Vpr-mediated Incorporation of UNG2 into HIV-1 Particles Is Required to Modulate the Virus Mutation Rate and for Replication in Macrophages*

Human immunodeficiency virus type 1 is able to infect nondividing cells, such as macrophages, and the viral Vpr protein has been shown to participate in this process. Here, we investigated the impact of the recruitment into virus particles of the nuclear form of uracil DNA glycosylase (UNG2), a cellular DNA repair enzyme, on the virus mutation rate and on replication in macrophages. We demonstrate that the interaction of Vpr with UNG2 led to virion incorporation of a catalytically active enzyme that is directly involved with Vpr in modulating the virus mutation rate. The lack of UNG in virions during virus replication in primary monocyte-derived macrophages further exacerbated virus mutant frequencies to an 18-fold increase compared with the 4-fold increase measured in actively dividing cells. Because the presence of UNG is also critical for efficient infection of macrophages, these observations extend the role of Vpr to another early step of the virus life cycle, e.g. viral DNA synthesis, that is essential for replication of human immunodeficiency virus type 1 in nondividing cells.

Human immunodeficiency virus type 1 (HIV-1) 1 Vpr is a 96-amino acid non-structural protein that is associated with virus particles and can accumulate at the nuclear envelope and in the nucleus of infected cells (1)(2)(3)(4). The incorporation of Vpr into particles requires a direct interaction with the p6 region of the Gag polyprotein precursor (5,6). Several independent biological activities have been attributed to Vpr during the HIV-1 life cycle. First, expression of Vpr alters the cell cycle progression by arresting cells in the G 2 phase (7-10). Second, Vpr influences the reverse transcription process of the viral DNA, and this can modulate the in vivo mutation rate of HIV-1 (11,12). Finally, Vpr is required for the infection of nondividing cells, and this requirement is related, at least in part, to its role in the nuclear translocation of the preintegration complex (PIC) containing the viral DNA. Vpr possesses an affinity for the components of the nuclear pore complex, and it has been proposed that Vpr may facilitate the nuclear translocation of the PIC across the nuclear envelope (4,(13)(14)(15)(16). Infection of nondividing, terminally differentiated macrophages and resting T cells represents a viral reservoir in the host that is crucial for subsequent virus spread to lymphoid organs and T-helper lymphocytes and finally for AIDS pathogenesis (17).
The HIV-1 Vpr protein has been found to interact with several cellular partners, including the uracil-DNA glycosylase (UNG) (18), a DNA-repair enzyme involved in the base excision repair pathway that specifically removes the RNA base uracil from DNA. Uracil can occur in DNA either by misincorporation of dUTP or by cytosine deamination (19). Two distinct forms of UNG are generated by alternative splicing and localize in mitochondria (UNG1) and in the nucleus (UNG2). Initially identified from a yeast two-hybrid screen using Vpr as bait (18), the interaction between Vpr and UNG was confirmed both in vitro and ex vivo in Vpr-expressing cells (11,18). Although the Trp residue in position 54 located in the exposed loop connecting the second and the third ␣-helix of HIV-1 Vpr has been shown critical to maintain the interaction with UNG, the Vpr-binding site was mapped within the common C-terminal part of UNG2 (11,18). The use of the peptide phage display or yeast twohybrid systems revealed that peptides able to bound Vpr had a common WXXF motif (20,21). Furthermore, UNG proteins contain such a motif in their C-terminal region, which may be necessary for the interaction with Vpr (20). Vpr has been found to specifically recruit the nuclear form of UNG into HIV-1 virions (UNG2, commonly named UNG in this report) (11). Although the viral integrase may also participate in this recruitment (22,23), the Vpr-dependent packaging of UNG2 into virions strikingly correlated with the ability of Vpr to influence the mutation rate of HIV-1 (24). This indicated that the interaction between Vpr and UNG2 may directly influence the reverse transcription accuracy and, thus, play a role in the modulation of the in vivo mutation rate of HIV-1 (11,12).
