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. Author manuscript; available in PMC: 2015 Jan 20.
Published in final edited form as: Virology. 2013 Dec 5;449:140–149. doi: 10.1016/j.virol.2013.10.037

Analysis of the N-terminal Positively Charged Residues of the Simian Immunodeficiency Virus Vif Reveals a Critical Amino Acid Required for the Antagonism of Rhesus APOBEC3D, G, and H

Kimberly Schmitt 1, Miki Katuwal 1, Yaqiong Wang 1, Cicy Li 1, Edward B Stephens 1
PMCID: PMC4104721  NIHMSID: NIHMS540678  PMID: 24418547

Abstract

Previous studies have shown that apolipoprotein B mRNA editing, enzyme catalytic, polypeptide G (APOBEC3G; hA3G) and F (APOBEC3F; hA3F) proteins interact with a nonlinear binding site located at the N-terminal region of the HIV-1 Vif protein. We have analyzed the role of 12 positively charged amino acids of the N-terminal region of the SIV Vif. Simian-human immunodeficiency viruses (SHIV) were constructed that expressed each of these amino acid substitutions. These viruses were examined for replication in the presence of rhesus macaque APOBEC3 proteins (rhA3A-rhA3H), incorporation of the different A3 proteins into virions, and replication in rhesus macaque PBMC. Similar to other studies, we found that K27 was essential for rhA3G activity and rhA3F but was not important for restriction of SHIVΔvif by rhA3A, rhA3D or rhA3H. Our results identified the arginine at position 14 of the SIV Vif as a critical residue for virus restriction by rhA3D, rhA3G and rhA3H.

INTRODUCTION

Select members of the apolipoprotein B mRNA editing, enzyme catalytic polypeptide 3 (APOBEC3; A3) gene family in primates represent an innate host defense system that can inhibit the replication of human immunodeficiency virus type 1 that does not express a Vif protein (Chiu and Greene, 2008; Goila-Gaur and Strebel, 2008; Harris et al., 2003; Jarmuz et al., 2002; Sheehy et al., 2002). The A3 family in humans and rhesus macaques consists seven members (A3A, A3B, A3C, A3D, A3F, A3G, and A3H) that are tandemly arranged on chromosome 22 or 10, respectively. All seven members either have one (A3A, A3C, A3H) or two (A3B, A3D, A3F, A3G) canonical cytidine deaminase motifs (H-x-E-x23-28-P-C-x2-4-C) (Betts et al., 1994; Dang et al., 2007; Jarmuz et al., 2002; Schmitt et al., 2011; Wedekind et al., 2003; Xie et al., 2004). Following the identification of A3G (formerly CEM15) as a potent inhibitor of the replication of Vif deficient HIV-1 (HIV-1Δvif ), it was found that in the absence of Vif A3G is incorporated and causes cytidine to uracil changes in the minus strand of single-stranded DNA during reverse transcription, ultimately leading to G-to-A mutations in the viral genome (Kao et al., 2003; Lecossier et al., 2003; Mangeat et al., 2003; Sheehy et al., 2002; Yu et al, 2004; Zhang et al., 2003). The Vif protein acts as an adaptor that binds the APOBEC3 proteins to the Cul5/ElonginB/C/rbx E3 ligase complex for ubiquitination and degradation by the proteosome (Conticello et al., 2003; Dussart et al., 2004; Kobayashi et al., 2005; Liu et al., 2004; Marin et al., 2003; Mehle et al., 2004; Sheehy et al., 2003; Stopak et al., 2003; Yu et al., 2003; 2004). In addition to A3G, other A3 proteins have been shown to restrict the replication of HIV-1Δvif . A3F is the most closely related protein to A3G at the amino acid level and has a tissue distribution similar to A3G (Wiegand et al., 2004; Zheng et al.,2004). When expressed exogenously, hA3F is incorporated into and restricts the replication of HIV-1Δvif virions, although the level of deamination is approximately 10 times less than A3G (Holmes et al., 2007; Wang et al., 2007). A3F has been found to be expressed at lower levels in human CD4+ T cells than A3G, causes less cytidine deamination of Vif-deficient HIV-1 and has less or no effect on its (Binka et al., 2012; Chaipan et al., 2013; Miyagi et al., 2010). A3B has been reported to have moderate activity against HIV-1Δvif but is expressed at low levels in natural HIV-1 cellular targets, so its role in restriction of HIV-1 is uncertain (Doehle and Cullen, 2005; Refsland et al., 2010; Yu et al., 2004). A3D was also identified to potently restrict HIV-1Δvif (Dang et al., 2007; Liddament et al., 2004; Weigand et al., 2004; Zheng et al., 2004). A3H has at least seven haplotypes with haplotype II being able to potently restrict HIV-1Δvif and HTLV-I replication (Dang et al., 2008; OhAinle et al., 2008; Ooms et al., 2010; 2012). A3A is neither incorporated into the viral nucleocapsid nor does it restrict the replication of HIV-1Δvif (Goila-Gaur et al., 2007). A3C is incorporated into the of HIV-1 virion and has been reported to cause limited cytidine deamination while other studies indicate that it has no effect on virus infectivity (Langlois et al., 2005; Hultquist et al., 2011).

Several domains have been identified in the N-terminal region of the HIV-1 Vif that are involved in the interaction with several human APOBEC3 proteins (Chen et al., 2009; Dang et al., 2007; 2010; Mehle et al., 2007; Pery et al., 2009; Russell and Pathak et al., 2007; Wichroski et al., 2005). Several studies that have used site-directed mutagenesis to identify amino acid residues that are critical for Vif neutralization of hA3G and hA3F. One study showed that deletion of amino acids 43-59 abolished interactions with A3G (Wichroski et al., 2005). In another study, the use of overlapping peptides found that a region from amino acids 33-88 formed a non-linear binding site for A3G (Mehle et al., 2007). Additionally, amino acid residues 14-17 and 40-44 were specifically found to effect the interactions with human A3F and A3G, respectively (Russell and Pathak, 2007). Other investigators showed that domains 23SLVx4Yx9Y38, 69YXXL72, 81LGxGxxIxW89 and 171EDRW174 were also involved in neutralizing hA3G and hA3F (Chen et al., 2009; Dang et al., 2010; Pery et al., 2009).

