Vpr.A3A Chimera Inhibits HIV Replication*

Several APOBEC3 proteins (A3F and A3G), that are cytidine deaminases restrict human immunodeficiency virus (HIV) replication in the absence of the viral infectivity factor (Vif) protein. However, Vif leads to their degradation and counteracts their effects. Another member, A3A, restricts some retrotransposons and another virus but not HIV. We reasoned that this failure was due to the lack of appropriate targeting. Thus, we fused A3A to another viral protein, Vpr, which binds p6 in Gag and is incorporated into viral cores. Indeed, the Vpr.A3A chimera but not A3A was found abundantly in the viral core. It also restricted potently the replication of HIV and simian immunodeficiency virus in the presence and absence of Vif. Because we identified a high frequency of G to A mutations in viral cDNAs, this antiviral activity was mediated by DNA editing. Interestingly, our fusion protein did not restrict murine leukemia virus, which does not incorporate Vpr. Thus, by targeting appropriately a potent single domain cytidine deaminase, we rendered HIV and simian immunodeficiency virus restriction resistant to Vif.

Innate immune defenses, which are poised to neutralize extracellular pathogens, are integral components of eukaryotic systems (1). Although some of these factors can interfere with the replication of HIV, 3 viral proteins can counteract them. The ability of members of the APOBEC3 (A3) family of cytidine deaminases (CDAs) to confer intrinsic immunity to retroviral infection was first recognized with the human A3G protein (2). A3G is packaged into newly synthesized viral particles from producer cells and acts in target cells to inhibit viral replication during reverse transcription. In humans, the APOBEC family includes AID, A1, A2, A3A-A3H, and A4 (3). All proteins in this family have one or two CDA motifs that contain one histidine, two cysteines, and one glutamic acid that are involved in the hydrolytic deamination of cytosines, thereby converting cytidine (C) to uridine (U) in a process called editing (for review in Ref. 3). Indeed, G to A changes observed in viral sequences from HIV-infected patients can be explained by direct C to U transitions in the minus-strand cDNA by CDAs (4). However, because mutant A3G proteins lacking the CDA activity retain substantial antiviral effects, the mechanism of this intrinsic restriction is not fully understood (5,6).
Although A3F and A3G can restrict the replication of HIV, this inhibition is overcome by the viral infectivity factor (Vif). Thus, primate lentiviruses have developed an escape strategy to prevent the incorporation of A3 proteins into progeny virions (7). Vif acts as an adaptor protein, which connects A3F and A3G to an E3 ubiquitin ligase, thereby inducing the polyubiquitylation and proteasomal degradation of these CDAs (8). As a consequence, A3F and A3G are unavailable for subsequent incorporation into viral particles, and their antiviral effects are absent in target cells. Anti-viral effects of A3 proteins are not limited to Vifdeficient forms of HIV but also extend to other lentiviruses, including SIV, the equine infectious anemia virus (EIAV), and more distant retroviruses such as the murine leukemia virus (MLV), foamy viruses, and the mouse mammary tumor virus as well as endogenous retroviruses and retrotransposons (9 -13).
In this study we wanted to create an A3 protein that can be targeted into the core of viral particles and is not degraded by Vif. To this end, we chose A3A, which contains a single copy of the CDA motif and is not active against HIV (13). However, it is catalytically active and inhibits endogenous long terminal repeat (LTR)-containing retroelements (MusDs and IAPs), non-LTR retroelements (LINEs and SINEs), and Alus (14 -19). Indeed, A3A inhibits the retrotransposition of IAPs more potently than do A3B or A3G (18). This CDA also inhibits the replication of the adeno-associated virus (parvovirus AAV) (19). To target A3A, we chose the viral protein R (Vpr), which binds p6 in Gag and is incorporated into the core of HIV particles (20 -23). Vpr is a small basic protein (14 kDa), whose functions include the import of preintegration complexes into the nucleus of cells and the delay of the cell cycle (for review in Ref. 24). Recently, several studies demonstrated that Vpr fusion proteins could be used as intravirion inactivating agents (22,23). In this study we constructed a Vpr.A3A chimera that was incorporated abundantly into the viral core where it restricted the replication of HIV and SIV in the presence and absence of Vif.

