Structural modulations of the envelope gp120 glycoprotein of human immunodeficiency virus type 1 upon oligomerization and differential V3 loop epitope exposure of isolates displaying distinct tropism upon virion-soluble receptor binding.

We investigated the binding of conformation-dependent anti-V2, anti-V3, and anti-CD4-binding site monoclonal antibodies to monomeric and virion-associated gp120 from human immunodeficiency virus type 1 isolates displaying marked differences in cell tropism. For all viruses examined, we found that the half-maximal binding values of the anti-V2 and anti-CD4-binding site antibodies with virion-associated gp120 were higher than those with monomeric gp120, but the maximum amount of antibodies bound was diminished only for one of the anti-V2 antibodies tested. These observations suggest that upon gp120 oligomerization, the V2 loop and CD4-binding site undergo conformational changes and that particular epitopes within these domains are occluded in the oligomeric gp120. In contrast, although the overall binding patterns and half-maximal binding values of the anti-V3 loop antibodies tested were similar with monomeric and oligomeric gp120, all the V3 loop epitopes examined were less accessible to antibody binding on the virion surface. This masking of the V3 loop is more pronounced for the primary-like macrophage-tropic isolates examined. Lastly, we observe that upon soluble receptor-virion binding, specific V3 loop epitopes that differ for viruses displaying different tropisms are exposed.     

The N276 Glycosylation Site Is Required for HIV-1 Neutralization by the CD4 Binding Site Specific HJ16 Monoclonal Antibody

Immunogen design for HIV-1 vaccines could be based on epitope identification of naturally occurring neutralizing antibodies in infected patients. A tier 2 neutralizing monoclonal antibody (mAb), HJ16 recognizes a new epitope in the CD4 binding site (CD4bs) region that only partially overlaps with the b12 epitope. We aimed to identify the critical binding site by resistance induction in a sensitive primary CRF02_AG strain. In four independent dose-escalation studies, the N276D mutation was consistently the only alteration found and it was confirmed to be responsible for resistance to HJ16 by site-directed mutagenesis in envelopes (envs) of the homologous CRF02_AG, as well as of a subtype A and a subtype C primary isolate. This mutation removes an N-linked glycosylation site. The effect of N276D was very selective, as it failed to confer resistance to a range of other entry inhibitors. Remarkably, sensitivity to the CD4bs VRC01 and VRC03 mAbs was increased in the N276D mutated viruses. These data indicate that binding of the CD4bs specific HJ16 mAb critically depends on the interaction with the N276-glycan, thus indicating that HJ16 is the first glycan dependent CD4bs-specific mAb.     

A novel human antibody against human immunodeficiency virus type 1 gp120 is V1, V2, and V3 loop dependent and helps delimit the epitope of the broadly neutralizing antibody immunoglobulin G1 b12

The V1/V2 and V3 loops are proximal to the CD4 binding site (CD4bs) of human immunodeficiency virus type 1 (HIV-1) gp120 and undergo conformational change upon CD4 receptor engagement by the HIV-1 envelope spike. Nearly all of the reported monoclonal antibodies (MAbs) against the CD4bs exhibit a very limited capacity to neutralize HIV-1. However, one such human MAb, immunoglobulin G1 (IgG1) b12, is uniquely able to neutralize primary isolates across subtypes with considerable potency. The molecular basis for the anti-HIV-1 activity of b12 is not fully understood but is relevant to vaccine design. Here we describe a novel human MAb, 4KG5, whose binding to monomeric gp120 is moderately enhanced by IgG1 b12. In sharp contrast, 4KG5 binding to gp120 is inhibited by soluble CD4 (sCD4) and by all other (n = 14) anti-CD4bs MAbs tested. 4KG5 is unable to recognize gp120 in which either V1, V2, or V3 has been deleted, and MAbs against the V2 or V3 loops inhibit the binding of 4KG5 to gp120. Moreover, 4KG5 is able to inhibit the binding of the CD4-induced MAbs 17b and X5 in the absence of sCD4, whereas 17b and X5 only weakly inhibit the binding of 4KG5 to gp120. Mutagenesis of gp120 provides further evidence of a discontinuous epitope of 4KG5 that is formed by the V1/V2 loop, the V3 loop, and a portion of the bridging sheet (C4). 4KG5 was isolated as a single-chain Fv from a phage display library constructed from the bone marrow of an HIV-1-seropositive subject (FDA2) whose serum neutralizes HIV-1 across subtypes. Despite its source, we observed no significant neutralization with 4KG5 against the autologous (R2) virus and several other strains of HIV-1. The results suggest a model in which antibody access to the CD4bs on the envelope spike of HIV-1 is restricted by the orientation and/or dynamics of the V1/V2 and V3 loops, and b12 avoids these restrictions.     