The simian immunodeficiency virus (SIV) and simian-human immunodeficiency virus (SHIV)/macaque models of infection have been used extensively to study different aspects of lentiviral pathogenesis and development of vaccines. Similar to the HIV-1 Vif, the SIV Vif has comparable YXXL, SLQYLA and HCCH domains (Luo et al., 2005; Pery et al., 2009; Schmitt et al., 2009; 2010; Yu et al., 2004). However, the N-terminal region of the SIV Vif (and other regions) have little sequence identity with the same region from the HIV-1 Vif, making it difficult to extrapolate findings to the Vif protein of SIV for testing in vivo. The SIV Vif has a region containing several positively charged amino acids that may be involved in electrostatic interactions with different rhesus macaque A3 proteins. In this study, we analyzed the importance of these positively charged residues on the exclusion of different rhesus macaque APOBEC3 proteins (rhA3A-H) from virions and on infectivity. We have identified a novel amino acid within the SIV Vif that is important for restriction by rhA3D, rhA3G, and rhA3H.

RESULTS

Expression and stability of the Vif mutants

Comparison of the amino acid sequence of the HIV-1 and SIVmac Vif proteins show that there was 18% sequence identity at the first 50 amino acid positions (Fig. 1). To determine which positively charged amino acids at the N-terminus of the protein were critical to Vif function (i.e., virus replication in the presence of the rhesus A3 proteins), we substituted the positively charged residues at positions 5, 6, 14, 18, 21, 23, 27, 30, 32, 34, 38, and 46 to alanines (Fig. 1). First, we examined the stability of the Vif mutant proteins in cells to insure that any observed effects on virus restriction were not due to an unstable Vif protein. For these experiments, an SIV Vif (human codon-optimized) with an N-terminal 3X-Flag-tag was used. 293 cells were transfected with vectors expressing each Vif protein for 48 h. Cells were radiolabeled with 35S-methionine and the radiolabel chased for 0 and 6 h. The cells were lysed and Flag-tagged proteins were immunoprecipitated using a rabbit polyclonal anti-Flag antibody. The results of this experiment showed that the twelve SIV Vif mutants were stable in 293 cells similar to the unmodified SIV Vif at the 6 h time point (Fig. 2).

Fig. 1.

Fig. 1

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Alignment of the amino terminal 50 amino acids of the HIV-1 and SIV Vif proteins. Underneath the SIV Vif sequence, the mutants analyzed in this study are indicated in bold.

Fig. 2.

Fig. 2

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Expression of the different mutant Vif proteins. Vectors expressing each Flag-tagged Vif mutant were transfected into 293 cells for 48 h. The cells were starved for methionine/cysteine and radiolabeled with 35S-methionine/cysteine for 60 minutes. The radiolabel was removed, the cells washed three times, and incubated in medium containing cold excess methionine/cysteine for either 0 or 6 h. The cells were lysed in 1X RIPA buffer and the FLAG-tag containing Vif proteins immunoprecipitated with a rabbit anti-Flag antibody. Panel A. Results of the pulse-chase analyses of cells transfected with vectors expressing the unmodified Vif, Δvif (first 27 amino acids), VifK5A, VifR6A, VifK14A, and VifR18A. Panel B. Results of the pulse-chase analyses of cells transfected with vectors expressing the VifK21A, VifH23A, VifK27A, VifK30A, VifK32A, VifK34A, VifK38A, and K46A. The time of the chase in hours is indicated above each lane.

Replication of SHIVs expressing mutant Vif proteins

We constructed SHIV viral genomes expressing Vif proteins with the amino acid substitutions in the N-terminal region as described above. The SHIV used, SHIVKU-2MC-4, has the tat, rev, vpu and env from HIV-1 (HXBc2) in the background of SIVmac239 and was derived from passage in rhesus macaques (Liu et al., 1999). Virus stocks were prepared in SupT1 cells, which do not express A3 proteins. Growth curves (based on p27 release) were established for each virus in SupT1 cells and results indicate that all viruses replicated in the SupT1 cells (Fig. 3A-B).

Fig. 3.

Fig. 3

Fig. 3

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Replication of SHIVs expressing each Vif mutant in SupT1 cells. SupT1 cells were inoculated with equivalent levels (25 ng) of SHIVKU-2MC4, SHIVΔvif, or viruses expressing each of the mutant Vif proteins. At 4 h post-inoculation, the medium was removed and replaced with fresh medium and incubated for 15 days. Replication was assessed by the level of p27 in the medium at specific days post-inoculation. The experiment was performed twice and p27 levels did not vary by more than 5%. Panel A. Replication of SHIVKU-2MC4, SHIVΔvif, and SHIVs expressing Vif mutants R5A, K6A, R14A, K18A, R21A, and H23A. Panel B. Replication of SHIVKU-2MC4, SHIVΔvif, and SHIVs expressing Vif mutants K27A, K30A, K32A, K34A, K38A, and K46A.

