Mapping of critical lysines for HIV-1 Vif function.
Previously, we created a lysine-free HIV-1 Vif mutant from pNL4-3 by replacing all 16 lysines with arginines (16K/R) and found that the mutant lost neutralizing activity against human A3G (5
). The sequence of the pNL4-3 vif
gene is shown in Fig. . To map lysines critical for Vif function, we created Vif lysine mutants and determined whether they lost activity against human A3G or A3F. Initially, two recombinant vif
genes were created from the WT and 16K/R vif
genes: 1-6K/R, with the N-terminal 6 lysines (K22, K26, K34, K36, K50, and K63) mutated to arginines, and 7-16K/R, with the C-terminal 10 lysines (K91, K92, K141, K155, K157, K160, K168, K176, K179, and K181) mutated to arginines. These two recombinant genes were cloned into pNL-A1, a pNL4-3-derived HIV-1 subgenomic vector, for vif
). Their activities were determined by measuring their abilities to rescue replication of HIV-1Δvif
in the presence of A3G or A3F. In the absence of Vif, the viruses were poorly infectious, indicating potent antiviral activity of both A3G and A3F (Fig. ). Consistently, the ΔSLQ mutant, in which the EloC-binding motif 144
Q was mutated to AAA, did not neutralize either A3G or A3F (Fig. ). As previously reported, the 16K/R mutant completely lost activity critical to A3G, and we found that it retained only partial activity against A3F (Fig. ). The 7-16K/R mutant retained full activity against both A3G and A3F (Fig. ), whereas the 1-6K/R mutant completely lost activity against A3G but not A3F (Fig. ). This result indicated that the N-terminal six lysines are important for Vif activity against A3G.
FIG. 1. Critical role of the HIV-1 Vif K26 residue in neutralizing A3G, but not A3F. (A) Amino acid sequence alignment of Vif proteins expressed from pNL4-3 and pNL-A1. The lysines are underlined and numbered. (B and C) Mutational analysis of Vif activity against (more ...)
To identity more critical lysines, we compared Vif protein sequences from pNL4-3 and pNL-A1. Among the N-terminal six lysines in pNL4-3, only three (K22, K26, and K34) exist in pNL-A1 (Fig. ). We mutated these three lysines to arginines and created a triple-lysine mutant, 3K/R. The 3K/R mutant, like 1-6K/R, failed to neutralize A3G but retained activity against A3F (Fig. ). Thus, K22, K26, and K34 are essential for Vif activity against A3G.
To compare these three lysines, we created three double-lysine mutants: K22-26/R, with both K22 and K26 changed to arginines; K22-34/R, with K22 and K34 changed to arginines; and K26-34/R, with K26 and K34 changed to arginines. All three mutants were still active against A3F, but K22-26/R and K26-34/R were completely inactive against A3G, while K22-34/R was only partially active against A3G (Fig. ). This result suggested that K26 is more critical than K22 and K34 for Vif to neutralize A3G.
We replaced each of these three lysines with alanine, aspartic acid, or arginine and created nine single-lysine mutants (Fig. ). Because aspartic acid has a negative charge, alanine has a neutral charge, and both arginine and lysine have positive charges, this mutagenesis allowed us to address whether the charges at these positions affect Vif function. Indeed, these mutants had very different phenotypes. The K22D mutant completely lost activity to neutralize A3G but not A3F, while the K22A and K22R mutants retained almost full activity against both proteins (Fig. ). The K34D mutant partially lost activity against both A3G and A3F, while the K34A and K34R mutants retained almost full activity (Fig. ). Interestingly, the K26A, K26D, and K26R mutations completely inactivated Vif activity against A3G, but not A3F (Fig. ). Because the reduction in Vif activity was more dramatic when mutations were introduced into K22 and K26, we conclude that K22 and K26 play more important roles than K34 in Vif function.
To validate these observations, we determined the levels of A3G and A3F protein expression in the presence of WT Vif or the Vif mutants in viral producer cells. The WT protein effectively reduced both A3G and A3F expression, although its effect on A3G was more potent than that on A3F (Fig. , lanes 1 and 2, and E, lanes 1 and 2). Mutants 16K/R, ΔSLQ, 3K/R, 1-6K/R, K22D, K26A, K26D, and K26R completely failed to reduce A3G protein expression; mutants K22-26/R, K26-34/R, and K34D partially reduced A3G protein expression; and mutants 7-16K/R, K22-34/R, K22A, K22R, K34A, and K34R fully reduced A3G expression, like the WT protein (Fig. ). In addition, mutants 16K/R, ΔSLQ, and K34D failed to reduce A3F expression, and the other mutants reduced A3F expression either as effectively as or more effectively than the WT Vif protein (Fig. ). In general, these results are in agreement with the previous results, further highlighting the important roles of K22 and K26 in Vif function.
