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. Author manuscript; available in PMC: 2012 May 14.
Published in final edited form as: Virology. 2010 Feb 11;400(1):86–92. doi: 10.1016/j.virol.2010.01.014

Induction of antibody mediated neutralization in SIVmac239 by a naturally acquired V3 mutation

Seth A Faith 1,2, Yingyun Wu 1, David Kuhrt 1,2, Jonathan D Steckbeck 1, Jodi K Craigo 1,3, Janice E Clements 4, Kelly Stefano Cole 1,2,*
PMCID: PMC3351114  NIHMSID: NIHMS180197  PMID: 20153009

Abstract

Achieving humoral immunity against human immunodeficiency virus (HIV) is a major obstacle in AIDS vaccine development. Despite eliciting robust humoral responses to HIV, exposed hosts rarely produce broadly neutralizing antibodies. The present study utilizes simian immunodeficiency virus (SIV) to identify viral epitopes that conferred antibody neutralization to clone SIV/17E-CL, an in vivo variant derived from neutralization resistant SIVmac239. Neutralization assays using rhesus macaque monoclonal antibodies were performed on viruses engineered to express single or multiple amino acid mutations. Results identified a single amino acid mutation, P334R, in the carboxy-terminal half of the V3 loop as a critical residue that induced neutralization while retaining normal glycoprotein expression on the surface of the virus. Furthermore, the R334 residue yielded neutralization sensitivity by antibodies recognizing diverse conformational and linear epitopes of gp120, suggesting that neutralization phenotype was a consequence of global structural changes of the envelope protein rather than a specific site epitope.

Introduction

Infection of nonhuman primates with simian immunodeficiency virus (SIV) provides a model for studying immune responses associated with HIV/AIDS in humans (Johnson and Hirsch, 1992). Both cellular and humoral immune responses have been correlated with protective immunity against SIV (Clements et al., 1995; Maecker and Maino, 2003; Paiardini et al., 2008; Sato and Johnson, 2007). Passive protection studies have further demonstrated that antibodies can provide protective immunity when present prior to or immediately preceding HIV or SIV challenge (Haigwood et al., 1996; Lewis et al., 1993; Mascola et al., 1999; Mascola et al., 2003; Mascola et al., 2000; Nishimura et al., 2002; Nishimura et al., 2003; Parren et al., 2001; Van Rompay et al., 1998). Unfortunately, these protective antibodies are infrequently observed in exposed hosts and are predominantly directed to intricate, conformationally dependent epitopes of the SIV envelope (env) proteins in regions with a propensity for mutation and subsequent immune evasion (Cole et al., 2001; Haigwood et al., 1992; Javaherian et al., 1994; Javaherian et al., 1992; Sato and Johnson, 2007). Therefore, identifying viral epitopes that play a role in antibody neutralization are important for our understanding of the immunogenic properties of SIV and HIV and will serve to enhance vaccine development.

Some limited HIV and SIV antibody neutralizing epitopes have been identified within the viral env gene (Pantophlet and Burton, 2006; Sato and Johnson, 2007). The HIV and SIV env gene produces a polyprotein (gp160) that is extensively modified by post-translational polysaccharide addition and is cleaved by host protease (furin) into two separate glycoproteins, gp120 [surface subunit (SU)] and gp41 [transmembrane subunit (TM)] (Luciw, 1996). On the surface of the virus, complete env complexes are comprised of a trimer of noncovalently linked heterodimers of gp120 and gp41. The role of these proteins in virus attachment and entry has been well characterized (Wyatt and Sodroski, 1998). Gp120 is involved with CD4 and co-receptor recognition and binding, while gp41 is responsible for forming the env trimer and mediating cell-virus fusion. Less well known is how neutralizing antibody responses to SIV and HIV are directed against the viral epitopes of the env proteins. Neutralizing antibody responses against SIV have been demonstrated to be associated with the gp41 cytoplasmic tail, ecto and transmembrane domains (Bonavia et al., 2005; Overholser et al., 2005; Puffer et al., 2004). Within gp120, V1/V2 (Johnson et al., 2002; Johnson et al., 2003; Puffer et al., 2004), V3 loop (Means et al., 2001; Palker et al., 1996; Pohlmann et al., 2004) and V4 regions (Choi et al., 1994; Kinsey et al., 1996) have also demonstrated significant roles in affecting virus neutralization by antibodies. Identifying and characterizing these determinants of neutralization in SIV has increased our understanding of the antigenic qualities of envelope proteins. However, more information is required to solve the precise molecular structure and antigenic qualities of these proteins.

