Alteration of a three-amino-acid sequence in HIV-1 Vif allows interaction with rhA3G.
A functional interaction of HIV-1 Vif with hA3G requires amino acid 128 of hA3G to be negative or neutral. Conversely, interaction of SIVagm Vif with agmA3G requires a positive charge at amino acid 128. We hypothesized that these charge requirements may result from the interaction of complementary charged amino acids in Vif and A3G. D128 in hA3G could interact with a positive charge in HIV-1 Vif, and K128 in agmA3G could interact with a negative charge in SIV Vif. To search for potential complementary amino acids in Vif that might interact with A3G amino acid 128, we searched known viral sequences to identify amino acid residues of Vif that were positively charged in HIV-1 and negative in SIV. In the SIVagm Vif sequences of the Los Alamos National Laboratory sequence database, there are seven conserved, negatively charged positions (E4, E17, E102, E104, D109, D/E122, and E215). Only two of the corresponding amino acids in HIV-1 Vif are positively charged (R4 and R15, corresponding to SIVagm Vif E4 and E17, respectively). HIV-1 Vif R15 is flanked by a nearby positive charge at R17, and these amino acids are conserved in HIV-1 Vif (R15 in >99% of isolates, and position 17 is either R or K in all sequenced viruses [82% R and 18% K]). We therefore focused on amino acids 14 to 17, which in HIV-1 Vif are DRMR. In SIVagm, positions 15 and 17 of Vif are neutral or negatively charged (Fig. ).
FIG. 1. A change of three amino acids in HIV-1 Vif allows functional interaction with rhA3G. (A) Alignment of HIV-1, SIVagm, and SIVmac Vif sequences. (B) Vif function of the indicated constructs was determined in a luciferase reporter virus assay by cotransfection (more ...)
To test the role of amino acids 4 and 14 to 17 of HIV-1 Vif in determining the species specificity of the interaction with A3G, mutant Vif expression vectors were generated and their function was tested in a single-cycle infection assay. Single-cycle viruses were generated by cotransfection of 293T cells with a Δvif HIV-1 luciferase reporter virus plasmid and a mutant or wild-type pcVif-HA expression vector. The pcVif plasmids expressed a Vif ORF that was codon optimized over its entire length to increase expression in the triple transfection (data not shown). A VSV-G expression vector was included in the transfection to generate infectious pseudotyped virus. The results of this analysis showed that a swap that included the R4 sequence (MEEEKR) maintained function against hA3G but did not counteract rhA3G or agmA3G. In contrast, the swap of the four-amino-acid sequence DRMR at positions 14 to 17 in the SERQ mutant (changes are in bold) was active against hA3G and rhA3G and partially active against agmA3G (Fig. ). Further analysis within positions 14 to 17 showed that changing only three of the four amino acids in the SEMQ mutant allowed interaction of HIV-1 Vif with rhA3G but not with agmA3G. Mutation of R14E and R17E to DEME was not sufficient to alter the interaction with A3G. Insertion of an E at position 17 in DRMER was also insufficient. Immunoblot analysis of the cell lysates showed that the mutant proteins were expressed comparably to wild-type HIV-1 Vif (Fig. , bottom). These findings suggest that positions 14 to 17 of HIV-1 Vif can influence the species specificity of the interaction with hA3G.
The role of this region in the interaction with rhA3G was tested with additional HIV-1 Vif mutants with changes at positions 14 to 17. Point mutations at each of the three positions (D14S, R15E, and R17Q) were tested (Fig. ). R15E failed to function on hA3G or rhA3G, while D14S and R15E were active against hA3G but not against rhA3G. Thus, single mutations were not sufficient to change the species specificity of the interaction. SEMR, DEMQ, and SRMQ double mutants were also tested (Fig. ). All double mutants remained at least in part active against hA3G. Only DEMQ was partially active on rhA3G. Immunoblot analysis confirmed the equivalent expression of each of the mutant Vif proteins (Fig. , bottom). Taken together, these findings mapped the minimal required change to amino acids 14, 15, and 17. R15 and R17 appear to be the most important.
FIG. 2. The charge of the amino acids in SEMQ determines the ability to interact with rhA3G. (A, B) The effects of single (A) and double (B) mutations in the HIV-1 Vif DRMR sequence were tested against hA3G (top) and rhA3G (bottom) as described in the legend (more ...) The charge at positions 14 to 17 is a determinant of the species specificity of the interaction with hA3G.