Incorporation of macaque rhA3A-H into SHIV, SHIV Δvif and SHIV vif mutants into viral particles

To determine if the seven rhA3 proteins were incorporated into SHIVΔvif, parental SHIVKU-2MC4, or SHIVs expressing the various Vif mutants, 293 cells were co-transfected with plasmids that expressed each of the tagged rhA3 proteins and each of the SHIV genomes. At 24 h, the cells were radiolabeled and the culture medium harvested at 36 h post-transfection. The virus was purified by ultracentrifugation as described in the Material and Methods section and immunoprecipitations were performed using an antibody against the HA-tag to determine if the A3 proteins were incorporated into virions. We chose radiolabeling and immunoprecipitations over immunoblots as they were more sensitive in detecting A3 proteins. Our results indicate that rhA3B but not rhA3C was incorporated into both SHIVΔvif and SHIVKU-2MC4 (Fig.4A). Since rhA3B or rhA3C did not affect the replication of SHIVΔvif and SHIVKU-2MC4 (see below in Table 1), we did not proceed with incorporation studies. Our results indicate that rhA3A was incorporated into SHIVΔvif but not SHIVKU-2MC4 or any of the SHIVs expressing the mutants Vif proteins (Fig. 4B). We found that rhA3D was incorporated into SHIVΔvif and SHIVVifR14A and to a lesser extent in SHIVVifR6A and SHIVVifR21A; however, the incorporation of rhA3D into SHIVs expressing the other Vif mutants were comparable to SHIVKU-2MC4 (Fig. 4C). RhA3F was found to be incorporated into SHIVKU-2MC4, SHIVΔvif, and to some extent in all of the SHIVs expressing the mutant Vif proteins, while rhA3G was incorporated into SHIVΔvif, SHIVVifR14A, and SHIVVifK27A (Fig. 4D-E) . Finally, rhA3H was incorporated into SHIVΔvif, and SHIVVifR14A in significant quantities (Fig. 4F). In summary, our results show that rhA3D, G, and H were readily incorporated into SHIVVifR14A, and that rhA3G was significantly incorporated into SHIVVifK27A. As we have previously reported, it is interesting to note that rhA3F was incorporated into all viruses tested (Schmitt et al., 2010).

Fig. 4.

Fig. 4

Fig. 4

Fig. 4

Fig. 4

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The incorporation of A3 proteins into viruses expressing each of the mutant Vif proteins. 293 cells were transfected with a plasmids expressing rhesus macaque tagged A3 proteins and genomes for SHIVKU-2MC4, SHIVΔvif, or the SHIVs expressing the different mutant Vif proteins. At 24 h post-transfection, the cells were starved for methionine/cysteine and radiolabeled with 35S-methionine/cysteine for 12 h. The culture medium was collected and the virus partially purified as described in the Materials and Methods section. The virus preparations were lysed in 1X RIPA buffer. Half of the samples were immunoprecipitated with an anti-HA serum to detect the HA-tag containing A3 proteins while the other half immunoprecipitated with anti-SHIV serum to detect the p27 protein. Samples were normalized for equivalent amounts of p27. A3 proteins were also immunoprecipitated from cell lysates with both anti-HA and anti-SHIV sera to show that both A3 and SHIV proteins were expressed in cells. For each panel, the upper gel represents the immunoprecipitation of A3 proteins from the cell lysates, the middle gel the A3 proteins in virus preparation and the lower gel the immunoprecipitation of p27 protein from the same virus preparation. Panel A. RhA3B and rhA3C. Panel B. RhA3A. Panel C. RhA3D. Panel D. RhA3F. Panel E. RhA3G. Panel F. RhA3H. The viral genomes or vectors transfected into cells are shown above the lanes.

Table 1.

Infectivity of SIV Vif N-Terminal Mutants

Vif ΔVif K5A R6A R14A R18A R21A H23A K27A K30A K32A K34A K38A K46A
RhA3A 100a 7* 86 44 90 47 44 76 86 100 78 46 93 88
RhA3B 100 100 79 80 66 100 76 100 100 100 100 95 100 100
RhA3C 100 91 100 63 60 100 53 100 100 100 48 70 70 62
RhA3D 100 2* 57 24* 0.7* 36 18* 48 100 66 75 35 92 93
RhA3F 100 7* 99 37 21* 100 14* 11* 19* 23* 13* 16* 100 84
RhA3G 100 4* 100 61 0.1* 100 57 77 1.0* 102 80 62 100 100
RhA3H 100 22* 65 100 1.0* 93 37 16* 79 25* 100 14* 98 70
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a

Infectivity of virus expressing the unmodified Vif was considered 100%. Those values that were statistically significant are marked with an (*). Infectivity was determined by transfecting 293 cells with the vectors with the viral genome and one expressing one of the rhA3 proteins, collecting virus at 48 h and assaying for infectivity using the TZM.bl assay.

Restriction of replication of SHIVs expressing mutant Vif proteins in the presence of the seven rhesus A3 proteins

We assessed the ability of the SHIVs expressing mutant Vif proteins to replicate in the presence of the seven rhesus A3 proteins. For these studies, we used N-terminally HA-tagged rhA3A-H and plasmids containing the SHIVKU-2MC4, SHIVΔvif, or the different Vif mutants viral genomes. All experiments were repeated at least six times and the mean shown in Table 1. Our results indicate that rhA3A, rhA3B and rhA3C did not restrict the replication of SHIVs containing the various Vif amino acid substitutions. Similar to previous studies, we found that the K27A mutant replicated poorly (1% of control) in the presence of rhA3G and in the presence of rhA3F (19% of control) but replicated well in the presence of rhA3A, rhA3D, rhA3F, and rhA3H. This indicates that the K27A mutation specifically resulted in sensitivity to rhA3G and to lesser extent rhA3F. A salient finding of the study was that rhA3D, rhA3G and rhA3H profoundly effected the replication of SHIVVifR14A. We also observed that rhA3D effected the replication of SHIVVifR6A and most of the Vif mutants with the exception of SHIVs expressing K5A, R14A, R18A, K38A, and K46A. RhA3G was found to effect the replication of three Vif mutants R14A, and K27A. It is important to note that the effect of K27 substitution on virus infectivity has previously been reported by other investigators (Chen et al., 2009; Dang et al., 2010). Finally, rhA3H was found to effect the replication of Vif mutants R14A, H23A, K30A, and K34A.