The present study was designed to identify the amino acid residues within the env gene that contribute to the antibody neutralization phenotype of SIV/17E-CL, a naturally derived clone of neutralization resistant SIVmac239 that acquired nine amino acid mutations in gp120 (V67M, K141R, T158A, K176N, Q217K, M309I, P334R, K340R and G382R) (Anderson et al., 1993; Regier and Desrosiers, 1990; Sharma et al., 1992). Our earlier investigations using surface plasmon resonance (SPR) determined that differences in association and dissociation kinetic rates of antibody with SIV/17E-CL env proteins were causative for the neutralization phenotype (Steckbeck et al., 2005). However, the region of the viral protein or the epitope(s) involved remained unknown. Here we investigated antibody mediated neutralization in vitro with viruses engineered to express amino acid residues from SIV/17E-CL gp120 within the SIVmac239 backbone. Results from these studies described a novel V3 epitope that conferred neutralization of SIVmac239 by monoclonal antibodies to both linear and conformational epitopes, suggesting that neutralization phenotype was the consequence of global structural changes of the env protein complex.

Materials and Methods

Cells

293T cells and SIV permissive TZM-bl cells (Wei et al., 2002), which express CD4, CCR5, CXCR4 and HIV/SIV inducible firefly luciferase, were maintained in Dulbecco’s modified Eagle’s medium (DMEM) media (Gibco-Invotrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS) (Atlanta Biologicals, Lawrenceville, GA).

Construction of mutant proviruses

Previously characterized provirus constructs SIVmac239 (Regier and Desrosiers, 1990) and SIVmac17E-CL (Anderson et al., 1993) were digested with Nhe1 and Bpu10I to isolate the encoding region of gp120 and a portion of the ectodomain of gp41 (fig 1). This region was ligated with homologous restriction sites into a CMV-promoter expression vector, TR600 (Bower et al., 2004), using T4 DNA ligase (New England Biolabs, Ipswich, MA) and recombinant DNA was synthesized in DH5α competent cells. The wild-type gp120 env sequences were mutagenized using the QuickChange XL Site Directed Mutagenesis kit (Stratagene, La Jolla, CA) according to manufacturer’s protocol and sequence specific primers. The mutant env constructs were ligated back into the SIV proviral backbone using Nhe1 and Bpu10I restriction sites and T4 DNA ligase and propagated in Stbl2 competent cells (Invitrogen, Carlsbad, CA). Transfection grade plasmids clones were verified by DNA sequencing at the University of Pittsburgh Genomics and Proteomics Core laboratory (Pittsburgh, PA) using an ABI 3730xl DNA analyzer (Applied Biosystems, Foster City, CA).

Figure 1.

Figure 1

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Design and characterization of SIV mutant viruses. Mutations in SIV/17E-CL are distributed throughout the gp120 env gene and cluster around the V1/V2 and V3 regions. The cloning strategy utilizes the Bpu10I restriction site, 5’ to the start codon of gp120, and the Nhe1 restriction site on the 3’ end of the ectodomain of gp41. All mutant constructs created infectious virus in vitro that activated LTR-luc expression in the TZM-bl. Viral titer stocks ranged from 102–105 TCID50 per ml.