EXPERIMENTAL PROCEDURES
Tissue Cultures-The human embryonic kidney cell line 293T and GHOST R3/X4/R5 cells were cultured in Dulbec-* This work was supported by research grants from the National Institutes of Health (to B. M. P). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1  co's modified Eagle's medium, 10% fetal bovine serum, 100 g/ml penicillin and streptomycin, and 2 mM glutamine at 37°C with 5% CO 2 . GHOST cells obtained from the AIDS Reference and Reagent Program have been engineered to stably express CD4 and the chemokine receptors CCR3, CXCR4, and CCR5 (25). The expression of these chemokine receptors was maintained by adding to the culture medium hygromycin (100 g/ml), Geneticin (500 g/ml), and puromycin (1 g/ml). Plasmids-HIV proviral plasmids pNL43-Luc and pNL43-Luc⌬Vif were described before (26). The full-length, envelopedefective SIVmac reporter virus (pSIVmac-Luc E Ϫ R Ϫ and pSIVmac-Luc E Ϫ R Ϫ ⌬vif) were kindly provided by Dr. Nathaniel Landau (27). HIV and SIVmac clones encoded luciferase in place of the nef gene. Expression vectors encoding the B-tropic MLV Gag-Pol proteins have been described previously (28). They were used to package MLV-based vectors encoding GFP in place of env but lacking Gag and Gag-Pol genes (pCNCG, Oxford Biomedica). The B-tropic MLV vectors were obtained from Dr. Paul D. Bieniasz. Reporter viruses lacking env were generated as vesicular stomatitis virus glycoprotein (VSV-G) pseudotypes. To construct the Vpr.A3A fusion protein, the human A3A was fused to the C terminus of Vpr in the context of the mammalian expression vector pcDNA3.1/V5-His TOPO (Invitrogen) (Fig. 1A). First, the vpr gene was amplified by PCR using a 5Ј-primer located before the initiation codon (ATG) of Vpr and 3Ј-primer at the end of the vpr gene of pNL43 proviral clone (Vpr forward primer, 5Ј-CAC CAT GGA ACA AGC CCC AGA AGA CCA A-3Ј; VprGlyClaI reverse primer, 5Ј-CCC ATC GAT GGG ACC ACC TCC GCC TGT CGA CAC CCA ATT CTG-3Ј). At the 3Ј site of Vpr gene, a linker of four glycines and the ClaI restriction site were introduced, and the VprGly fragment was cloned into pcDNA3.1-V5 TOPO. Next, a PCR-amplified A3A DNA fragment flanked by ClaI and XbaI restriction sites at 5Ј and 3Ј termini, respectively (ClaIA3Af, 5Ј-CCC ATC GAT GGG ATG GAA GCC AGC CCA GCA TCC GGG-3Ј; A3AXbaIrev, 5Ј-GCT CTA GAG TTT CCC TGA TGG CCC GCA GCC TCC CAC TCA-3Ј) was amplified from pKA3A vector previously described (18), digested with ClaI and XbaI enzymes, and ligated into pcDNA3.1-VprGly vector digested with the same enzymes. In addition, other plasmid constructions were used as controls. The Vpr.Nef fusion protein (pEF.Myc.Vpr.Nef) was described before (29). The Vpr expressing vector was constructed as described above, and the Vpr gene was inserted in the pcDNA3.1-V5 TOPO (pcDNA3.1 Vpr). Mammalian expression vectors for human A3C, A3F, and A3G (26) were subcloned into pcDNA3.1D/V5-His-TOPO. The pkA3A vector expressing hA3A HA-tagged protein was a gift of Dr. Brian Cullen. All constructions were checked by restriction enzyme digestion and DNA sequencing with cytomegalovirus and bovine growth hormone primers.