Replication of SHIVs expressing mutant Vif proteins in rhesus PBMC

We showed above that each of the viral genomes were capable of replicating in SupT1 cells. Therefore, we determined if the above Vif mutants were capable of replicating in rhesus PBMCs (Fig. 5A-B). PBMC were isolated, stimulated and then inoculated with 104 TCID50 of virus for 4 h. The inoculum was removed, cells washed, and then incubated in fresh medium containing 50 ng of IL-2. Medium was removed every other day for up to 15 days and analyzed for p27. The results of this experiment are shown in Fig. 5A-B. The results indicate that the mutants SHIVVifK5A, SHIVVifR6A, SHIVVifR18A, and SHIVVifR21A replicated in rhesus PBMC to levels similar to the parental SHIVKU-2MC4, while SHIVVifR14A and SHIVVifR27A and SHIVVifK30A did not replicate to any significant extent in rhesus PBMC (Fig. 5A-B). Additionally, viruses such as SHIVVifH23A, SHIVVifK32A, SHIVVifK34A, SHIVVifK38A, and SHIVVifK46A also replicated in the rhesus PBMC but at reduced levels.

Fig. 5.

Fig. 5

Fig. 5

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Rhesus macaque PBMC growth curves of SHIVs expressing Vif mutants. Blood from uninfected naive rhesus macaques was used to isolate PBMC on Ficoll-Hypaque gradients and stimulated for 2 days in medium containing Conconavlin A (10 μg/ml) and IL-2 (50 ng/ml). The cells were washed three times, and the cells were inoculated with equivalent levels of SHIVKU-2MC4, SHIVΔvif, or SHIVs expressing each of the Vif mutants for 4 h. The cells were again washed to remove the residual inoculum and incubated in fresh medium containing 50 ng/ml IL-2 for 15 days. Fresh medium was added every three days containing 50 ng/ml IL-2. Panel A. Replication of SHIVKU-2MC4, SHIVΔvif, and SHIVs expressing the Vif mutants R5A, K6A, R14A, R18A, R21A, and H23A. Panel B. Replication of SHIVKU-2MC4, SHIVΔvif, and SHIVs expressing the Vif mutants K27A, K30A, K32A, K34A, K38A, and K46A.

VifR14A binds less efficiently with rhA3D, rhA3G, and rhA3H while VifK27A binds less efficiently with rhA3G

SHIVs expressing VifR14A incorporated rhA3D, G, and H into virus particles whereas a SHIV expressing VifK27A had increased rhA3G incorporated into virus particles. Therefore co-immunoprecipitation experiments were used to determine if these Vif proteins still interacted with rhA3D, G, and H. For these experiments we used Flag-tagged SIV Vif proteins (SIVVifwt, VifR14A, VifK27A, control FLAG-tag vector, and a VifΔ17-50 in which amino acids 17-50 were deleted and does not bind rhA3D,G, and H) and HA-tagged rhA3D, rhA3G, or rhA3H proteins. Each of the Flag-SIV Vif proteins were co-transfected with one of the rhA3 proteins into 293 cells. At 48 h, cells were lysed in a non-ionic detergent, the Flag-tagged proteins immunoprecipitated with an anti-Flag rabbit antibody, and the complexes were collected on anti-rabbit coated magnetic beads. The immunoprecipitates were analyzed for the presence of HA-rhA3 proteins using an antibody against the HA-tag. Our results indicated that wtVif readily co-immunoprecipitated rhA3G while VifK27A and VifR14A co-immunoprecipitated significantly less rhA3G (Fig. 6). We also found that VifR14A co-immunoprecipitated less rhA3D and H compared to wtVif. These results suggest that both of these mutants interact less with rhA3G, rhA3D, and rhA3H.

Fig. 6.

Fig. 6

Fig. 6

Fig. 6

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Co-immunoprecipitation experiments reveal that VifR14A interacts with rhA3D, rhA3G, or rhA3H and VifK27A interacts with rhA3G at reduced efficiencies. 293 cells were transfected with vectors pSIVVif, pSIVΔvif, pSIVVifR14A, pSIVVifK27A, pSIVVifK38A, or pSIVVifΔ17-50 and vectors expressing HA-rhA3G, HA-rhA3D, or HA-rhA3H. At 48 h post-transfection, the cells were washed and lysed in a buffer containing 1% Triton X-100 for 30 min on ice and 30 min at room temperature. Cell lysates were clarified by centrifugation in a microfuge, and the complexes immunoprecipitated using an anti-FLAG antibody on anti-rabbit IgG-coated magnetic Dynabeads (Life Technologies) overnight at 4°C. The beads were washed three times with the buffer containing 1% Triton X-100 for 5 min at 4°C. The bound proteins were boiled in 50 Cl of 1X sample reducing buffer and proteins were separated by SDS-PAGE. Western blot analysis was performed using a rabbit polyclonal anti-HA primary antibody (sc-805, Santa Cruz Biotechnology) to detect HA-tagged A3 proteins and a rabbit polyclonal anti-FLAG primary antibody (F7425, Sigma) to detect the Vif proteins. The lysates containing the non-immunoprecipitated proteins were collected and made 1X with sample reducing buffer, boiled and subjected to Western blot analysis using anti-FLAG (for Vif) and anti-HA (for A3 proteins). For panels A-C, the upper blot represents the bound fractions co-immunoprecipitated with the anti-FLAG antibody and detected with either the rabbit polyclonal anti-FLAG antibody to detect the Vif proteins or the rabbit polyclonal anti-HA antibody to detect the HA-A3 proteins, respectively. The lower blot represents the unbound fractions from the co-immunoprecipitation with the rabbit polyclonal anti-FLAG antibody and detected with either the rabbit polyclonal anti-HA antibody for the HA-A3 proteins or the rabbit polyclonal anti-FLAG for the Vif proteins. Panel A. Co-immunoprecipitation of VifR14A and VifK27A with rhA3D. Panel B. Co-immunoprecipitation of VifR14A and VifK27A with rhA3G. Panel C. Co-immunoprecipitation of VifR14A and VifK27A with rhA3H.