Production of viruses

SIVmac239, SIV/17E-CL and mutant proviral DNA was transiently transfected as previously described (Bower et al., 2004). Briefly, 8µg of proviral DNA was conjugated with 24 µl Lipofectamine 2000 (Invitrogen) in Opti-MEM media (Invitrogen) and incubated with 6–7 × 106 293T cells for 5 hours at 37°C. The media was replaced with 10ml of fresh Opti-MEM and the cultures were incubated an additional 48 hours at 37°C. Cell free supernatants were harvested and frozen at −80°C prior to experimental analysis. Virus titer was determined by serially diluting viral supernatants in DMEM media and culturing with TZM-bl cells (104 cells per well) in 96 well plates (BD-Falcon, San Jose, CA). Following an incubation of 48 hours, supernatants were removed and the cells were lysed with 50 µl cell lysis buffer (Promega, Madison, WI) for 15 minutes at room temperature. Lysates (40 µL) were transferred to white luminescence plates (USA Scientific, Ocala, Fl) and assayed for luciferase production on a Lmax II luminometer (Molecular Devices, Union City, CA) with 25 µl of Luciferase Assay Substrate (Promega) per well. The TCID50 was determined using the Reed and Meunch method (Poli and Fauci, 2004), with a positive cutoff value 2.5 times that of the negative samples.

Virus neutralization determination

MAbs (table 1) were serially diluted 5-fold in DMEM containing 10% FBS in sterile 96 well V-bottom plates (Sarstedt, Newton, NC) for a final assay concentration range of 10 - 0.00013 µg/ml. Virus was diluted in DMEM with 10% FBS to yield an MOI of 0.01, mixed with diluted MAb and pre-incubated 1h at 37°C. The virus-MAb mixture was added to TZM-bl cells (104 cells per well) in 96 well plates (BD-Falcon) and cultured in the presence of 20 µg/ml DEAE-Dextran (Amersham Biosciences, Piscataway, NJ) for 48 hours. SIV infection was assayed by luciferase production as described above. Neutralizing titers are reported as the IC50, the MAb concentration (µg/ml) that neutralized 50% of the virus infection, using the method of Karber (Karber, 1931). All values represent the averages of at least two independent experiments assayed in duplicate.

Table 1.

Monoclonal antibody phenotypes: epitope specificity and SIV neutralization

MAb Binding groupa Putative
binding domainb 		Epitope
conformationc 		Neutralizing antibody titer (µg/ml)d
SIV/17E-CL SIVmac239
3.8E I N' terminus conformational >25 >25
3.10A II V1 linear >25 >25
5.5B III V2 linear >25 >25
3.11H IV V3-cys loop linear 0.070 >25
1.11A V CCR5 bs conformational 0.125 >25
4.7A V CCR5 bs conformational 0.021 >25
3.11E VIa CCR5 bs conformational 0.038 >25
3.2C VIb V4 conformational 0.015 >25
E31 VIb V4 conformational 0.039 >25
1.10A VII V4 conformational 0.009 >25
C26 VII V4 conformational 0.006 >25
3B3 VIII Unknown conformational >25 >25
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a

Competition groups were previously defined by cross-competition ELISA(Cole et al. 2001)

b

Putative binding domains are based on previously published data (Cole et al. 2001)

c

Epitope conformation was previously determined by reducing/nonreducing immunoblot (Cole et al. 2001)

d

Neutralization antibody titers were previously assayed and reported as the concentration of MAb that inhibited 50% infection (IC50) using the TZM-bl assay (Steckbeck et al. 2005)

Western blot analysis

To quantitate envelope content of each SIV mutant, viral supernatants were pelleted over a 10% glycerol cushion at 19,000 × g for 1.5 h at 4°C, denatured by boiling in reducing buffer and resolved by SDS-PAGE using Tris-HCl 5 and 12.5% gels (Bio-Rad, Hercules, CA). Viral proteins were transferred to polyvinylidine difluoride membrane and probed for gp120 env using a MAb directed against a linear epitope in V3 not affected by the mutations in this study [3.11H, (Cole et. al. 2001)] and p27gag using MAb 2F12 (AIDS Research and Reference Reagent Program). Band densities of gp120 env and p27gag were determined using ImageJ software (National Institutes of Health, Bethesda, MD). Each immunoblot contained a positive control of SIVmac239, thus allowing for calculations of the gp120/p27gag ratio for each mutant virus in relation to the parental strain SIVmac239.