In this report, we further investigated the specific contribution of UNG2 incorporated into HIV-1 particles in the early phase of the virus life cycle. To address this question, we developed an experimental system in which UNG2 was incorporated into virus particles independently of Vpr by expressing UNG2 as a chimeric protein fused to the C-terminal extremity of the VprW54R mutant, a Vpr variant that fails to recruit UNG2 into virions and to influence the virus mutation rate even though it is incorporated as efficiently as the wild type Vpr protein (11). The VprW54R-UNG fusion was efficiently incorporated into HIV-1 virions and restored a mutation rate equivalent to that observed with wild type Vpr. Because we showed that VprW54R variant specifically influenced HIV-1 replication in monocyte-derived macrophages, these results support the conclusions that the Vpr-dependent recruitment of UNG2 into virions is directly involved in the modulation of the HIV-1 mutation rate and is required for efficient virus replication in non-dividing cells such as macrophages.

Retroviral Vectors and Expression
Plasmids-Most of the retroviral vectors and yeast and mammalian expression plasmids used in this study have been described previously (5,11) except plasmids for the expression in bacteria of wild type (WT) and mutated forms of UNG2 fused to the glutathione S-transferase (GST) and for expression in mammalian cells of UNG fused to the C terminus of the wild type or W54R Vpr proteins. The 231 WXXF 234 motif found within the nuclear UNG2 form (numbering according to Haug et al. (25)) was mutated by PCR-mediated site-directed mutagenesis using specific primers containing the desired mutations to obtain the UNG W231A/F234G -mutated form. The PCR product was then cloned back into the EcoRI-XhoI restriction sites of pGadGE (5) and the pGEX-4T1 vectors (Amersham Biosciences) to obtain plasmids for expression of UNG W231A/F234G fused either to the Gal4 activation domain or to the GST in yeast and in bacteria, respectively. To construct the plasmids for expression of the Vpr-UNG and VprW54R-UNG fusions, the Vpr-and VprW54R-coding sequences were, respectively, amplified by PCR using a specific set of primers to delete the stop codon, and the PCR products were cloned back into the BamHI-HindIII restrictions sites of the pAS1B plasmid (11). The UNG-coding sequence was then amplified by PCR to create a HindIII site at the 5Ј end, and the products were inserted in-frame into the HindIII-XhoI sites of either the pAS1B-Vpr-or the pAS1B-VprW54R-digested plasmids.
Yeast Two-hybrid Assay-The HF7c yeast reporter strain containing the Gal4-inducible gene, HIS3, was cotransformed with vectors for expression of the indicated Gal4 DNA binding domain and Gal4 activation domain hybrids and plated on selective medium lacking tryptophan and leucine as reported (5). Double transformants were patched on the same medium and replica-plated on selective medium lacking tryptophan, leucine, and histidine for auxotrophy analysis.
In Vitro Binding Assay-The GST-UNG fusions were expressed in Escherichia coli BL21 cells (Invitrogen) after induction with 0.5 mM isopropyl-1-thio-␤-galactopyranoside for 3 h at 30°C. Bacterial pellets were resuspended in phosphate-buffered saline containing 2 mM EDTA, 2 mM dithiothreitol, and an antiprotease mixture (Sigma). After 1 h of incubation at 4°C with 0.12% lysozyme, bacterial lysis was completed by adding 1% Triton X-100, 10 mM MgCl 2 , 10 g/ml RNase A, and 20 g/ml DNase I. Lysates were centrifuged at 60,000 ϫ g for 30 min at 4°C. Supernatants were incubated with glutathione-Sepharose beads (Amersham Biosciences) for 1 h at 4°C. Beads were washed 3 times with 1 M NaCl containing 0.5% Triton X-100 and then with phosphatebuffered saline. The concentration of the fusion proteins was estimated on a SDS-PAGE gel stained with Coomassie Blue G-250 (Bio-Rad) and determined relative to a range of bovine serum albumin standard. Cell lysates from 6 ϫ 10 6 HeLa cells expressing HA-Vpr (WT or W54R) were prepared and incubated with purified GST or GST-UNG proteins as previously described (4). Bound proteins were then resolved by SDS-PAGE and then analyzed by Western blotting as described (4).
