the MOI and cell density. We used a lower MOI to infect the MDDC than did Manel et al. (33). Conditions in vivo may influence the balance between the enhancing and inhibitory activities of infected MDDC.

It is remarkable that HIV-1 is sensitive to the Vpx-targeted restriction factor yet lacks Vpx to counteract it (18, 20). This role does not appear to have been subsumed by Vpr, which does not have the potency of Vpx in increasing MDDC infection. The absence of Vpx may serve to avoid the activation of an innate immune response, as suggested by Manel et al. (33). For SIV and HIV-2, both of which encode Vpx, it is possible that transfer from MDDC and MDM to T cells plays a greater role. In HIV-1 replication, virus spread may be driven more by T cell-to-T cell transmission, particularly once coreceptor usage expands to CXCR4.

The p6 chimeric viruses described here provide a useful tool with which to study the role of Vpx in virus replication. In addition, they provide a novel approach to vaccine and lentiviral vector design. Lentiviral vectors that package Vpx could provide a means to increase the transduction of MDDC and MDM. Because MDDC present antigens to T cells, the chimeric p6 virus might be used to enhance T cell responses to lentiviral vector-encoded immunogens.