PGV04, an HIV-1 gp120 CD4 binding site antibody, is broad and potent in neutralization but does not induce conformational changes characteristic of CD4

Recently, several broadly neutralizing monoclonal antibodies (bnMAbs) directed to the CD4-binding site (CD4bs) of gp120 have been isolated from HIV-1-positive donors. These include VRC01, 3BNC117, and NIH45-46, all of which are capable of neutralizing about 90% of circulating HIV-1 isolates and all of which induce conformational changes in the HIV-1 gp120 monomer similar to those induced by the CD4 receptor. In this study, we characterize PGV04 (also known as VRC-PG04), a MAb with potency and breadth that rivals those of the prototypic VRC01 and 3BNC117. When screened on a large panel of viruses, the neutralizing profile of PGV04 was distinct from those of CD4, b12, and VRC01. Furthermore, the ability of PGV04 to neutralize pseudovirus containing single alanine substitutions exhibited a pattern distinct from those of the other CD4bs MAbs. In particular, substitutions D279A, I420A, and I423A were found to abrogate PGV04 neutralization. In contrast to VRC01, PGV04 did not enhance the binding of 17b or X5 to their epitopes (the CD4-induced [CD4i] site) in the coreceptor region on the gp120 monomer. Furthermore, in contrast to CD4, none of the anti-CD4bs MAbs induced the expression of the 17b epitope on cell surface-expressed cleaved Env trimers. We conclude that potent CD4bs bnMAbs can display differences in the way they recognize and access the CD4bs and that mimicry of CD4, as assessed by inducing conformational changes in monomeric gp120 that lead to enhanced exposure of the CD4i site, is not uniquely correlated with effective neutralization at the site of CD4 binding on HIV-1.     

A Variable Region 3 (V3) Mutation Determines a Global Neutralization Phenotype and CD4-Independent Infectivity of a Human Immunodeficiency Virus Type 1 Envelope Associated with a Broadly Cross-Reactive, Primary Virus-Neutralizing Antibody Response

The human serum human immunodeficiency virus type 1 (HIV-1)-neutralizing serum 2 (HNS2) neutralizes many primary isolates of different clades of HIV-1, and virus expressing envelope from the same donor, clone R2, is neutralized cross-reactively by HIV-immune human sera. The basis for this cross-reactivity was investigated. It was found that a rare mutation in the proximal limb of variable region 3 (V3), 313-4 PM, caused virus pseudotyped with the R2 envelope to be highly sensitive to neutralization by monoclonal antibodies (MAbs) directed against conformation-sensitive epitopes at the tip of the V3 loop, such as 19b, and moderately sensitive to MAbs against CD4 binding site (CD4bs) and CD4-induced (CD4i) epitopes, soluble CD4 (sCD4), and HNS2. In addition, introduction of this sequence by mutagenesis caused enhanced sensitivity to neutralization by 19b, anti-CD4i MAb, and HNS2 in three other primary HIV-1 envelopes and by anti-CD4bs MAb and sCD4 in one of the three. The 313-4 PM sequence also conferred increased infectivity for CD4(+) CCR5(+) cells and the ability to infect CCR5(+) cells upon all of these four and two of these four HIV-1 envelopes, respectively. Neutralization of R2 by HNS2 was substantially inhibited by the cyclized R2 V3 35-mer synthetic peptide. Similarly, the peptide also had some lesser efficacy in blocking neutralization of R2 by other sera or of neutralization of other primary viruses by HNS2. Together, these results indicate that the unusual V3 mutation in the R2 clone accounts for its uncommon neutralization sensitivity phenotype and its capacity to mediate CD4-independent infection, both of which could relate to immunogenicity and the neutralizing activity of HNS2. This is also the first primary HIV-1 isolate envelope glycoprotein found to be competent for CD4-independent infection.     