Results

Synthesis of mutant viruses

The envelope gene of SIV/17E-CL differs from SIVmac239 by 9 amino acids in gp120 (V67M, K141R, T158A, K176N, Q217K, M309I, P334R, K340R and G382R), see figure 1 (Anderson et al., 1993). Single and multiple rounds of site directed mutagenesis (see materials and methods) were used to generate amino acid mutations in the gp120 env SIVmac239 backbone according to a scheme that represents mutation sets in and around the V1,V2 or V3 regions of SIV/17E-CL (fig 1). Viral stocks were generated from recombinant virus construct in mammalian tissues as described in the materials and methods section. Synthesis of infectious virus was verified from every engineered construct and yielded viral stock titers ranging from 102–105 TCID50 per ml (data not shown).

Envelope expression in SIV mutants

Previous reports suggest that mutations in SIV gp120 that reduce envelope incorporation into viral membranes and/or increase env shedding from the viral membrane result in enhanced sensitivity to antibody mediated neutralization (Klasse and Moore, 1996; Yuste et al., 2005). We previously demonstrated that SIV/17E-CL env expression is similar to SIVmac239 and is therefore not a factor for neutralization (Steckbeck et al., 2005). To determine whether the mutant viruses studied here displayed altered env expression, we assessed the quantity of gp120 present in mutant viruses. Figure 2a demonstrated that with the exception of 239-A158, 239-NK-IRRR, 239-NK and 239-N176, all mutant viruses expressed levels of gp120 env similar to SIVmac239. To measure the level of gp120 env in each mutant clone, band densities from duplicate experiments were determined for gp120 env and p27gag and the ratios were compared to the parental virus, SIVmac239. Using this semi-quantitative analysis, four mutant viruses (239-A158, 239-NKIRRR, 239-NK and 239-N176) displayed significant decreases in gp120 expression (35, 4.9, 17 and 11% respectively) (fig 2b). Interestingly, while 239-A158 displayed reduced gp120 expression, 239-MRA and 239-MRA-IRRR, which also contained the T158A mutation, did not express a reduced gp120 env phenotype. So, the significant reduction of env by T158A only occurred when T158A was expressed alone (239-A158). Figure 2b also demonstrated that mutant viruses with the K176N mutation (239-NKIRRR, 239-NK and 239-N176) expressed significantly lower levels of gp120 env. Thus, the analysis of gp120 expression showed that env content was unperturbed in most mutants and significant variability was limited to few mutants with distinct point mutations.

Figure 2.

Figure 2

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Analysis of gp120 expression in mutant viruses. (a) Virus supernatants were pelleted and denatured viral proteins were immunodetected for gp120 env (MAb 3.11H) and structural protein p27gag (MAb 2F12). (b) Band densities were determined and the gp120/ p27gag ratio of each mutant was calculated. The data reported is the percent change in mean gp120/ p27gag ratio (±S.D.) compared to SIVmac239 from two independent experiments.

Lastly, the immunoblot analysis revealed one further observation. Figure 2a showed that gp120 env from mutants with the A158 residue (239-MRA, 239-A158 and 239-MRA-IRRR) resolved at a lower molecular weight than SIVmac239 and mutants with T158. This migration pattern of the env proteins would suggest that the T158A mutation resulted in a loss of an N-linked glycosylation residue, as expected by the change in amino acid sequence, N-C-T →N-C-A.

Analysis of V1/V2 region residues for neutralization sensitivity to monoclonal antibodies