Viral Production and Infectivity Assay-HIV viruses were produced by transfection of pNL43 Luc vectors (8 g) with 4 g of each vector expressing APOBEC, Vpr, or Vpr.Nef fusion proteins. Reporter viruses (SIVmac and B-Tropic MLV) were generated as VSV-G pseudotypes by co-transfection of 293T cells in 10-cm dishes with 8 g of proviral plasmid DNA, 4 g of VSV-G, and 4 g of APOBEC expression vector or the controls plasmids (pcDNA3.1 Vpr or pEF.Myc.Vpr.Nef) using FuGENE 6 transfection reagent (Roche Applied Science). B-tropic MLV viruses were generated using an MLV-based vector encoding GFP in place of env, an expression vector B-tropic MLV Gag-Pol protein (28), the pVSV-G vector and the A3 and control vectors in the proportion 1:1:0.5:0.5, respectively. Viruses in the supernatant were collected after 48 h of transfection and filtered through a 0.45-m filter, and the viral titers were measured with an enzyme-linked immunosorbent assay kit against p24 (Roche Applied Science) or p27 (RETRO-TEK; ZeptoMetrix). Equal amounts of HIV-1 and SIVmac were used to infect GHOST-R3/X4/R5 cells in six-well plates. 24 h later cells were lysed in CAT lysis buffer (Promega). After removing the nuclei, the cytosolic fraction was used to determine the luciferase activity using a luciferase assay kit (Promega). MLV infectivity assays were done in six-well plates seeded with GHOST-R3/X4/R5 cells per well the day before infection. 48 h post-infection, the target cells were assayed for GFP expression by fluorescence microscopy, then harvested by trypsinization and resuspended in 1ϫ phosphatebuffered saline. An aliquot was used for flow cytometry (FACSCalibur, BD Biosciences), and the data were analyzed using CellQuest software (BD Biosciences Immunocytometry Systems). Uninfected control cells were also processed as described above. Infectivity assays were done in triplicate; each assay was performed three times with similar results. Values are presented as the percentage of infectivity relative to the virus infectivity in the absence of A3 vectors.
A3 Encapsulation and Isolation of HIV Cores-Normalized amounts of virus were pelleted by ultracentrifugation through 20% sucrose cushion at 45,000 rpm for 1 h (Beckman), 4°C. Cell lysates and pelleted virions were solubilized in 10 l of HEPES lysis buffer. Virion cores were obtained using previously published methods (30). Briefly, viral pellets from the cushion of 20% sucrose ultracentrifugation were resuspended in phosphate-buffered saline (3.5 l/ml of initial culture volume). Subsequently, 40 l of fresh virus suspension was mixed with an equal volume of 200 mM NaCl, 100 mM MOPS (pH 7.0), and virions were lysed for 2 min at room temperature by adding Triton X-100 to a final concentration of 0.5%. HIV cores were recovered by centrifugation in a microcentrifuge at full speed (14,000 rpm) for 8 min at 4°C. Pellets were washed twice with 100 mM NaCl, 50 mM MOPS (pH 7.0) and resuspended in 8 l of the same buffer. Cores and supernatants were processed immediately for further analysis. Cores and supernatants were then analyzed by immunoblotting to evaluate the efficiency of core isolation. The HIV reverse transcriptase (RT) (8C4), HIV gp41 (no. 50 -69), and HIV p24 monoclonal antibodies (183-H12-5C) were obtained from National Institutes of Health AIDS Research and Reference Reagent Program. To examine A3 and Vpr packaging, purified virions were subjected to SDS-PAGE and Western blotting using ␣-V5-horseradish peroxidase (HRP; Invitrogen) for Vpr.A3A, Vpr, A3G, and A3F proteins and ␣-HA monoclonal antibody (Sigma-Aldrich) for A3A protein and the appropriate HRP-conjugated secondary antibodies followed by detection with ECL reagents (Amersham Biosciences). FEBRUARY 1, 2008 • VOLUME 283 • NUMBER 5

JOURNAL OF BIOLOGICAL CHEMISTRY 2519
Detection of Hypermutation in Viral DNA-35-mm subconfluent monolayers of GHOST R3/X4/R5 cells were infected with 4 ng of p24 wild-type HIV virus produced in the presence or absence of the Vpr.A3A fusion protein in 12 h. Total cellular DNA was purified using the DNeasy kit (Qiagen), treated with DpnI for 2 h at 37°C to digest any residual plasmid DNA, and subjected to high fidelity PCR (Expand High Fidelity PCR sys-tem, Roche Applied Science) using HIV-specific oligonucleotides U3 R (forward) 5Ј-CAG ATA TCC ACT GAC CTT TGG-3Ј and U3 R (reverse) 5Ј-GAG GCT TAA GCA GTG GGT TC-3Ј. The PCR products were cloned into pCR4-TOPO (TOPO TA cloning kit for sequencing, Invitrogen) and used to transfect Escherichia coli (DH5␣). Plasmids were purified from at least 10 individual colonies, and the insert (417-bp) was sequenced using T3 and T7 primers. The nucleotide sequences of independent clones were aligned and analyzed using Hypermut software (31).