Assay for Incorporation of Vpr-UNG Fusions into HIV-1 Particles-Incorporation of the Vpr-UNG fusions into HIV-1 virions was analyzed as previously described (5) using a virion packaging assay in which HA-tagged fusions were expressed in trans in virus-producing cells.
UNG Catalytic Assay-A catalytic assay was developed to detect the incorporation of UNG into virus particles. Briefly, we cotransfected (Superfect, Qiagen) a vpr-defective HIV-1 vector and Vpr and UNG expression plasmids derived from pAS1B (11) into 293T cells. After 48 h, cell culture supernatants were collected, and virions were concentrated by ultracentrifugation as previously described (5). Virions were resuspended as described (5), and 1 g of crude viral protein was used in the UNG assay. The DNA oligonucleotide 5Ј-TTTTTTTTTTT-TUTTTTTTTTTTTT-3Ј used for the UNG assay was chosen based on previous studies (26) and was obtained from Sigma-Genosys (The Woodlands, TX). Uracil-DNA glycosylase inhibitor was obtained from New England Biolabs (Beverly, MA). Assays of UNG activity were done with the single-stranded DNA oligonucleotide substrate and were performed in UNG reaction buffer (20 mM Tris-HCl, 1 mM EDTA, 1 mM dithiothreitol, pH 8.0) at 37°C for 1 h. Apurinic sites were cleaved by adding 1 ⁄2 volume of 0.5 M NaOH and 1 ⁄2 volume of 30 mM EDTA and then boiling for 30 min (27). Samples were then applied to a nondenaturing 20% polyacrylamide gel with electrophoresis at 60 V for 3.5 h or were applied to a denaturing 19% polyacrylamide gel run at 400 V for 2 h. Gels were stained with SYBR Gold (Molecular Probes, Eugene, OR), and nucleic acids were visualized with an ultraviolet transilluminator.
Analysis of HIV-1 Mutant Frequencies-The HIV-1 vectors used in these studies have been previously described (11,12,28). To produce vector virus, the HIV vectors were complemented in trans with pSVgagpol-rre-MPMV, the amphotropic murine leukemia virus env expression plasmid, pSV-A-MLV-env, and a Vpr expression plasmid derived from pAS1B (11). HIV-1 vector and expression plasmids were transfected into HeLa cells by using Superfect (Qiagen). Infection of HeLa target cells was also done by cocultivation of virus-producing cells with target cells (29). The protocols for analysis of mutant frequencies have been previously described with minor modifications (12,28,30). Specifically, infected peripheral blood mononuclear cells (PBMCs) and monocytes-derived macrophages (MDMs) were not placed under drug selection, and after purification of proviral DNA with the lac repressor protein, the vector cassette containing the mutation target was PCR amplified before analysis of mutant frequencies in E. coli.
Isolation and Infection of PBMCs and MDMs-PBMCs from HIV-1 seronegative donors were isolated by Ficoll-Hypaque density centrifugation. Phytohemagglutinin-stimulated cells were plated in RPMI, 10% fetal calf serum containing 10 units/ml interleukin-2 and streptomycin (100 g/ml). Peripheral blood monocytes were isolated from fresh PB-MCs by adherence to plastic at 37°C. After overnight culture the adherent cells were removed from plates by gentle scraping, and residual T-lymphocytes were further depleted using anti-CD2 immunomagnetic beads. Cells were cultured in medium without added growth factors. Mature macrophages were derived by culturing the purified monocytes 7-14 days in RPMI, 10% fetal calf serum without additional cytokines. Cells typically became enlarged or spindle shaped with extended processes.