Each mutant virus was examined for neutralization sensitivity using the TZM-bl assay (Steckbeck et al., 2005). Neutralization studies were divided into study groups of mutants according to region, and are summarized in fig 1. Data from V1 region mutants showed that the 239-MRA virus (mutations V67M, K141R and T158A) was not neutralized by MAb at the highest concentration (10µg/ml) (table 2). The 239-M67 (V67M) and 239-R141 (K141R) mutants alone were also not sensitive to neutralization (table 2). However, the 239-A158 mutant (T158A) yielded a clone that was partially sensitive to neutralization by MAbs that recognize epitopes on the carboxy-terminal half of gp120. Four of the eight neutralizing antibodies yielded mean IC50 values (0.64 - 0.037 µg/ml, table 2) within one log10 deviation from the IC50 values observed in SIV/17E-CL (table 1). Interestingly, the 239-NKIRRR mutant that contains the V2 and V3 mutations and none of the V1 mutations demonstrated a neutralization profile more comparable to SIV/17E-CL (tables 1) with IC50 values ranging from 0.39 to 0.003 µg/ml (table 2). Therefore, the single point mutation T158A in V1 conferred partial neutralization sensitivity in only 239-A158, while determinants outside of V1 imparted full neutralization.

Table 2.

Neutralization phenotype resulting from V1 and V2 mutations a

Monoclonal
Antibody		 Binding
Domain		 V1 V2
Mac239 17E-CL 239-MRA 239-M67 239-R141 239-A158 239-NKIRRR 239-NK 239-N176 239-K217 239-MRA-IRRR
3.8E N' terminus >10 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10
3.10A V1 >10 >10 >10 >10 >10 >10 6.43 (0.81) >10 >10 >10 >10
5.5B V2 >10 >10 >10 >10 >10 >10 1.18 (0.45) >10 >10 >10 >10
3.11H V3-cys loop >10 0.046 (0.02) >10 >10 8.30 (2.4) 0.510 (0.41) 0.028 (0.02) 1.01 (0.43) 1.11 (1.1) >10 0.260 (0.18)
1.11A CCR5 bs >10 0.075 (0.05) >10 >10 >10 0.143 (0.14) 0.039 (0.01) 0.215 (0.01) 0.857 (0.90) >10 0.673 (0.31)
4.7A CCR5 bs >10 0.016 (0.01) >10 >10 >10 0.300 (0.27) 0.017 (0.01) 0.530 (0.32) 0.233 (0.15) >10 0.300 (0.16)
3.11E CCR5 bs >10 0.024 (0.02) >10 >10 8.65 (1.9) 0.640 (0.65) 0.010 (0.01) 1.24 (1.1) 0.154 (0.19) >10 0.220 (0.07)
3.2C V4 >10 0.022 (0.01) >10 >10 >10 0.225 (0.22) 0.029 (0.01) 0.273 (0.16) 0.331 (0.29) >10 0.133 (0.07)
E31 V4 >10 0.029 (0.02) >10 >10 >10 0.175 (0.02) 0.023 (0.01) 0.195 (0.02) 0.042 (0.01) >10 0.520 (0.28)
1.10A V4 >10 0.005 (0.004) >10 >10 5.25 (3.7) 0.026 (0.02) 0.002 (0.001) 0.084 (0.01) 0.029 (0.03) 5.49 (4.1) 0.033 (0.01)
C26 V4 >10 0.004 (0.003) >10 >10 >10 0.037 (0.003) 0.003 (0.001) 0.089 (0.03) 0.031 (0.04) >10 0.026 (0.01)
3B3 Unknown >10 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10
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a

IC50 neutralization titers are reported as the mean concentration of MAb (µg/ml) (±S.D.) from at least two independent experiments. Values in bold type represent mean IC50 values that do not exceed 1 log10 variance from the IC50 of SIV/17E-CL.

Next, mutations in the V2 region were examined. The 239-NK virus containing both the K176N and Q217K mutations was partially sensitive to neutralization (table 2), albeit to a lesser degree than SIV/17E-CL (table 1). Only two of the eight neutralizing antibodies displayed IC50 values within a log10 deviation of SIV/17E-CL. Further analysis of the V2 mutations was performed with clones 239-N176 (K176N) and 239-K217 (Q217K). 239-N176 was sensitive to antibody neutralization (IC50 values 1.11 to 0.031 µg/ml), while 239-K217 was not neutralized by the antibodies tested (table 2). Clone 239-MRA-IRRR lacking the V2 region mutations, but containing the V1 and V3 mutations was more sensitive to neutralization than the 239-NK virus (table 2), with IC50 values ranging from 0.67 to 0.026 µg/ml. Therefore, the data of the V2 region analysis showed that the K176N mutation was sufficient to confer partial neutralization in SIV/17E-CL, but that the V3 region contained factors for neutralization as well.