Identification of a lysine critical for SIV Vif function.
To further explore the functions of K22 and K26, we compared the N-terminal amino acid sequences of Vif proteins from different primate lentiviruses. Vif sequences from all subtypes of HIV-1, SIVcpz, HIV-2, SIVmac239, and three different SIVagm strains, SIVagmTan, SIVagm9063, and SIVagmSab, were initially selected (Fig. ). It was found that both K22 and K26 were located in a highly conserved 21WKSLVK26 motif in HIV-1 Vif (22WHSLIK27 in HIV-2 and SIVmac239 and 23WxxxVK28 in SIVagm). Further analyses indicated that a consensus motif, W[N/K/H]SL[V/I]K, is present in >90% of 2,742 Vif sequences from HIV-1, HIV-2, and SIVmac, and it is present in ~75% of 113 Vif sequences from SIVcpz (Table ). Clear species-specific motifs were observed for HIV-1 (W[N/K]SLVK), HIV-2 (WHSL[V/I]K), SIVmac (WHSLIK), and SIVcpz (W[N/H]SL[I/V]K), while SIVagm showed more variation (Table ). We therefore summarized these sequences as a general consensus motif, WxSLVK.
FIG. 2. Critical roles of SIVmac Vif K27 and SIVagm Vif K28 residues in neutralizing A3G, but not A3F. (A) N-terminal amino acid sequence alignment of Vif proteins from HIV-1, HIV-2, SIVcpz, SIVmac239, and three SIVagm isolates. Three lysines (K22, K26, and K34) (more ...)
Motif sequences in HIV and SIV strainsa
Since the K26 (or K27 in SIVmac and K28 in SIVagm) residue is highly conserved, we decided to continue to study the role of this residue in SIV Vif function. A K27A or K28A mutation was introduced into the SIVmac239 or SIVagm9063 vif
gene, respectively, and the genes were subsequently cloned into the pNL-A1 vector for expression. Both SIVmac Vif K27A and SIVagm Vif K28A were expressed at levels comparable to those of the WT proteins after transfection into 293T cells (Fig. ). SIVmac Vif K27A almost completely lost activity against human A3G, macA3G, and agmA3G and partially lost activity against human A3F and agmA3F (Fig. ). As previously reported, macA3F had very low antiviral activity (48
), and it was impossible to compare the abilities of WT and K27A Vifs to neutralize macA3F (Fig. ). The SIVagm Vif K28A mutant partially lost activity against agmA3G and completely lost activity against agmA3F (Fig. ). In general, the K27A or K28A mutation in SIV Vif tended to selectively inactivate the A3G protein. Thus, we conclude that, like the K26 in HIV-1 Vif, the K27 or K28 in SIV Vif is more important in protecting against A3G than A3F.
Role of SLV in Vif function.
We found that SL[V/I] residues are also conserved, except in SIVagm (Fig. and Table ). We next addressed how they could regulate Vif function. All three SLV residues in HIV-1 Vif (or SLI in SIVmac) were mutated to alanines, creating a ΔSLV mutant for HIV-1 Vif and ΔSLI for SIVmac Vif. These mutants were expressed at levels comparable to those of their WT proteins after transfection into 293T cells (Fig. ). However, ΔSLV and ΔSLI failed to neutralize both A3G and A3F proteins from humans, and ΔSLI failed to neutralize both A3G and A3F proteins from humans, MAC, and AGM (Fig. ). Thus, we conclude that, unlike K22 and K26, the SLV residues are critical for Vif neutralization of both A3G and A3F.
FIG. 3. Critical roles of SLV/I residues in Vif function. (A) Expression of HIV-1 Vif ΔSLV and SIVmacVif ΔSLI mutants in 293T cells. (B) Activities of ΔSLV and ΔSLI mutants determined as for Fig. . The standard (more ...) Actions of K22, SLV, and K26 in Vif activity.
How do the K22, SLV, and K26 residues regulate HIV-1 Vif function? One important question was whether they could regulate A3G or A3F binding and degradation. As noted above, the 14DRMR17 domain of Vif binds to A3F and the 69YxxL72 domain of Vif binds to both A3G and A3F. We introduced an M16A or Y69A mutation into HIV-1 Vif to disrupt the DRMR or YxxL motif, respectively. We had confirmed that the M16A mutant failed to neutralize A3F and Y69A failed to neutralize both A3F and A3G (data not presented). These two mutants were used as controls in Vif/A3 protein binding experiments.