Vpr.A3A Chimera Blocks the Replication of HIV and Is Incorporated into Viral
Particles-To target A3A into the cores of HIV, we constructed a number of plasmid effectors that are depicted in Fig. 1A (effectors). For the expression of the Vpr.A3A chimera, A3A was subcloned from pkA3A expression vector, and Vpr was amplified by PCR from the HIV-1 NL4-3 provirus (Fig. 1B,  targets). They were ligated in-frame and introduced into the pcDNA3.1-V5 expression vector. Thus, our fusion protein also contained the V5 epitope tag at its C terminus. Control plasmid effectors, e.g. those expressing the Vpr.Nef chimera, or just Vpr or A3A alone are also presented in Fig. 1A. To determine whether the Vpr.A3A chimera possessed any antiviral activity, we used the wild type (WT) (NL43Luc, HIV) and mutant HIV-1 NL4-3 lacking Vif (NL43⌬VifLuc, HIV⌬Vif) luciferase proviral targets (Fig. 1B, targets). These targets were co-expressed with different A3 proteins or Vpr in 293T cells. Supernatants were harvested and purified, and amounts of viral particles were determined by p24 capture enzyme-linked immunosorbent assay. Equal amounts of different HIVs were then used to infect GHOST R3/X4/R5 (GHOST) cells. The infectivity of our viruses was determined by measurements of intracellular luciferase activities 2 days later. As presented in Fig. 2A, lane 2, the Vpr.A3A chimera blocked the WT HIV and mutant HIV⌬Vif to background levels (greater than 10-fold reduction compared with the control; lane 1). In contrast, A3A had no effect on the WT HIV or mutant HIV⌬Vif (Fig. 2A, lane 3). The reduced infectivity of viruses which were produced in the presence of A3G and A3C was consistent with previous reports (13). Briefly, A3G was sufficient to reduce the infectivity of only HIV⌬Vif to close to background levels ( Fig. 2A, lane 5). A3C weakly inhibited only the mutant HIV⌬Vif (1.6-fold) ( Fig.  2A, lane 4). Moreover, the presence of Vpr alone did not influence viral infectivity (Fig. 2A, lane 6). To exclude the possibility that the reduction of viral infectivity was due to a Vpr-based fusion protein, we also co-expressed the virus and the Vpr.Nef chimera (Fig. 2A, lane 7). Again this fusion protein did not interfere with viral replication, validating our results with the Vpr.A3A chimera. We conclude that the

Vpr.A3A Inhibits HIV
Vpr.A3A chimera blocks the replication of HIV and is not inactivated by Vif.
To evaluate the incorporation efficiencies of A3A and the Vpr.A3A chimera, the same aliquots of viruses and producer cells were subjected to Western blotting. Virions were pelleted through a 20% sucrose cushion and normalized to levels of p24, and their protein profiles were examined in parallel (Fig. 2B). A3A and the Vpr.A3A chimera were identified using ␣-HA and ␣-V5 antibodies, respectively. Our fusion protein had an apparent molecular mass of 40 kDa, consistent with the combined sizes of Vpr and A3A (Fig. 2B, lanes 3 and 4). Moreover, Western blotting confirmed that our chimera was packaged efficiently into WT HIV and mutant HIV⌬Vif particles (Fig. 2B,  upper panel, lanes 3 and 4). Interestingly, A3A was also incorporated into progeny virions (Fig. 2B, upper panel,  lanes 5 and 6). However, it did not affect the infectivity of HIV (Fig. 2A, lane 3). Most likely, this incorporation was outside the ribonucleoprotein complex or the core (30). Equal loading of virions on these blots was confirmed with antibodies against p24 (Fig. 2B, middle panel, lanes 1-6).