Virus infection of primary cells was done using virus produced from 293T cells transfected (Superfect, Qiagen) with T-cell tropic or macrophage-tropic HIV-1 molecular clones expressing either WT or mutant Vpr. CAp24 equivalent amounts of virus produced from infected cells was used for infection of primary cells and corresponded to a multiplicity of infection of about 0.05. After overnight incubation at 37°C, the cells were washed twice and placed in either RPMI, 10% fetal calf serum (macrophages, resting T-cells) or medium supplemented with 10 units/ml interleukin-2 (stimulated PBMCs/stimulated T-cells). Sampling of cell culture supernatants was done immediately after washing (day 0) and on subsequent time points. Amounts of CAp24 produced were determined by enzyme-linked immunosorbent assay.

Characterization of the Determinants Involved in the Interaction between Vpr and UNG2-Previous studies have established a correlation between the property of Vpr to interact
with UNG and to influence the HIV-1 mutation rate (11,24). In particular, a VprW54R variant (see Fig. 1B) that failed to bind UNG in a yeast two-hybrid assay also failed to recruit UNG into HIV-1 virions and was not able to complement a vpr null mutant HIV-1 in a mutation rate assay (11). To further characterize the respective molecular determinants of Vpr and UNG involved in the interaction, we developed an in vitro binding assay using recombinant UNG expressed in E. coli in fusion with the glutathione S-transferase (GST-UNG). Because it has been reported that Vpr binding is related to the presence of a WXXF motif found within the C-terminal part of UNG2 (amino acids 231-234), a GST-UNG W231A/F234G mutant was included as a control of specificity in this in vitro binding assay. Purified recombinant GST-UNG and GST-UNG W231A/F234G fusions were immobilized on GSH-Sepharose beads and then incubated with lysates from cells expressing either HA-tagged WT Vpr or VprW54R. Bound proteins were analyzed by West-ern blotting with anti-HA (Fig. 1A). HA-Vpr specifically bound to GST-UNG but not to GST alone. In contrast, VprW54R was not retained on GST-UNG, and neither WT Vpr nor VprW54R bound to GST-UNG W231A/F234G . These results were in complete agreement with the yeast two-hybrid data reported in Fig. 1, B and C. Only WT UNG, but not UNG W231A/F234G , fused to Gal4 activation domain interacted with Vpr fused to Gal4 DNA binding domain, as indicated by growth of the HF7c yeast reporter strain on medium without histidine. As expected, the VprW54R variant failed to interact with UNG in the twohybrid assay. Together, these observations provide further data in support that the Trp in position 54 of Vpr is critical to maintain the interaction with UNG and that the 231 WXXF 234 motif of UNG2 is involved in binding to Vpr.
UNG-associated Enzymatic Activity Is Recovered from HIV-1 Particles-To determine whether the UNG incorporated into virions was catalytically active, we developed a simple assay for UNG activity using a 25-base T homopolymer oligonucleotide substrate containing a single uracil residue located at position 12 (see Fig. 2A). In the presence of UNG activity, the uracil residue is excised and leaves the phosphodiester backbone. Heating the sample can destroy the backbone at that position, resulting in a 12-base product that can be visualized on a nondenaturing polyacrylamide gel. UNG activity from purified HIV-1 virions was, thus, determined by transiently transfecting 293T cells with an HIV-1 vector with either Vpr or VprW54R in combination with UNG expression plasmids. HIV-1 virions were collected 48 h later, concentrated, and used in the UNG activity assay ( Fig. 2A). Expression of Vpr in virus-producing cells led to UNG enzymatic activity from purified virions. This indicates that the fully active endogenous UNG was incorporated into HIV-1 virus particles, but expression of Vpr in combination with a HA-tagged UNG form led to the detection of a higher level of UNG activity into virions. In contrast, expression in virus-producing cells of the VprW54R mutant alone or in combination with UNG was not associated with the detection of enzymatic activity from virions. Similarly, very low levels of UNG activity were detected from virions produced from cells co-expressing Vpr and a mutated UNG W231A/F234G form that does not interact with Vpr (see Fig.  1). As shown in Fig. 2B, no activity was detected when the assays were performed in the presence of a specific inhibitor of UNG (uracil-DNA glycosylase inhibitor) in the reaction mixtures (31), demonstrating that the activity detected into virions is related to a specific recruitment of UNG. These results show that Vpr-mediated recruitment of UNG results in the presence of catalytically active enzyme into HIV-1 virions.