Analysis of V3 region residues for neutralization sensitivity to monoclonal antibodies

Lastly we evaluated mutations in and around the V3 region (M309I, P334R, K340R and G382R) for neutralization sensitivity. Mutant clone 239-IRRR, expressing all four point mutations around V3, showed IC50 values equivalent to SIV/17E-CL, ranging from 0.31 to 0.006 µg/ml (table 3). To elucidate the critical neutralizing mutations in V3, additional single point mutants were generated and assayed for neutralization. The 239-I309 (M309I), 239-R340 (K340R) and 239-R382 (G382R) mutants were resistant to neutralization by all MAbs tested (table 3). Of these clones, only 239-R334 (P334R) demonstrated a neutralization phenotype equivalent to 239-IRRR and SIV/17E-CL (table 3). To determine the contribution of the neighboring mutations that also introduced an arginine (R) in and around V3 (K340R and G382R), combination mutant clones were assayed for neutralization. The data from clones 239-RRR, 239-R334R340 and 239-R340R382 reinforced the observation of the P334R mutation as the neutralizing determinant, as only those clones containing the R334 residue (239-RRR and 239-R334R340) yielded a neutralization phenotype equal to 239-IRRR (table 3). Furthermore, all mutant clones assayed that contained P334R (239-NKIRRR, 239-MRA-IRRR, 239-IRRR, 239-R334, 239-RRR and 239-R334R340) were neutralization sensitive (table 2 and table 3) with IC50 values for MAbs that were equivalent to SIV/17E-CL.

Table 3.

Neutralization phenotype resulting from V3 mutations a

Monoclonal
Antibody		 Binding
Domain		 V3
Mac239 17E-CL 239-IRRR 239-I309 239-R334 239-R340 239-R382 239-RRR 239-R334R340 239-R340R382
3.8E N' terminus >10 >10 >10 >10 >10 >10 >10 >10 >10 >10
3.10A V1 >10 >10 3.58 (0.39) >10 6.43 (2.8) >10 >10 7.13 (4.9) >10 >10
5.5B V2 >10 >10 9.08 (1.85) >10 5.58 (4.7) 8.90 (2.2) >10 >10 >10 >10
3.11H V3-cys loop >10 0.046 (0.02) 0.313 (0.38) >10 0.111 (0.06) 8.98 (2.0) 9.36 (1.1) 0.086 (0.06) 0.435 (0.50) >10
1.11A CCR5 bs >10 0.075 (0.05) 0.228 (0.24) >10 0.110 (0.07) >10 >10 0.069 (0.03) 0.200 (0.12) >10
4.7A CCR5 bs >10 0.016 (0.01) 0.056 (0.07) >10 0.051 (0.03) >10 >10 0.042 (0.02) 0.075 (0.02) >10
3.11E CCR5 bs >10 0.024 (0.02) 0.059 (0.07) >10 0.062 (0.04) 7.85 (4.3) 7.68 (2.7) 0.050 (0.02) 0.072 (0.05) >10
3.2C V4 >10 0.022 (0.01) 0.014 (0.02) >10 0.048 (0.03) 7.10 (3.6) 7.76 (4.4) 0.016 (0.01) 0.060 (0.04) >10
E31 V4 >10 0.029 (0.02) 0.010 (0.13) >10 0.101 (0.03) >10 >10 0.161 (0.15) 0.050 (0.01) >10
1.10A V4 >10 0.005 (0.004) 0.005 (0.004) >10 0.004 (0.002) 6.73 (2.8) 7.08 (3.6) 0.004 (0.002) 0.023 (0.03) 7.80 (3.8)
C26 V4 >10 0.004 (0.003) 0.006 (0.007) >10 0.004 (0.004) 7.85 (4.3) 9.06 (1.6) 0.006 (0.002) 0.010 (0.01) >10
3B3 Unknown >10 >10 >10 >10 >10 >10 >10 >10 >10 >10
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a

IC50 neutralization titers are reported as the mean concentration of MAb (µg/ml) (±S.D.) from at least two independent experiments. Values in bold type represent mean IC50 values that do not exceed 1 log10 variance from the IC50 of SIV/17E-CL.