A3F or A3G fusion proteins with a FLAG-HA tag were coexpressed with different Vif mutants in 293T cells, and a similar GFP expression vector was included as an additional negative control. The proteins were pulled down by the anti-FLAG antibody-conjugated beads, and bead-associated proteins were determined by Western blotting. GFP did not bind to the WT Vif proteins, whereas both A3F and A3G bound to the WT Vif proteins (Fig. , lanes 6 and 7, and B, lane 1). As expected, the M16A mutant bound to A3G, whereas the Y69A mutant bound poorly to A3G (Fig. , lanes 3 and 5). We previously reported that the binding of the 16K/R mutant to A3G was slightly reduced (5
), and we found that its binding to A3F was also slightly reduced (Fig. , lane 8). The ΔSLV mutant bound to A3F and A3G as efficiently as did the WT Vif (Fig. , lane 9, and B, lane 2), and the K26R mutant still bound to A3F (Fig. , lane 10). We further compared how the mutations at amino acid position 22, 26, or 34 affected Vif binding to A3G. Mutants K22D, K26A, and K26D completely lost binding ability to A3G, whereas mutants K22A, K22R, K26R, K34A, K34D, and K34R still bound to A3G (Fig. , lanes 6 to 14). In general, these binding results were consistent with those from Vif neutralization assays, although it was unclear why ΔSLV and K26R failed to neutralize A3G or A3F.
FIG. 4. Mechanism of K22, SLV, K26, and K34 residues in regulating Vif function. (A and B) Interaction of Vif mutants with A3F or A3G. The indicated Vif proteins were coexpressed with GFP, A3F, or A3G protein with a FLAG-HA tag in 293T cells. The proteins were (more ...)
Next, we determined whether the mutations affect the degradation of A3F and A3G. We determined A3F and A3G protein levels in both viral producer cells and virions in the presence of Vif mutants. HIV-1 proviral clones containing a ΔSLV, K22A, K26A, or K34A mutation in the vif gene were coexpressed with A3F or A3G proteins in 293T cells; viral particles were collected and purified from these cell cultures; and A3F and A3G levels were determined by Western blotting. The WT, K22A, and K34A Vif proteins effectively decreased A3F and A3G levels in both cells and virions (Fig. , lanes 2, 4, 6, 8, 10, and 12); the ΔSLV mutant did not decrease A3G and A3F expression in cells or virions (Fig. , lanes 3 and 9); and the K26A mutant could decrease only A3F, but not A3G, expression in cells and virions (Fig. , lanes 5 and 11). These results are consistent with those from viral replication assays (Fig. ) and confirmed that these residues regulate A3F and A3G neutralization via protein degradation and virion exclusion.
Replication of HIV-1 with K26R and ΔSLV mutations in human T-cell lines.
To further evaluate the functions of these residues, human T cells were infected with HIV-1 bearing either a K26R or a ΔSLV mutation in the vif gene, and viral growth curves were determined. As a comparison, viruses with either an M16A or a Y69A mutation in vif were also included. One permissive cell line (CEM-SS) and three nonpermissive cell lines (H9, Hut-78, and PM1), as well as human PBMC, were selected. These cells were infected with equal amounts of each virus, and viral replication was determined by measuring the p24Gag concentration in the supernatant using an ELISA kit. As expected, in CEM-SS cells, the WT and vif-defective viruses and four vif mutant viruses (M16A, K26R, ΔSLV, and Y69A) all grew well, because viruses do not need a functional vif gene in this cell line (Fig. ). Moreover, we were able to detect all these different Vif proteins in CEM-SS cells by Western blotting on days 6 and 9 postinfection, indicating that they were well expressed during viral replication (Fig. ). In contrast, in Hut-78, H9, and PM1 cells and PBMC, only the WT virus replicated well, whereas the ΔVif virus showed approximately a 2-log-unit reduction in viral production, because viruses need a functional vif gene in these cell lines (Fig. ). In addition, viral replication was inhibited by both M16A and K26R mutations, and the K26R mutation caused a more profound inhibition than the M16A mutation. Because the M16A and K26R mutants did not neutralize A3F or A3G, respectively, this result confirmed that A3G has stronger antiviral activity than A3F. The replication of HIV-1 with a Y69A or ΔSLV mutation was further reduced to the Δvif HIV-1 levels, confirming that these two vif genes failed to neutralize both A3F and A3G. Thus, these results were consistent with those from viral replication assays (Fig. ) and further confirmed the importance of SLV and K26 in Vif function.
FIG. 5. Replication of HIV-1 with mutations in the vif gene in human T-cell lines. (A) Replication in a permissive cell line. CEM-SS cells were infected with equal amounts of six different HIV-1 strains (WT, ΔVif, M16A, K26R, ΔSLV, and Y69A). (more ...)