Incorporation of Vpr-UNG Fusion Proteins into HIV-1 Particles-To gain further insight on the functional role of the recruitment of UNG into HIV-1 virions, we took advantage of the VprW54R mutant to specifically incorporate UNG without requiring the interaction with Vpr by expressing UNG as a fusion to the C-terminus of VprW54R. We previously reported that this Vpr mutant failed to recruit UNG2 into virions even though it is efficiently incorporated into virions (11). The virion incorporation of the VprW54R-UNG fusion was first analyzed  (lanes 1 and 3) or 3 g (lanes 2 and 4) of plasmids for expression of either HA-tagged Vpr (lanes 1 and 2) or VprW54R (lanes 3 and 4). Lysates from transfected cells were incubated with equal amounts of GST, GST-UNG, or GST-UNG W231A/F234G immobilized on GSH-Sepharose beads, as indicated at the bottom. Bound proteins were resolved by SDS-PAGE and immunoblotted with an anti-HA antibody. One-tenth of the input of the cell lysate from the transfected cells used for the binding assay was run on the right panel. B and C, two-hybrid analysis of the Vpr binding to UNG. HF7c reporter strain expressing either wild type Vpr or VprW54R fused to Gal4 DNA binding domain (Gal4BD) in combination with either wild type UNG or UNG W231A/F234G fused to Gal4 activation domain (Gal4AD) was analyzed for histidine auxotrophy. Double transformants were patched on selective medium with histidine (ϩHis) and then replica-plated on medium without histidine (ϪHis). Growth in the absence of histidine indicates interaction between hybrid proteins. Each patch represents an independent transformant. using a packaging assay in which the fusion was expressed in trans in virus-producing cells. 293T cells were cotransfected with the HIV-1 vector lacking the vpr gene in combination with the VprW54R-UNG expression plasmid. Cell-and virion-associated Vpr-UNG fusions were then assayed by immunoblotting (Fig. 3A). The VprW54R-UNG fusion as well as the wild type Vpr-UNG fusion used as a control were well expressed in virusproducing cells (upper panels), and both fusions were detected from virions purified from the supernatant of transfected cells (lower panels). Using the same enzymatic assay as described above (see Fig. 2A), we checked that the VprW54R-UNG and Vpr-UNG fusions incorporated into virions were catalytically active (Fig. 3B). These results indicate that the Vpr-UNG fusion proteins are efficiently incorporated into HIV-1 particles and retain UNG enzymatic activity.
The Vpr-UNG Fusion Proteins Can Influence the HIV-1 Mutant Frequency-The VprW54R-UNG fusion, therefore, represents a valuable tool for analyzing the direct contribution of UNG to the modulation of the virus mutation rate. We, thus, used an HIV-1 mutation rate assay to determine whether the Vpr-UNG fusions could complement a vpr-defective HIV-1 for mutant frequencies (11). Briefly, the plasmids for expression of Vpr-UNG fusions were transiently cotransfected with helper packaging plasmids into cells containing a single integrated HIV-1 vector provirus containing the lacZ gene as a mutation target. The viruses produced were then used to infect permissive cells, which allowed for a determination of the virus mutant frequency per round of replication (Fig. 4). In contrast to the 4-fold increase in mutant frequency observed by transcomplementation with the VprW54R mutant, the wild type fusion but also the VprW54R-UNG fusion gave rise to virus mutant frequencies equivalent to that observed by complementation with the wild type Vpr protein. These data show that the VprW54R-UNG fusion can rescue the defective phenotype of VprW54R and modulate HIV-1 mutant frequency as efficiently as Vpr, demonstrating that the recruitment of UNG into virions is directly responsible for this Vpr function.