Discussion

This study analyzed the antibody neutralization phenotypes resulting from single and multiple point mutations in SIVmac239 gp120 representing mutations derived from SIV/17E-CL. Using a broad panel of rhesus MAbs with linear and conformational epitopes to multiple domains throughout SIV gp120, we sought to identify the mutation(s) responsible for inducing antibody mediated neutralization seen in SIV/17E-CL. The first mutation identified, T158A, only influenced neutralization when expressed alone, as seen in virus 239-A158 (table 2). Interestingly, the mutation of threonine 158 to alanine removes a potential N-linked glycosylation site from SIVmac239 (g6) by disrupting the N-linked glycosylation signal (Asn-X-Ser/Thr), N-C-T →N-C-A. Previous research suggests that the carbohydrates around V1/V2 and V3 shield epitopes on env because removal of glycosylation sites in SIV often enhances antibody neutralization (Chackerian, Rudensey, and Overbaugh, 1997; Cole et al., 2004; Reitter and Desrosiers, 1998; Reitter, Means, and Desrosiers, 1998). In our study, the T158A Δg6 mutation induced a neutralizing phenotype when expressed alone (239-A158), but showed no effect when other mutations were expressed in cis, 239-MRA (V67M, K141R and T158A). A more significant observation of the 239-A158 (T158A) mutant was a significant reduction in gp120 env/p27gag ratio compared to SIVmac239 (figure 2b). Reduced envelope content in the virion has been correlated with neutralization sensitivity of HIV and SIV (Klasse and Moore, 1996; Yuste et al., 2005). Therefore, reduced env protein expression would explain the neutralization sensitivity of the 239-A158 clone, yet does not dictate the neutralization phenotype of SIV/17E-CL which does not have lowered env expression.

Next, a K176N mutation amino-terminal to V2 was observed to induce monoclonal antibody mediated neutralization in vitro, albeit to a lesser degree than SIV/17E-CL (table 1 and table 2). As reported previously, V1/V2 plays a role in neutralization, given that mutations and deletions of V1/V2 induce a neutralizing phenotype (Johnson et al., 2002; Johnson et al., 2003; Puffer et al., 2004). Neutralization sensitivity has been reported for SIVmac316 that expresses a mutation at amino acid 176 in gp120 (Johnson et al., 2003; Means et al., 2001). Furthermore, the MERT cluster of mutations (V67M, K176E, G382R K573T) are implicated in the macrophage tropic phenotype associated with SIVmac316 (Mori et al., 1992). Macrophage tropism is often associated with antibody neutralization sensitivity in SIV (Means et al., 2001; Puffer et al., 2002; Rudensey et al., 1998). Indeed, SIV/17E-CL is macrophage tropic (Mankowski et al., 1997), however the V67M and G382R mutations had no effect on neutralization sensitivity in this study (table 2 and table 3). More importantly the K176N mutation yielded significant reductions in envelope expression (fig 2). As may be the case for the T158A mutation, the atypically lowered expression of the glycoproteins on the viral membrane of the SIV K176N mutant induced the observed antibody neutralization phenotype.