Variants of human immunodeficiency virus type 1 that efficiently use CCR5 lacking the tyrosine-sulfated amino terminus have adaptive mutations in gp120, including loss of a functional N-glycan

By selecting the R5 human immunodeficiency virus type 1 (HIV-1) strain JR-CSF for efficient use of a CCR5 coreceptor with a badly damaged amino terminus [i.e., CCR5(Y14N)], we previously isolated variants that weakly utilize CCR5(Delta18), a low-affinity mutant lacking the normal tyrosine sulfate-containing amino-terminal region of the coreceptor. These previously isolated HIV-1(JR-CSF) variants contained adaptive mutations situated exclusively in the V3 loop of their gp120 envelope glycoproteins. We now have weaned the virus from all dependency on the CCR5 amino terminus by performing additional selections with HeLa-CD4 cells that express only a low concentration of CCR5(Delta18). The adapted variants had additional mutations in their V3 loops, as well as one in the V2 stem (S193N) and four alternative mutations in the V4 loop that eliminated the same N-linked oligosaccharide from position N403. Assays using pseudotyped viruses suggested that these new gp120 mutations all made strong contributions to use of CCR5(Delta18) by accelerating a rate-limiting CCR5-dependent conformational change in gp41 rather than by increasing viral affinity for this damaged coreceptor. Consistent with this interpretation, loss of the V4 N-glycan at position N403 also enhanced HIV-1 use of a different low-affinity CCR5 coreceptor with a mutation in extracellular loop 2 (ECL2) [i.e., CCR5(G163R)], whereas the double mutant CCR5(Delta18,G163R) was inactive. We conclude that loss of the N-glycan at position N403 helps to convert the HIV-1 envelope into a hair-trigger form that no longer requires strong interactions with both the CCR5 amino terminus and ECL2 but efficiently uses either site alone. These results demonstrate a novel functional role for a gp120 N-linked oligosaccharide and a high degree of adaptability in coreceptor usage by HIV-1.     

HIV-1 Neutralizing Antibodies Display Dual Recognition of the Primary and Coreceptor Binding Sites and Preferential Binding to Fully Cleaved Envelope Glycoproteins

The gp120 CD4 binding site (CD4bs) and coreceptor binding site (CoRbs) are two functionally conserved elements of the HIV-1 envelope glycoproteins (Env). We previously defined the presence of CD4bs-neutralizing antibodies in the serum of an HIV-1-infected individual and subsequently isolated the CD4bs-specific monoclonal antibodies (MAbs) VRC01 and VRC03 from the memory B cell population. Since this donor''s serum also appeared to contain neutralizing antibodies to the CoRbs, we employed a differential fluorescence-activated cell sorter (FACS)-based sorting strategy using an Env trimer possessing a CoRbs knockout mutation (I420R) to isolate specific B cells. The MAb VRC06 was recovered from these cells, and its genetic sequence allowed us to identify a clonal relative termed VRC06b, which was isolated from a prior cell sort using a resurfaced core gp120 probe and its cognate CD4bs knockout mutant. VRC06 and VRC06b neutralized 22% and 44% of viruses tested, respectively. Epitope mapping studies revealed that the two MAbs were sensitive to mutations in both the gp120 CoRbs and the CD4bs and could cross-block binding of both CD4bs and CoRbs MAbs to gp120. Fine mapping indicated contacts within the gp120 bridging sheet and the base of the third major variable region (V3), which are elements of the CoRbs. Cell surface binding assays demonstrated preferential recognition of fully cleaved Env trimers over uncleaved trimers. Thus, VRC06 and VRC06b are Env trimer precursor cleavage-sensitive neutralizing MAbs that bind to a region of gp120 that overlaps both the primary and the secondary HIV-1 receptor binding sites.     