Recruitment of UNG into Virions Is Essential for Efficient Replication of HIV-1 in Macrophages-Because data presented
here as well as a previous study demonstrate that the recruitment of UNG into HIV-1 virions plays an important role at an early step in HIV-1 replication (e.g. viral DNA synthesis) (11), we tested whether this recruitment had a direct impact on virus replication in primary target cells of HIV-1. The W54R mutation was introduced into the vpr gene of either a T-cell tropic (NL4 -3) or a macrophage tropic (YU-2) HIV-1 molecular clone. Wild type and Vpr mutant proviruses were transfected into 293T cells, and cell culture supernatant was harvested, adjusted for equal amounts of CAp24 antigen, and used to infect PBMCs or MDMs. Virus production was then monitored by measuring the CAp24 antigen every 3 days (Fig. 5). Whereas HIV-1-expressing Vprwt replicated efficiently in MDMs with a rapid increase in CAp24 antigen at 9 days postinfection and peaking at 15 days, HIV-1-expressing VprW54R had a significant replication defect, with only low levels of CAp24 detected 9 days after infection (Fig. 5A). In contrast, viruses expressing Vprwt or VprW54R both efficiently replicated in PBMCs (Fig.  5B). In summary, these data show that VprW54R mutation can influence HIV-1 replication in MDMs and not in PBMCs, indicating that the Vpr-dependent incorporation of UNG is important for virus replication in non-dividing cells.
Influence of Vpr on HIV-1 Mutant Frequency in Monocytederived Macrophages-Because the replication defect of HIV-1 expressing the VprW54R mutant was specifically apparent in non-dividing cells, we finally analyzed in MDMs the influence of virion-associated UNG on virus mutant frequencies. The same HIV-1 mutation rate assay was used, but viruses containing the lacZ␣ peptide gene as a mutation target were then used FIG. 2. UNG-associated enzymatic activity recovered from HIV-1 particles. A, analysis of UNG activity into virions. Virions produced from cells expressing either wild type Vpr or VprW54R alone or in combination with wild type UNG or UNG W231A/F234G were collected from cell supernatants and prepared as described under "Experimental Procedures." Assays of UNG activity were performed with a 25-bp single-stranded DNA oligonucleotide substrate containing a uracil base at position 13 (shown on the right), and apurinic sites were then cleaved by adding 0.5 M NaOH and 30 mM EDTA and boiling for 30 min. The samples were run on a 20% polyacrylamide denaturing gel. Gels were stained with SYBR Gold, and nucleic acids were visualized with an ultraviolet transilluminator. After the alkaline and heat treatment, the deoxyribose phosphate backbone was hydrolyzed to form two 12-bp fragment products (shown on the right). The control lane contains untreated DNA substrate, whereas the ⌬Vpr lane corresponds to purified virions produced from cells transfected with vpr-defective HIV-1 vector alone as indicated under "Experimental Procedures." B, analysis of UNG activity in the presence of uracil-DNA glycosylase inhibitor (UGI). Activity was assayed as in A, but uracil-DNA glycosylase inhibitor was added to the reaction mixture where indicated. The samples were then analyzed as described in A.
to infect MDMs to determine whether the VprW54R mutant could complement a vpr-defective HIV-1. As reported in Table  I, complementation with Vprwt or with the previously characterized VprR90K (32) mutant that efficiently interacts with UNG led to an average mutation frequency (0.006 and 0.007 mutation/cycle, respectively), which is equivalent to that observed when HeLa cells were the targets for infection (see Fig.  4, and Mansky et al. (11)). However, VprW54R as well as the lack of Vpr expression (⌬Vpr) led to a 16 -18-fold increase in virus mutant frequencies (averages were 0.109 and 0.098, respectively) compared with that observed with Vprwt during infection of MDMs. This increase is about 4 -5 times higher than the increase in virus mutant frequencies observed when HeLa cells were used as target cells (11). Again, trans-complementation with either the VprW54R-UNG or Vpr-UNG fusions led to a mutation rate comparable with that obtained with Vpr alone, demonstrating that UNG fused to VprW54R restores a normal mutation phenotype. These observations indicate that virus mutant frequencies are significantly higher in MDMs when UNG is not packaged into HIV-1 virions and confirm that the recruitment of UNG is directly responsible for this Vpr function.