VifR14A and VifK27A are incorporated into viral nucleocapsid complexes in the presence of rhA3G

As the data above showed, rhA3D, G, and H interacted less with VifR14A and VifK27A and interacted less with rhA3G. Next, we determined if rhA3G was incorporated into the core at levels that differed from the wild type Vif. At 48 h, the virus was collected from the culture medium, pelleted and resuspended in either PBS or PBS with 1% Triton X-100. Samples were loaded on a 20/60% step gradient and subjected to ultracentrifugation for 6 h. On these gradients intact virus was located at fraction 2 while the nucleocapsids were located in fraction 3. Fractions were collected, divided into three samples and immunoprecipitated with appropriate antibodies against p27 (for viral Gag), HA (for A3 proteins), and SHIV (for Env). As expected, cells transfected with vectors expressing the SHIVΔvif genome and rhA3G had significant rhA3G incorporated into virions compared to cells transfected with parental SHIVKU-2MC4 (Fig. 7A-B). Similar to SHIVΔvif, rhA3G was detected in intact virus or nucleocapsid complexes (Triton-X-100 treated virus) from cells transfected with vectors containing the SHIVVifR14A or SHIVVifK27A genomes and rhA3G (Fig. 7C-D). Thus, the pulldown experiments (Fig. 6) are in agreement with the incorporation data (Fig. 7).

Fig 7.

Fig 7

Fig 7

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Incorporation of rhA3G into the nucleocapsids of SHIVKU-2MC4, SHIVΔvif, SHIVVifR14A and SHIVVifK27A. 293 cells were transfected with plasmids containing the viral genomes (SHIVVifR14A, SHIVVifK27A, SHIVΔvif, or SHIVKU-2MC4) and rhA3G. At 24 h, the cells were starved and radiolabeled with 35S-methionine/cysteine for 24 h. The culture medium was harvested, and the virus concentrated using ultracentrifugation. The virus was then subjected to ultracentrifugation on sucrose step gradients and fractions S1-S4 collected as described in the Materials and Methods. Each fraction was analyzed for the presence of rhA3G (upper gel), p27 (middle gel) and envelope gp120 (lower gel). Panel A. SHIVKU-2MC4. Panel B. SHIVΔvif. Panel C. SHIVVifR14A. Panel D. SHIVVifK27A.

DISCUSSION

Analysis of the N-terminal sequence of Vif from SIVmac239 (SIVsmm/HIV-2 group) and HIV-1 (SIVcpz/HIV-1 group) Vif reveals little sequence identity (approximately 18%) between HIV-1 and SIV Vif proteins at the N-terminus (Fig. 1). Having constructed a full complement of HA-tagged rhesus macaque A3 proteins, we asked if any of the positively charged residues at the N-terminus of SIV Vif affected the anti-viral properties of the seven rhesus A3 proteins. We were especially interested in amino acids 27 to 38 that had five lysine residues spaced every two to four residues (27KYLKYKTKDLQK38). Twelve site-directed mutants in the SIV Vif were constructed and expressed in the context of simian-human immunodeficiency virus (SHIV). Our studies here indicate that SHIVs expressing Vif mutants K5A, R18A, K38A, and K46A had no significant effect on SHIVΔvif infectivity in the presence of the seven rhA3 protein. We found that the other six Vif mutants resulted in small reductions in infectivity that varied with the rhA3 protein. Of these amino acids, R21A was common to four rhA3 proteins and R6A was common to three A3 proteins. Our results from these 10 mutants suggest that amino acids 6-34 may form a surface that is involved in Vif interactions with rhA3D, rhA3F, rhA3G, and rhA3H.

Two amino acids analyzed in this study, K27 and R14, had the greatest impact on virus replication. Previous studies had shown that the K26 residue of the HIV-1 and the K27 residue of SIV Vif were critical for interaction with hA3G and its degradation (Chen et al., 2009; Dang et al., 2009). Our results here confirm this finding as rhA3G was significantly incorporated into SHIVVifK27A, resulting in a 100-fold and 5-fold reduction in infectivity of this virus in the presence of rhA3G and rhA3F, respectively. However, we found that rhA3A, rhA3B, rhA3C, rhA3D, and rhA3H had no effect on the infectivity on SHIVVifK27A. This emphasizes the key role that this amino acid has on the interactions with human and rhesus A3G and would explain its highly conserved nature among most SIV Vif proteins (Chen et al., 2009). The finding that rhA3F was incorporated into all of the virus mutants was not surprising as we previously showed that rhA3F was incorporated into parental SHIVKU2MC4 (Schmitt et al., 2010). Our analysis of the replication of the 12 mutants in rhesus PBMC suggests that both rhA3F and rhA3G were involved in the virus restriction. The key mutant here was SHIVVifK30A, which did not replicate in rhesus PBMC. The data suggest that that rhA3G was not alone and sufficient for virus restriction is based on our findings that rhA3G did not restrict SHIVVifK30A in our 293 assays while rhA3F restricted SHIVVifK30A 5-fold.