In this study, the most significant changes in antibody mediated neutralization were observed in the carboxy-terminal portion of the V3 loop at residue 334 (table 3). All mutants assayed that contained R334 (239-NKIRRR, 239-MRA-IRRR, 239-IRRR, 239-R334, 239-RRR and 239-R334R340) were neutralized by MAbs at concentrations equivalent to SIV/17E-CL while retaining normal protein expression of env (fig. 2). Although impressive, it is not so surprising given the documented importance that HIV and SIV V3 has in regard to viral tropism and neutralization phenotypes (Hartley et al., 2005). Evidence thus far suggests that the V3 loop may be split into two domains each with a different role in determining phenotype. The amino-terminal portion is suggested to directly interact with neutralizing antibodies, while the carboxy-terminal portion is weakly or non-immunogenic and may interact with other domains of the gp120 monomer (Benichou et al., 1993; Huang et al., 2005; McBride et al., 1993; Miller, Murphey-Corb, and Montelaro, 1992; Palker et al., 1996; Pohlmann et al., 2004; Rosen et al., 2005; Rosen, Samson, and Anglister, 2008). Changes in the carboxy-terminal domain of V3 are suspected to influence phenotype via structural changes of the individual monomers or the complete trimer (Huang et al., 2005; Rosen, Samson, and Anglister, 2008; Rosen et al., 2006). Our data supports this theory in part because the neutralizing phenotype obtained by the P334R mutation was apparent not only for a V3 binding antibody (3.11H), but for the entire panel of MAbs recognizing both linear and conformational epitopes clustered throughout the gp120 molecule (table 3).

One particular model in HIV could explain structure changes associated with R334. The “11/25” rule states that in HIV-1 negative/neutral charges at residues 11 or 25 in the V3 loop confer CCR5 co-receptor usage and macrophage tropism, while positive charges yield CXCR4 co-receptor usage and T-lymphocyte or dual tropism phenotype (De Jong et al., 1992; Fouchier et al., 1992; Resch, Hoffman, and Swanstrom, 2001). The corresponding amino acids in T-lymphotrophic SIVmac239 V3 are 11 (P306) and 25 (P334). Interestingly, Rosen et al. (Rosen, Samson, and Anglister, 2008) recently demonstrated that the 25th amino acid in HIV-1 V3, residue 322, displays electrostatic interaction with residue 440 in the C4 domain. The 440 residue in C4 was mapped as the second amino acid following a conserved YAPP domain, which is also present in SIVmac as a YLPP domain. Interestingly, the second amino acid carboxy-terminal to this domain in SIV is a negatively charge glutamic acid E454. If SIV structure is homologous to HIV-1, the R334 and E454 would theoretically interact with electrostatic attraction and yield structure and phenotype of macrophage tropic viruses. Indeed, studies of SIV V3 mutations found that changing P334 to uncharged amino acids leucine or glutamine did not confer macrophage tropism in SIVmac239 (Kirchhoff, Mori, and Desrosiers, 1994; Pohlmann et al., 2004), yet SIV/17E-CL (R334) is a macrophage tropic virus (Mankowski et al., 1997). Therefore, it is feasible that the 11/25 rule for positivity applies to HIV-1, but could more broadly reflect the necessity for position specific electrostatic interactions between V3 and other regions of SIV gp120, such as V4.

This study indentified the P334R mutation in the C’-terminal portion of the V3 loop to be the main determinant of neutralization in SIV/17E-CL. Given that this residue affected neutralization by MAbs recognizing diverse conformational and linear epitopes in gp120 and that the V3 region has been observed to be associated with viral tropism and neutralization phenotypes, it is likely that the neutralization sensitive phenotype of SIV/17E-CL is a result of the structural changes of the envelope protein elicited by the P334R mutation. Given the currently limited data describing V3 structure within the context of gp41/gp120 trimers, this report supports the importance of further studies on the V3 region to assist in developing effective therapeutics and preventative strategies against HIV AIDS.

Acknowledgments

This work was supported by the University of Pittsburgh Center for Vaccine Research and funding from a National Institutes of Health grant (AI 52058) to KC.

Footnotes

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Competing Interests

The authors declare that they have no competing interests.

Contributor Information

Seth A. Faith, Email: saf31@pitt.edu.

Yingyun Wu, Email: ywu@novavax.com.

David Kuhrt, Email: dmk26@pitt.edu.

Jonathan D. Steckbeck, Email: jds170@pitt.edu.

Jodi K. Craigo, Email: craigoj@pitt.edu.

Janice E. Clements, Email: jclement@bs.jhmi.edu.

Kelly Stefano Cole, Email: stefcole@pitt.edu.

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