DISCUSSION
The observations described in this report provide important new insights into the functional role for the interaction of the Vpr auxiliary protein from HIV-1 and the cellular UNG2 protein, an enzyme implicated in DNA repair. We demonstrate that the Vpr-dependent incorporation of UNG into HIV-1 particles is directly responsible for the role of Vpr in the in vivo modulation of the virus mutation rate (11,12). Moreover, our results show that the incorporation of UNG into virions is critical for efficient replication of HIV-1 in primary non-dividing cells such as macrophages. This observation parallels the involvement of Vpr in the nuclear import of viral DNA in non-dividing cells (33) and extends its role to another early step of the virus life cycle (e.g. viral DNA synthesis) essential for replication of HIV-1 in non-dividing cells. Several lines of evidence reported here support these conclusions. First, although the UNG binding-deficient VprW54R variant failed to influence the virus mutation rate, a VprW54R-UNG fusion was able to influence HIV-1 mutant frequencies in a manner equivalent to that of wild type Vpr. Second, when the VprW54R variant was introduced into infectious HIV-1 molecular clones, replication in MDMs was significantly diminished, whereas virus replication in PBMCs was not altered. Finally, the lack of UNG virion incorporation during virus replication in macrophages further exacerbated HIV-1 mutant frequencies compared with that measured in actively dividing cells.
Using both yeast two-hybrid and biochemical approaches, we confirm that substitution of the Trp 231 and/or Phe 234 residues of the WXXF motif of UNG alters its binding to Vpr, whereas mutation of the Trp 54 residue of HIV-1 Vpr abolishes binding to UNG. Moreover, no UNG activity was detected into purified virions trans-complemented with either wild type Vpr and UNG W231A/F234G or VprW54R and wild type UNG, indicating that the enzymatic activity detected into virus particles is strictly related to the direct interaction that takes place in virus-producing cells between Vpr and UNG. Currently, three distinct cellular partners of Vpr contain a WXXF motif including TFIIB (34), the adenine nucleotide translocator (35), and UNG (20). The Trp 54 residue of HIV-1 is crucial both for binding to UNG and then its recruitment into viral particles (11,32) but does not participate in the interaction of Vpr with the viral Gag precursor in virus-producing cells, which allows for the incorporation of Vpr into virions (5,11). The three-dimensional structure of the complete Vpr polypeptide was recently solved and confirms that Trp 54 is localized between the second and third ␣-helix. This suggests that this residue is easily accessible for protein-protein interactions with UNG (36) and that substitution of Trp 54 does not modify or alter the overall conformation of the HIV-1 Vpr protein. Indeed, the VprW54R mutant is still able to induce a G 2 arrest of the cell cycle (32) and efficiently localizes at the nuclear envelope through interaction with the human CG1 nucleoporin (data not shown and Ref. 4).
We, therefore, took advantage of the VprW54R mutant, which failed to incorporate UNG into virions (11), to generate a Vpr-UNG fusion protein that allows for an evaluation of the specific role(s) of UNG recruitment into viral particles on the early steps of HIV-1 infection. The VprW54R-UNG fusion is efficiently incorporated into virions, and enzymatic assays performed from purified virions show that the UNG fused to VprW54R was still catalytically active. These results confirm that Vpr can efficiently target proteins within HIV-1 particles without affecting the catalytic properties of the cargo (20,(37)(38)(39). Moreover, the observation that the Vpr-UNG fusions can restore the mutant frequency phenotype indicates that Vpr and the virion-associated UNG are directly responsible for the modulation of the virus mutant frequency in vivo. The enhanced alteration in virus mutant frequencies observed in primary macrophages when UNG was not incorporated into virions shows that this phenotype may have greater biological relevance in nondividing cells than in actively dividing cells. It is of particular interest to note that viruses lacking Vpr or expressing the VprW54R mutant display analogous mutation rate, indicating that no other viral proteins can rescue this Vpr phenotype. Although it was proposed that the viral integrase was also able to mediate interaction with UNG (22,23), our results argue that Vpr is the main viral determinant that allows for the incorporation of cellular UNG into virus particles. However, preliminary results obtained from in vitro binding assays suggest that both Vpr and integrase associate with UNG to form a trimeric complex (data not shown), but further analyses are needed to document the dynamic of interactions between UNG, Vpr, and integrase as well as reverse transcriptase (23) both in virus-producing cells and then in target cells.