Previous studies showed that the substitution of the aspartic acid and arginine residues at positions 14 and 15 of the HIV-1 Vif with alanines resulted in sensitivity to hA3F, hA3D, and hA3H (Russell and Pathak, 2007; Zhang, et al., 2008; Zhen et al., 2010). A salient finding of our study was that a single amino acid substitution in the SIV Vif, R14A, resulted in a 100-fold or greater reduction in the infectivity in the presence rhA3D, rhA3G, and rhA3H and a 5-fold reduction in infectivity in the presence of rhA3F. Additionally, we found decreased interactions of Vif14A with rhA3D, G, and H as determined using co-immunoprecipitations. Comparison of the amino acid sequences of the N-terminus of HIV-1 and SIV Vif reveal significant differences in this region. The HIV-1 Vif has an arginine (position 15) in the context of 11WQVDRMRIK19 while the R14 of the SIV Vif is flanked by two proline residues (11PTWRIPER18). The finding that the rhA3D, rhA3G, and rhA3H severely affected the infectivity of SHIVVifR14A is a unique finding suggesting a central role for this amino acid in the SIV Vif interactions with these three rhesus A3 proteins. Recently, two groups demonstrated that HIV-1 Vif (and SIV Vif) interact with the ubiquitously expressed host cell protein known as core-binding factor subunit beta (CBF-β), which is part of a heterodimeric core-binding transcription factor (PEBP2/CBF). The CBF-β protein is important for regulating host genes involved in hematopoeisis and osteogenesis (Jäger et al., 2011; Zhang et al., 2011). In one study, it was shown that amino acids at the N-terminus of Vif were important for binding to CBF-β, particularly the tryptophan residues at positions 21 and 38 (Zhang et al., 2011). Therefore, it will be of interest to determine if R14 is important in CBF-β binding to the SIV Vif protein.

In another study that examined the importance of the HIV-1 Vif K26 in restriction, investigators found decreased VifK26A interactions with A3G based on the concentrations of the Triton-X-100 (0.1 versus 1.0%) used in co-immunoprecipitations (Chen et al., 2009). In our studies with R14A and K27A proteins (Fig. 6) we show that both Vif mutants were easily detected in our co-immunoprecipitations using 1.0% Triton-X-100, which may reflect different affinities for the HIV-1 Vif/hA3G and SIV Vif/rhA3G interactions.

The only studies performed to assess the role of Vif in vivo have used either Vif-deleted SIV or SHIVs with the highly conserved SLQYLA and HCCH domains mutated (Desrosiers et al., 1998; Schmitt et al., 2009, 2010). We have recently constructed a novel SHIV with the K27 deleted (SHIVVifΔK27), which like SHIVVifK27A is restricted by rhA3G and to a much less extent by rhA3F. It will be of interest to determine if inoculation SHIVVifΔK27 into macaques results in persistent infections in the macaque. We envision several possible scenarios. In the first scenario, though unlikely, inoculation of SHIVVifΔK27 into macaques could result in viral loads and disease that is similar to the parental virus. In the second scenario, if inoculated macaques develop no significant CD4+ T cell loss and have low or transient viral loads, we can conclude that these two rhA3 proteins are all that is required for SIV/SHIV restriction in vivo. In a third scenario, if inoculation of SHIVVifΔK27 into macaques results in moderate viral loads (and perhaps associated with CD4+ T cell loss) it would suggest that tissue reservoirs may exist for the virus where rhA3G and/or rhA3F are not expressed or expressed at low levels. One possible reservoir for this virus is the thymus. It is possible that A3G and A3F expression is developmentally regulated during the differentiation of immature thymocytes into CD4+ and CD8+ T cells. With pathogenic X4 SHIV, there is a near complete destruction of the thymus during the initial weeks of infection (Joag et al., 1996). Previous studies have shown that CXCR4 is expressed on double negative 1 (DN1) to double positive cells (DP), representing many of the subsets of immature thymocytes, while CCR5 is generally expressed on maturing CD4+ CD8- subsets (Ara et al., 2003; Plotkin et al., 2003; Trampont et al., 2010). Thus, it will be of interest to determine if SHIVVifΔK27 can replicate in one or more subsets of thymocytes during differentiation. While it seems intuitive that the first scenario is most likely, it has not been formally shown in vivo.

MATERIALS AND METHODS

Cells, plasmids, and viruses

The C8166, and SupT1 lymphocyte cell lines were used for transfections and as indicator cells to measure infectivity and cytopathicity of the viruses used in this study. Both cell lines were maintained in RPMI-1640, supplemented with 10mM Hepes buffer pH 7.3, 2 mM glutamine, 5 μg per ml gentamicin and 10% fetal bovine serum (R10FBS). 293 and TZM.bl cells were used for transfections and as an indicator cell line for infectivity assays, respectively (Platt et al., 1998; 2009; Takeuchi et al., 2008; Wei et al., 2002; Derdeyn et al., 2000). Rhesus PBMC were isolated from SIV/SHIV-negative donors on Ficoll-Hypaque gradients. The derivation of SHIVKU-2MC4 has been previously described (Liu et al., 1999). The plasmid expressing rhA3A has been previously reported (Genbank accession JF831054; Schmitt et al., 2011). For expression of the rhesus C (Genbank accession EU381233), D (Genbank accession JF714488), and H (Genbank accession DQ507277) proteins, genes were synthesized by GenScript and subcloned into the pcDNA3.1(+) vector with HA-tags at N-terminus. The rhA3B was based on our analysis of the from the rhesus macaque genome, was synthesized by GenScript and cloned into pcDNA3.1(+). Sequence analysis shows that this rhA3D clone has an aspartic acid at position 316, which was recently shown to be important for restriction of HIV-1 (McDougle et al., 2013). The pcDNA3.1(+)-rhA3G-HA and pcDNA3.1(+)-rhA3F-V5 were kindly provided by Nathaniel Landau (New York Universtiy of School of Medicine, New York, NY). All stocks of APOBEC3 plasmids were prepared by growing bacteria at 30°C.