To further probe the role of positions 14 to 17, we tested the dependence of charge on Vif function. The SDMQ and SHMQ mutants tested the requirement for a negative charge at position 15. The AEMQ mutant tested the importance of amino acid 14; the SEMA and SEMN mutants tested position 17; and the AAMA mutant tested positions 14, 15, and 17. All of the mutants interacted at least partially with hA3G (Fig. , upper part). SDMQ but not SHMQ interacted with rhA3G, suggesting a requirement for a negative charge at position 15 (Fig. , lower part). AEMQ, SEMA, and SEMN also interacted with rhA3G, suggesting that the identity of amino acids 14 and 17 is not critical but that they cannot be negative. AAMA was inactive against rhA3G, further demonstrating the importance of a negative charge at position 15. Taken together, these results suggest that the charge at amino acids 14 to 17 influences the interaction with rhA3G. Amino acid 15 must be negative, and amino acid 17 must be neutral.
The SEMQ mutation allows interaction of HIV-1 Vif with hA3G mutated at amino acid 128.
The requirement for positive charges at positions 14 and 17 in HIV-1 Vif suggested an interaction with a negative charge on hA3G. Because of its role in determining the interaction with Vif, amino acid 128 was considered a likely candidate interaction site on hA3G. To determine whether this might be the case, we tested whether SEMQ HIV-1 Vif would interact with D128K hA3G. As expected, DRMR did not. Interestingly, SEMQ interacted with D128K mutant hA3G (Fig. , right part). These results are consistent with a direct interaction of hA3G D128 with DRMR in Vif. In this analysis, the Vif mutations were introduced directly into the HIV-1 reporter construct. Because of the overlap with pol, the SEMQ mutation introduced three amino acid changes in IN (see Fig. S1 in the supplemental material). However, these did not interfere with the production of infectious reporter virus. In addition, the analysis showed that the Vif mutations were functional as expressed in cis in the context of the virus, arguing that the previous results were not simply the result of overexpression from the codon-optimized Vif expression vector.
FIG. 3. SEMQ mutant Vif in cis is active against hA3G, rhA3G, and D128K hA3G. (A) The infectivity of Δvif mutant HIV-1 Luc, wild-type HIV-1 Luc, SEMQ mutant HIV-1 Vif Luc, and Δvif mutant HIV-1 Luc cotransfected with SIVmac Vif was tested against (more ...)
To determine the relative efficiency with which the SEMQ mutation altered the Vif phenotype, the reporter viruses were produced from 293T cells cotransfected with hA3G, rhA3G, and D128K mutant hA3G expression vectors over a range of plasmid ratios. Reporter viruses that expressed wild-type HIV-1 Vif, SEMQ Vif, and SIVmac Vif were all similarly resistant to hA3G and differed from Δvif mutant virus, which was sensitive (Fig. , left part). A reporter virus that expressed HIV-1 Vif was inhibited by rhA3G (Fig. , middle part), in contrast to viruses that expressed SEMQ Vif and SIVmac Vif, which were resistant. Viruses that expressed HIV-1 Vif, SEMQ mutant HIV-1 Vif, and SIVmac Vif were all similarly resistant to D128K mutant hA3G (Fig. , right part). Taken together, the results show that the SEMQ mutation allows HIV-1 Vif to counteract rhA3G and D128K mutant hA3G with an efficiency comparable to that of SIVmac Vif. These results show that the SEMQ gene is functional as expressed by the virus and that the alteration to IN was not deleterious.
SEMQ mutant Vif reduces A3G virion encapsidation.
To biochemically assess SEMQ mutant Vif function, its effect on A3G virion encapsidation and steady-state intracellular level was measured. 293T cells were transfected with Δvif mutant HIV-1, wild-type HIV-1, or Δvif mutant HIV-1 plus pcSIVmac-vif or SEMQ mutant HIV-1 Vif and an A3G expression vector. The virions were pelleted, and their A3G content was quantitated by immunoblot analysis. hA3G was present in Δvif virions but not in controls that lacked viral DNA or that expressed SIVmac Vif or SEMQ mutant Vif (Fig. , upper part). The cell lysates showed that SIVmac Vif and SEMQ mutant Vif reduced the steady-state level of hA3G (Fig. , lower part). HIV-1 Vif and SIVmac Vif were detected with anti-Vif and anti-V5 epitope tag MAbs, respectively. SIVmac Vif appeared to be present at higher levels, probably because it was supplemented in trans. HIV-1 Vif did not exclude rhA3G from virions (Fig. , upper part). In contrast, SIVmac Vif and SEMQ mutant Vif effectively prevented the encapsidation of rhA3G. SIVmac Vif and SEMQ mutant Vif correspondingly reduced steady-state rhA3G levels in the cell lysates (Fig. , lower part). Similarly, HIV-1 Vif did not exclude D128K mutant hA3G from virions but SIVmac and SEMQ mutant Vif were effective and reduced the steady-state levels of D128K mutant hA3G in the cell lysates (Fig. , upper and lower parts). SEMQ mutant Vif was expressed at amounts comparable to wild-type Vif in each panel, suggesting that the effect was not caused by differences in levels of Vif.