HIV-1 and other lentiviruses are unusual among retroviruses in their ability to infect resting or terminally differentiated cells. Vpr from HIV-1 has been related to facilitate nuclear import of the viral DNA in such non-dividing cells (33). In this report, we have identified that the virion incorporation of UNG via Vpr also contributes to the ability of HIV-1 to replicate in primary macrophages, assigning another critical role of Vpr during the viral life cycle. This implies that UNG is a cellular factor that plays an important role in the early steps of the HIV-1 replication cycle (i.e. viral DNA synthesis). In agreement, it has been recently reported that the misincorporation of uracil into minus-strand viral DNA affects the initiation of the plus strand DNA synthesis in vitro (40). These results suggest that UNG is likely recruited into HIV-1 particles to subsequently minimize the detrimental accumulation of uracil FIG. 5. The VprW54R mutation specifically influences HIV-1 replication in monocyte-derived macrophages. The W54R mutation was introduced into the vpr gene of either a T-cell tropic (NL4 -3) or a macrophage tropic (YU-2) HIV-1 molecular clone. Wild type (Vprwt, circles) and mutated (VprW54R, triangles) viruses produced in cell-free supernatant of 293T cells transfected with proviral DNAs were harvested, adjusted for equal amounts of CAp24 antigen, and used to infect MDMs (A) or PBMCs (B). Virus production was then monitored by measuring the CAp24 antigen every 3 days. into the newly synthesized proviral DNA. Although further work is needed to explain the precise mechanism for how UNG catalytic activity may specifically influence HIV-1 replication in macrophages, it is noteworthy that such nondividing cells express low levels of UNG and contain relatively high levels of dUTP (41). Similarly, most non-primate lentiviruses, such as feline immunodeficiency virus, caprine-arthritis-encephalitis virus, and equine infectious anemia, have also developed an efficient strategy to reduce accumulation of uracil into viral DNA. These lentiviruses encode and package a dUTP pyrophosphatase (for review, see Refs. 19 and 41), an enzyme that hydrolyzes dUTP to dUMP, into virus particles and, thus, maintains a low level of dUTP. Interestingly, replication of feline immunodeficiency virus, caprine-arthritis-encephalitis virus, or equine infectious anemia, which lacks functional a dUTP pyrophosphatase activity, is severely affected in nondividing host cells (e.g. primary macrophages) (42)(43)(44). Altogether, these results indicate that uracil misincorporation in viral DNA strands during reverse transcription is deleterious for the ongoing steps of the virus life cycle. The presence of a viral dUTP pyrophosphatase or a cellular UNG will prevent these detrimental effects for replication of non-primate and primate lentiviruses in macrophages, respectively. It is intriguing to note that two viral auxiliary proteins from HIV-1, Vpr and Vif, act in the same way to contribute in the fidelity of the synthesis of the viral DNA from the RNA template but using two different mechanisms. The Vif protein forms a complex with the cellular deaminase apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3G (CEM15), preventing its encapsidation into virions (45)(46)(47)(48)(49), whereas Vpr binds the DNA repair enzyme, UNG, to recruit it into the particles where it could start to exert its activity. It is tempting to speculate that action of both viral proteins may influence the mutation rate during the course of HIV-1 infection, and their balance may play a key role during disease progression in infected individuals.
In summary, this report provides strong evidence for a direct role of the cellular UNG2 incorporated into HIV-1 virions in the modulation of virus mutation rate. The requirement of UNG2 incorporation by Vpr for efficient virus replication in macrophages implies that the interaction between Vpr and UNG2 could represent an attractive target for antiviral intervention.