The stability of the Vif mutant proteins

The stability of the engineered mutations in the human codon-optomized SIV Vif were assessed by transfecting 293 cells in a 6 well plate with 3 μg of vectors expressing p3xFLAG-Vif, p3xFLAG-CVif, p3xFLAG-VifK5A, p3xFLAG-VifR6A, p3xFLAG-VifR14A, p3xFLAG-VifR18A, p3xFLAG-VifR21A, p3xFLAG-VifH23A, p3xFLAG-VifK27A, p3xFLAG- VifK30A, p3xFLAG-VifK32A, p3xFLAG-VifK34A, p3xFLAG-VifK38A, or p3xFLAG-VifK46A using TurboFect (Fermentas) and the manufacturer's instructions. After 48 h, the cells were starved for 2 h in medium without methionine/cysteine and then radiolabeled with 300 μCi (per well) of 35S-methionine/cysteine for 1 h. The cells were then chased for 0 and 6 h. At these time points, the cells were lysed in 1X RIPA and the nuclei was removed by centrifugation. The Flag-tagged proteins were immunoprecipitated using a rabbit polyclonal antibody (Sigma-Aldrich, St. Louis, MO) overnight at 4CC. The samples were resuspended in 2X sample buffer, boiled for 5 minutes, run on a 12%-SDS PAGE gel, and visualized by autoradiography.

Construction of SHIVs with amino acid substitutions in the SIV Vif protein

For the construction of various SHIVs expressing mutant Vif proteins, the PacI/SphI fragment (nucleotides 4,609 to 5,901) from the p5’-SHIV4 was subcloned into the pSI vector at Pac I and Sph I sites. Oligonucleotide site-directed mutagenesis was used to change the following amino acids to alanines: K5, R6, R14, R18, R21, H23, K27, K30A, K32, K34, K38, and K46. All substitutions were made using the Quick-Change Mutagenesis Kit (Stratagene) following the manufacturer's instructions. The resulting PacI/SphI fragment was digested, isolated, and subcloned into full length SHIVKU-2MC4 (Liu et al., 1999). The resulting plasmid was sequenced to ensure that the desired mutations were introduced as expected. The full-length plasmids with the K5A, R6A, R14A, R18A, R21A, H23A, K27A, K30A, K32A, K34A, K38, and K46 mutations were designated as SHIVVifK5A, SHIVVifR6A, SHIVVifR14A, SHIVVifR21A, SHIVVifH23A, SHIVVifK27A, SHIVVifK30A, SHIVVifK34A, SHIVVifK38A, and SHIVVifR46A, respectively. Plasmids with full length genomes were transfected into SupT1 cells using procedures previously described and stocks of each virus were prepared, titered on SupT1 cells, and stored at -86CC (Hout et al., 2004; 2005; Stephens et al., 2002). The construction of SHIVΔvif has been previously described (Schmitt et al., 2010). The preparation and titration of this viral stock was the same as described above. The full-length plasmid was transfected into SupT1 cells as described above, stocks prepared, titered on SupT1 cells, and stored at -86CC.

Analysis of the replication of Vif mutants in SupT1 cells and macaque PBMC

SupT1 (A3G/F negative) cells were inoculated with equivalent levels (25 ng) of parental SHIVKU-2MC4, each SHIV expressing a Vif mutant, or SHIVΔvif for 4 h at 37°C. At 4 h, cells were centrifuged, washed 3 times to remove the inoculum and incubated in fresh medium at 37°C for up to 15 days. Aliquots of culture supernatants were obtained at 0, 1, 3, 5, 7, 9, 11, 13, and 15 days post-inoculation and the levels of p27 assessed using commercial p27 antigen capture kits (Zeptometrix).

We also assessed the replication of SHIVKU-2MC4, the various SHIVs expressing mutant Vif proteins, or SHIVΔvif in rhesus PBMC. For replication in rhesus macaque PBMC cultures, blood was obtained from uninfected rhesus macaques, and the PBMC separated on Ficoll-Hypaque gradients. The PBMC were stimulated in Con A (10 μg/ml) and IL-2 (50 ng/ml) for 48 h. The cells were washed three times and inoculated with 25 ng of virus for 6 h at 37°C. The inoculum was washed off and the cells incubated in medium containing 50 ng/ ml of IL-2. Aliquots of culture supernatants were obtained at 0, 1, 3, 5, 7, 9, 11, 13 and 15 days post-inoculation and the levels of p27 released into the culture medium assessed using commercial p27 antigen capture kits (Zeptometrix).

SHIV restriction assays

SHIV, SHIVCvif, or the mutant SHIVs (1 μg) described above were co-transfected with plasmids expressing rhA3A, rhA3B, rhA3C, rhA3D, rhA3F, rhA3G, or rhA3H (0.5 μg) using PEI (Fermentas) in a 12-well plate. At 48 h post-transfection, the culture medium was harvested and clarified by low speed centrifugation. Equivalent amounts of p27 were serially diluted using 10-fold dilutions from 101 to 106 and used to inoculate TZM-bl cells in a 96 well plate. At 48 h post-inoculation, the media was removed, the cells were washed with 1x PBS (pH 7.4), and the monolayer was fixed using 1% formaldehyde-0.2% glutaraldehyde in 1x PBS. The cells were washed three times and incubated in a solution containing 4 mM potassium ferrocyanide, 4 mM potassium ferricyanide, 4 mM magnesium chloride, and 0.4 mg of X-gal per ml for 2 h at 37CC. The reaction was stopped and the infectious units (IU) per ml were calculated (Derdeyn et al., 2000; Wei et al., 2002). All samples were run at least in triplicate.