These results indicate that the Vpr.A3A fusion protein is incorporated efficiently into viral particles where it restricts the infectivity of HIV. In contrast, because A3A had no effect, this finding suggests that the targeting via Vpr was responsible for the antiviral effects of our chimera.
Vpr.A3A Chimera Induces Extensive G to A Changes in the Viral Genome-Because the Vpr.A3A chimera blocked viral replication, we wanted to know if DNA editing was involved. To this end, we analyzed sequences of viral reverse transcripts from newly infected cells. WT HIV was co-expressed with the Vpr.A3A chimera in 293T cells, normalized to levels of p24, treated with DpnI to remove contaminating plasmid DNA, and then used to infect GHOST cells. 12 h later a fragment of U3 R was amplified and cloned. Nucleotide sequences were determined for at least 10 independent clones from each infection.
In cells infected with the WT HIV in the presence of the Vpr.A3A chimera, we found a total of 62 G t A transitions distributed over 125 dG residues in 10 sequences (4170 bp) yielding a mutation frequency of 1.5 per 100 bases (Fig. 3A, left  panel, black bars). Only a single non-G-t-〈 change was found in the same 10 sequences (Fig. 3A, left panel, lollipop). Levels of mutations were high in comparison to those with the WT HIV in the absence of the Vpr.A3A chimera and were similar to those described previously (32). Indeed, we found just one change (T to C) in 10 sequences analyzed (4170 bp) for a mutation frequency of 0.02 per 100 bases (Fig.  3A, right panel, lollipop).
To reveal the base preference for this DNA editing, we analyzed an additional 62 G to A mutations generated in the presence of the Vpr.A3A chimera. The spectrum of mutations revealed that our chimera targets dC residues that are preceded by dC or dT (Fig. 3B, rows C and T). This target specificity agrees with previous reports, where A3A mutated dCs preceded by dC or dT (19). We conclude that the Vpr.A3A chimera has the ability to promote editing of cytidines in the minus strand of viral reverse transcripts and, consequently, to inhibit the replication of HIV.
Vpr.A3A Chimera Inhibits SIV Infection-These findings raised the possibility that the Vpr.A3A chimera could restrict the replication of other retroviruses. To investigate this notion, we evaluated the effects of our chimera on the replication of SIV. In these experiments we produced WT SIVmac-Luc-E Ϫ R Ϫ (SIV) and mutant pSIVmac-Luc-E Ϫ R Ϫ ⌬vif (SIV⌬Vif) viruses in the presence of the Vpr.A3A chimera and other proteins as in Fig. 2A. Luciferase reporter viruses were pseudotyped with the VSV-G Env in the presence of different A3 proteins in 293T cells. As with HIV, the infectivity of these viruses was determined by infecting GHOST cells. Equivalent amounts of viruses, as determined by p27 capture enzymelinked immunosorbent assay, were used, and luciferase activities were measured 2 days later. In addition to inhibiting the infectivity of HIV, the Vpr.A3A chimera also inhibited potently the infection by SIV (Fig. 4A). As presented in lane 2 of Fig. 4A, viral infectivity was inhibited 6-fold by our chimera in the presence and absence of Vif. In contrast, the FIGURE 3. Vpr.A3A chimera causes extensive editing of viral genomes. WT HIV was produced in the presence of the Vpr.A3A chimera or the empty vector and used to infect GHOST cells. 24 h later, DNA was isolated, and a fragment in the U3 R region from the viral cDNA was amplified. After cloning into the TA vector, viral cDNAs were sequenced, and these sequences were aligned. A, profiles of mutations in 10 representative 412-bp fragments of HIV produced in the presence (left panel) and absence (right panel) of the Vpr.A3A chimera. Each G to A transition is denoted by a vertical bar, and the one non-G to A change is indicated by a lollipop. Mutations in each clone (numbers 1-10) were compared with the original HIV-1 NL4-3 sequence. Nucleotide sequences were analyzed using Hypermut software. B, surrounding base preference for the CDA activity of the Vpr.A3A chimera. All 62 C to T transitions in the minus strand, which were produced in the presence of our chimera, were compared, and frequencies of bases at their 5Ј (left, negative numbers) and 3Ј (right, positive numbers) ends were calculated as percentages. n represents the number of independent C to T transitions. FEBRUARY 1, 2008 • VOLUME 283 • NUMBER 5

JOURNAL OF BIOLOGICAL CHEMISTRY 2521
co-expression of A3A had no effect (Fig. 4A, lane 3). A3C exhibited more potent antiviral effects on the mutant SIV⌬Vif than on the mutant HIV⌬Vif, as reported previously (33). Again, the presence of Vpr and the Vpr.Nef chimera had no effect, validating the antiviral restriction of the Vpr.A3A chimera on SIV.