FIG. 4. SEMQ mutant Vif excludes hA3G, rhA3G, and D128K mutant hA3G from virions. (A) Encapsidation of hA3G (left), rhA3G (middle), and D128K mutant hA3G (right) was tested in wild-type HIV-1 (lane 1), Δvif mutant HIV-1 (lane 2), Δvif mutant HIV-1 (more ...) HIV-1 with the SEMQ mutation replicates in human cells that stably express rhA3G.
To test whether introduction of the SEMQ mutation would allow HIV-1 to productively replicate in cells that express rhA3G, the mutation was introduced into replication-competent NL4-3. The SEMQ mutant NL4-3 virus, along with control Δvif mutant and wild-type NL4-3, was used to infect HOS.T4.X4 cells that stably expressed CD4/CXCR4 and hA3G or rhA3G or that lacked A3G. Clones of HOS cells that expressed moderate (clone 8) or high (clone 5) levels of rhA3G were used (Fig. ). Virus replication kinetics was measured over 2 weeks. The three viruses replicated similarly in cells that lacked A3G (Fig. ). On cells that express hA3G, the wild-type and SEMQ mutant viruses replicated but the Δvif mutant virus replicated poorly (Fig. ). On clone 8 and clone 5 rhA3G cells, the SEMQ mutant virus replicated efficiently, unlike the Δvif mutant and wild-type viruses, which failed to replicate (Fig. ). These results show that the SEMQ mutation allows efficient replication of HIV-1 on cells that expressed relatively high levels of rhA3G.
FIG. 5. Viral replication kinetics in cells expressing hA3G or rhA3G. (A) hA3G and rhA3G stably expressed by HOS.T4.X4 cells derived by retroviral vector transduction were detected by an immunoblot assay and probing with an anti-HA MAb. The parental cells lack (more ...) rhA3F does not block HIV-1 replication.
hA3F has previously been shown to block Δvif
mutant HIV-1 infectivity in single-round reporter virus assays (14
). To test whether A3F restricts HIV-1 in a species-specific manner similar to that of A3G, we cloned rhA3F (see Fig. S2 in the supplemental material) and tested its activity against wild-type, Δvif
mutant, and SEMQ mutant HIV-1 reporter viruses. As expected, hA3F inhibited Δvif
mutant but not wild-type HIV-1 (14
). In contrast, hA3F inhibited the SEMQ mutant virus. This suggested that the SEMQ mutation caused Vif to lose its ability to counteract hA3F (Fig. ). rhA3F inhibited wild-type HIV-1 and the SEMQ mutant. hA3F and rhA3F inhibited Δvif
mutant SIVmac, and this was partially overcome by SIVmac Vif, consistent with a recent report by Zennou and Bieniasz (32
) which showed that SIVmac Vif was partially active against rhA3F. Our data suggested that the interaction between Vif and A3F is species specific and that SEMQ mutant Vif fails to interact with rhA3F.
FIG. 6. rhA3F does not block HIV-1 replication. (A) The antiviral effects of hA3F and rhA3F were tested in a single-cycle luciferase reporter virus assay. Virus was produced by cotransfection of wild-type, Δvif mutant, or SEMQ mutant NL-Luc or wild-type (more ...)
To determine whether rhA3F blocks the replication of HIV-1, we infected HOS cells that stably express CD4/CXCR4 and rhA3F with replication-competent wild-type, Δvif mutant, or SEMQ mutant NL4-3 or NL4-3(Vifmac), an HIV-1 strain that contains an engineered SIVmac vif gene in the nef position. The HOS cells expressed moderate (C1) or high (C4) levels of rhA3F (Fig. ). All viruses replicated well in the parental HOS.CD4.CXCR4 cell line (Fig. ). Surprisingly, intermediate and high levels of rhA3F had no effect on the replication kinetics. Some differences in peak p24 production were evident among the HOS cell clones, but these were caused by differences in coreceptor expression (data not shown). Taken together, these data demonstrate that although rhA3F is active against HIV-1 in the single-cycle reporter virus assay, it does not block virus replication under more physiological conditions.