RhA3 protein incorporation assays

We determined if the seven rhA3 proteins were incorporated into the various SHIVs expressing the different Vif mutants. Plasmids of viruses (derived from SHIVKU-2MC4) expressing each of the mutant Vif proteins described above were used to transfect 293 cells along with plasmids expressing either HA-tagged rhA3A, B, C, D, F, G, or H. At 36 h post-transfection, cells were starved for 2 h in medium without methionine/cysteine and then radiolabeled with 300 μCi (per well) of 35S-methionine/cysteine for 12 h. At 36 h, virus containing supernatants were harvested and clarified by low speed centrifugation. The clarified supernatant was then subjected to ultracentrifugation to pellet the virus (SW41 rotor, 38,000 rpm, 1 h, 4°C). The pellet was resuspended in PBS (pH 7.4) and layered on a 20/60% sucrose step gradient and again subject to ultracentrifugation (SW55Ti, 38,000 rpm, 1 h). The virus (at the interface) was harvested, pelleted again by ultracentrifugation described above, and resuspended in 200 μl of 1x PBS (pH 7.4). An aliquot was saved to determine p27 content by commercial antigen capture assays and the remaining sample divided in half and immunoprecipitated overnight at 4°C with either anti-SHIV to detect the viral proteins or a rabbit polyclonal HA antibody (sc-805; Santa Cruz) to detect HA-rhA3G, D, C, B, and H or a mouse monoclonal V5 antibody (Sigma-Aldrich) to detect V5-rhA3F. The samples were resuspended in 2x sample buffer, boiled for 5 minutes, run on a 12% SDS-PAGE gel, and visualized by autoradiographic techniques. All samples were loaded using equivalent amounts of p27. The cells were lysed in 1x RIPA, the nuclei were removed by centrifugation, the lysates were divided in half, and the samples were probed for either the HA or V5-tagged A3 proteins or with anti-SHIV to detect the p27 protein as described above.

Co-immunoprecipitation experiments

We determined of the SIV Vif and various Vif mutants would co-immunoprecipitate rhA3G, rhA3D, or rhA3H. 293 cells were transfected with 2μl of p3XFLAG-SIVVif, p3XFLAG-SIVΔvif, p3XFLAG-SIVVifR14A, p3XFLAG-SIVVifK27A, p3XFLAG-VifK38A, p3XFLAG-SIVVifK46A, or p3XFLAG-SIVVifΔ17-50 and 6C/l of either pHA-rhA3G, pHA-rhA3D, or pHA-rhA3H. At 48 h post-transfection, the cells were washed in cold 1X PBS and lysed in buffer (50mM Tris-HCl, pH7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, protease inhibitor cocktail) for 30 min on ice and 30 min at room temperature. Cell lysates were cleared by centrifugation at 16,000 x g at 41C for 10 min. The resulting supernatants were added to anti-rabbit IgG-coated magnetic Dynabeads (Invitrogen Dynal, Oslo, Norway) and immunoprecipitated with rabbit anti-FLAG antibody (Sigma-Aldrich) overnight at 41C. The beads were washed three times with buffer (50 mM Tris-HCl, pH 7.4, 250 mM NaCl, 1 mM EDTA, 1% Triton X-100, protease inhibitor cocktail) for 5 min at 4°C. The bound proteins were eluted with 50 Cl of 1X sample reducing buffer (63 mM Tris-HCl, pH6.8, 10% glycerol, 2% SDS, 0.0025% bromophenol blue) and boiled for 5 min. The proteins were separated by SDS-PAGE and subjected to Western blot analysis to detect the HA-tagged A3 proteins using a rabbit polyclonal anti-HA primary antibody (sc-805, Santa Cruz) and the Vif proteins detected using a rabbit polyclonal anti-FLAG antibody (F7425, Sigma-Aldrich). The lysates containing the non-immunoprecipitated proteins were collected and made 1X with sample reducing buffer, boiled, and subjected to Western blot analysis for A3 and Vif proteins as described above.

The incorporation of rhA3G into the viral nucleocapsids

In order to assess the incorporation of rhA3G proteins in the NPC, 293 cells seeded into a 6-well plate and transfected with a vector expressing rhA3G (3 μg/well) for 24 h. The cells were then transfected with SHIVΔvif and vectors expressing either p3xFLAGSIV-Vif, p3xFLAG-SIVVifR14A, p3xFLAG-SIV-VifK27A, or p3xFLAG-SIV-VifΔ17-50 for an additional 24 h. The cells were then starved for methionine/cysteine for 2 h and radiolabeled with 300μCi of 35S-methionine/cysteine for an additional 24 h. The virus-containing supernatants were harvested, cleared by low speed centrifugation, and concentrated by pelleting the virus through a 20% sucrose cushion using ultracentrifugation (SW41 rotor, 247,000xg, 1h, 4°C). The concentrated viral pellets were resuspended in 1x PBS (pH 7.4) and used on sucrose step-gradients with or without Triton-X-100. The sucrose step gradients were prepared as follows: 2.0 ml of a 60% sucrose solution (w/v, in 1x PBS) was placed into the bottom of a SW55Ti ultracentrifuge tube and overlaid with 2.0 ml of a 20% sucrose solution (w/v, in 1x PBS). Immediately prior to the addition of concentrated virus stocks, the step gradients were overlaid with 100 μl of either 1x PBS or 1% Triton-X-100 in 1x PBS. This procedure minimized the amount of time the detergent was exposed to the virions. Samples were then centrifuged in a SW55Ti rotor (247,000xg for 6 hours at 4°C). Four fractions of 1.0 ml each were collected from the top of the gradient with fraction 1 containing soluble proteins, fraction 2 a buffer of 20% sucrose that separates soluble proteins from the viral particles or viral cores, fraction 3 that includes the interphase of 20%:60% sucrose where viral particles and viral cores accumulate, and fraction 4 containing the 60% sucrose fraction. Gradient fractions were divided in halves or thirds, diluted to 1 ml with 1x RIPA buffer and subjected to immunoprecipitation assays using an HA-specific antibody (sc-805, Santa Cruz) for the A3 proteins, a Flag-specific antibody (F7425, Sigma-Aldrich) for the Vif protein or anti-SHIV serum for the capsid and matrix proteins.

Research Highlights.

  1. We mutated positively charged amino acid at the amino terminus of the SIV Vif.

  2. The lysine at position 27 of the SIV Vif was important for antagonism of rhesus APOBEC3G and APOBEC3F.

  3. The arginine at position 14 of the SIV Vif was important for antagonism of rhesus APOBEC3DE, G and F.

ACKNOWLEDGMENTS

The work reported here were partially supported by NIH grants to E.B.S. We also thank the KUMC Biotechnology Support Facility for the sequence analyses.

Footnotes

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