To extend our analyses, we also investigated the incorporation of A3A proteins into SIV particles. In Fig. 4B, Western blotting of producer cells and virions from supernatants revealed the presence of A3A and the Vpr.A3A chimera in producer cells and viral particles (Fig. 4B, top and bottom panel,  lanes 3-6). Input viruses are presented in the middle panel and were identified by antibodies against p27. We conclude that the incorporation of our chimera into viral particles restricts the infectivity of SIV irrespective of Vif, suggesting that the Vpr.A3A fusion protein also possesses broad antiviral activity against other primate lentiviruses.
Vpr.A3A Chimera Does Not Restrict the Replication of MLV-Results of antiviral restriction of HIV and SIV by the Vpr.A3A chimera were somewhat surprising in light of effects of A3A, which had no effect on the infectivity of these viruses. Thus, we wanted to examine our chimera with MLV, which does not incorporate Vpr (34). To determine whether MLV is sensitive to the Vpr.A3A fusion protein, VSV-G-pseudotyped B-tropic MLV-enhanced GFP reporter viruses were co-expressed with different A3 proteins in 293T cells (28). Virions were harvested, normalized to p30, and used to infect GHOST cells as with HIV and SIV (Figs. 2 and 4). To follow their infectivity, the number of GFP-positive cells was determined by FACS. As presented in Fig. 5A, lanes 2 and 3, neither A3A nor our chimera inhibited the infection by MLV. Controls with other A3 proteins (Fig. 5A,  lanes 4 and 5) demonstrated a potent suppression of MLV infection by A3G but no A3F (13). Moreover, Vpr alone had no effect (Fig. 5A, lane 6). Thus, neither the Vpr.A3A chimera nor A3A restricted the replication of MLV.
Next, we wanted to determine whether MLV could package A3A proteins into progeny virions. For this purpose we performed high speed centrifugation through a sucrose cushion to collect viral particles. Western blotting of producer cells and lysed virions identified A3G, A3F, Vpr, and the Vpr.A3A chimera with ␣-V5 and A3A with ␣-HA antibodies. As presented in Fig. 5B, bottom panel, lanes 2-5 and 7), all proteins were expressed at high levels in producer cells. As previously reported, MLV virions also packaged A3G, consistent with their sensitivity to this protein (Fig. 5B, upper panel, lane 4) (35). However, the incorporation of A3F into viral particles was reduced, which explains the relative absence of its antiviral effects (Fig. 5B, upper panel, lane 3). Interestingly, both FIGURE 4. Vpr.A3A chimera restricts the replication and is incorporated into SIV. A, Vpr.A3A chimera inhibits the replication of SIV. SIV and SIV⌬Vif luciferase reporter viruses were co-expressed with indicated A3 proteins in 293T cells. After normalization to p27 enzyme-linked immunosorbent assay, equal amounts of viruses were used to infect GHOST cells. The infectivity was determined by quantitation of the luciferase activity as in Fig. 2A. These results were obtained from three independent experiments with errors denoted as in Fig. 2A. B, A3A and the Vpr.A3A chimera are packaged into SIV particles. Presented are Western blots of transfected 293T cells (bottom) and viral particles obtained by ultracentrifugation of cell culture supernatants (upper and middle panels). A3A and the Vpr.A3A chimera were detected via their epitope tags as in Fig. 2B. Equal loading of virions was confirmed with ␣-p27 antibodies. Other markings are as in Fig. 2.

Vpr.A3A Inhibits HIV
A3A and our chimera were incorporated to the same extent as A3G into MLV particles (Fig. 5B, upper panel, lanes 2 and  7). However, because it was not found alone in these progeny virions, Vpr did not mediate this incorporation (Fig. 5B, upper panel, lane 5). By probing for p30, we also confirmed that the same amounts of viral particles were used in these experiments (Fig. 5B, middle panel). We conclude that although neither protein affected the infectivity of MLV, A3A and the Vpr.A3A chimera were incorporated efficiently into MLV particles.
Vpr.A3A Chimera but Not A3A Is Incorporated into the Core of HIV-We hypothesized that A3A is loosely associated with viral particles and that Vpr targeting carries the Vpr.A3A chimera into the viral core. There, the A3A subunit can act as a CDA and restrict viral infectivity. To prove this notion experimentally, virions were produced in the presence of increasing concentrations of our A3A proteins and pelleted through a sucrose cushion (30). Thus, we co-expressed increasing amounts of A3A and Vpr.A3A fusion proteins with a constant amount of WT HIV. Equivalent amounts of viruses were used to infect GHOST cells. In Fig. 6A, we observed that the Vpr.A3A chimera already restricted potently viral infectivity at a ratio of input plasmids of 1 (Vpr.A3A) to 12 (HIV) (lane 3). In contrast, A3A had no effect even at much higher concentrations (Fig. 6A, lane 6). Viruses from these experiments were isolated by pelleting through a sucrose cushion, resuspended, and solubilized with Triton X-100. This treatment released Env (gp41) into the supernatant and retained p24 and RT in the core (Fig. 6, B and C) (30). To determine the efficiency of our fractionation, we compared the composition of proteins in viral cores to those of input viruses by Western blotting (Fig. 6, B and C, compare all panels). Of note, RT was enriched in the core, and gp41 was found only in the supernatant, which indicates that our fractionation was successful (Fig. 6, B and C, lanes 6 -15). Moreover, the Vpr.A3A chimera was packaged into the viral core (Fig. 6B, lanes 12-15). Importantly, it was found in the supernatant only in viruses that were produced in the presence of high concentrations of our chimera (Fig. 6B, lanes 9 and 10). In sharp contrast, A3A was absent from the viral core (Fig. 6C,  lanes 12-15). These findings indicate that whereas A3A is loosely associated with viral particles, the Vpr.A3A chimera is incorporated efficiently into the viral core, where it blocks the infection by HIV.

DISCUSSION
In this study we wanted to simplify HIV restriction by A3 proteins. By using Vpr to target a potent CDA into HIV, we were able to convert an innocent bystander into a powerful inhibitor of viral replication. Indeed, the Vpr.A3A chimera restricted the replication of all primate lentiviruses and was not sensitive to Vif. Our chimera also edited viral cDNAs in target cells. Moreover, the Vpr.A3A chimera had no effect against MLV, whose Gag does not bind Vpr. Finally, whereas A3A was found to be loosely associated with viral particles, the targeting by Vpr led to the incorporation of the Vpr.A3A chimera into the viral core. Thus, we targeted a single CDA domain A3 protein to restrict the replication of primate lentiviruses even in the presence of Vif.
These results were somewhat surprising in light of the effects of A3A alone. Moreover, because A3A and our chimera were found abundantly in HIV, SIV, and MLV particles, their differences were not caused by virion exclusion. More likely, whereas Vpr targeted A3A into the core and possibly into the ribonucleoprotein, A3A alone was only loosely associated with viral particles (36). In support of this notion, recent work suggested different distributions of high and low molecular mass A3G complexes in HIV particles, where only the latter inhibited viral replication in target cells (30). Indeed, several studies already described the presence of A3A in viral particles (19). Our results are also in good agreement with a very recent study by Goila-Gaur et al. (37). Using a different strategy, they fused A3A to the N terminus of A3G (A3G.A3A chimera). However, although also targeted into viral cores, this fusion protein restricted the replication of HIV only in the absence of Vif. Thus, not only did FIGURE 6. Vpr.A3A but not A3A is incorporated into the viral core. A, effects of different ratios of effector plasmids on the infectivity of HIV. WT HIV was generated from 293T cells co-transfected with a fixed amount of the HIV proviral plasmid (6 g) and increasing concentrations of plasmids encoding A3A or the Vpr.A3A chimera (1, 0 g; 2, 0.24 g; 3, 0.5 g; 4, 1 g; 5, 3 g; 6, 6 g). The infectivity of viruses, which were produced in the presence of A3A and the Vpr.A3A chimera, is indicated as gray and black bars, respectively. All viruses were normalized by p24 enzyme-linked immunosorbent assay. Equivalent viral titers were used to infect GHOST cells. Results were obtained from three independent experiments. B and C, only the Vpr.A3A chimera but not A3A is incorporated into the viral core. Aliquots of viruses, which were used in A, were solubilized by brief Triton X-100 treatment. Viral cores contained p24 and RT and supernatants contained gp41 and p24. Triangles represent increasing amounts of the Vpr.A3A chimera (B) or A3A (C) in co-transfections. In panels B and C, viruses before Triton X-100 treatment are presented on the left and, after treatment and fractionation, on the right. Arrows point to informative proteins that were revealed by Western blotting. FEBRUARY 1, 2008 • VOLUME 283 • NUMBER 5 this chimera behave more like WT A3G and A3F proteins in terms of trafficking, incorporating into viral cores, but it remained sensitive to Vif (37). However, it did extend the notion that single domain CDAs are portable and can modify nucleic acids when targeted appropriately.

Vpr.A3A Inhibits HIV
Thus, precise targeting of A3 into viral particles was critical for its antiviral effects. Moreover, we observed abundant editing of viral cDNAs, which means that A3A was enzymatically active in our chimera. However, we cannot exclude the possibility that the Vpr.A3A fusion protein also affected other parameters during reverse transcription, some of which have been highlighted recently with enzymatically inactive A3F and A3G proteins. They include interference with primer tRNA processing and removal as well as decreased synthesis of viral cDNA (38). Although some of these effects could have resulted from the overexpression of these mutant A3 proteins (6), these effects will be studied with appropriate mutations of the A3A moiety in our chimera in the future. Nevertheless, we were able to achieve effective inhibition of HIV and SIV replication with the simple targeting of A3A to p6 in Gag. Because neither A3A nor Vpr alone is found in high molecular mass complexes, stress granules, or P bodies, it is likely that these compartments play less important roles in the cell biology of this restriction (18,36,39,40).
Importantly, the Vpr.A3A chimera was not destroyed or blocked by Vif. Thus, WT HIV and SIV as well as mutant HIV⌬Vif and SIV⌬Vif were inhibited equivalently. Thus, one could now contemplate using our fusion protein as an inhibitor of infection by primate lentiviruses. Indeed, cells expressing the Vpr.A3A fusion protein should not support the propagation of these viruses.
Finally, our chimera might also inhibit incoming viruses, as has been observed with A3G in resting T cells and A3F and A3G in dendritic cells (2,41,42). Thus, its ability to bind p6 in Gag might facilitate such a restriction in target cells. Supporting this notion, after the uncoating step and during the formation of the preintegration complex, subunits of Gag are exposed, where TRIM5 ␣ is able to interact with p24 (43)(44)(45)(46)(47). Reverse transcription also commences at this step. Because the Vpr.A3A chimera inhibited SIV, such therapeutic strategies could be tried first in the monkey model of AIDS in rhesus macaques.