R5 Clade C SHIV Strains with Tier 1 or 2 Neutralization Sensitivity: Tools to Dissect Env Evolution and to Develop AIDS Vaccines in Primate Models

Background

HIV-1 clade C (HIV-C) predominates worldwide, and anti-HIV-C vaccines are urgently needed. Neutralizing antibody (nAb) responses are considered important but have proved difficult to elicit. Although some current immunogens elicit antibodies that neutralize highly neutralization-sensitive (tier 1) HIV strains, most circulating HIVs exhibiting a less sensitive (tier 2) phenotype are not neutralized. Thus, both tier 1 and 2 viruses are needed for vaccine discovery in nonhuman primate models.

Methodology/Principal Findings

We constructed a tier 1 simian-human immunodeficiency virus, SHIV-1157ipEL, by inserting an “early,” recently transmitted HIV-C env into the SHIV-1157ipd3N4 backbone [1] encoding a “late” form of the same env, which had evolved in a SHIV-infected rhesus monkey (RM) with AIDS. SHIV-1157ipEL was rapidly passaged to yield SHIV-1157ipEL-p, which remained exclusively R5-tropic and had a tier 1 phenotype, in contrast to “late” SHIV-1157ipd3N4 (tier 2). After 5 weekly low-dose intrarectal exposures, SHIV-1157ipEL-p systemically infected 16 out of 17 RM with high peak viral RNA loads and depleted gut CD4⁺ T cells. SHIV-1157ipEL-p and SHIV-1157ipd3N4 env genes diverge mostly in V1/V2. Molecular modeling revealed a possible mechanism for the increased neutralization resistance of SHIV-1157ipd3N4 Env: V2 loops hindering access to the CD4 binding site, shown experimentally with nAb b12. Similar mutations have been linked to decreased neutralization sensitivity in HIV-C strains isolated from humans over time, indicating parallel HIV-C Env evolution in humans and RM.

Conclusions/Significance

SHIV-1157ipEL-p, the first tier 1 R5 clade C SHIV, and SHIV-1157ipd3N4, its tier 2 counterpart, represent biologically relevant tools for anti-HIV-C vaccine development in primates.     

Macrophage tropism determinants of human immunodeficiency virus type 1 in vivo.

Strains of human immunodeficiency virus type 1 differ in their abilities to infect and replicate in primary human macrophages. Chimeric clones were constructed from a provirus unable to infect macrophages (NLHX) and envelope sequences (V3 loop) of viruses derived without cultivation from brain (YU2 and w1-1c1) or spleen (w2-1b4) tissues. The substituted V3 loop sequences in each case were sufficient to confer upon NLHX the ability to infect macrophages. Furthermore, an envelope domain immediately N terminal to the V3 loop also was found to modulate the level of replication in macrophages. These results demonstrate that an envelope determinant derived directly from patients with AIDS confers HIV-1 tropism for macrophages.     

Relationship of the human immunodeficiency virus type 1 gp120 third variable loop to a component of the CD4 binding site in the fourth conserved region.

Neutralizing antibodies that recognize the human immunodeficiency virus gp120 exterior envelope glycoprotein and are directed against either the third variable (V3) loop or conserved, discontinuous epitopes overlapping the CD4 binding region have been described. Here we report several observations that suggest a structural relationship between the V3 loop and amino acids in the fourth conserved (C4) gp120 region that constitute part of the CD4 binding site and the conserved neutralization epitopes. Treatment of the gp120 glycoprotein with ionic detergents resulted in a V3 loop-dependent masking of both linear C4 epitopes and discontinuous neutralization epitopes overlapping the CD4 binding site. Increased recognition of the native gp120 glycoprotein by an anti-V3 loop monoclonal antibody, 9284, resulted from from single amino acid changes either in the base of the V3 loop or in the gp120 C4 region. These amino acid changes also resulted in increased exposure of conserved epitopes overlapping the CD4 binding region. The replication-competent subset of these mutants exhibited increased sensitivity to neutralization by antibody 9284 and anti-CD4 binding site antibodies. The implied relationship of the V3 loop, which mediates post-receptor binding steps in virus entry, and components of the CD4 binding region may be important for the interaction of these functional gp120 domains and for the observed cooperativity of neutralizing antibodies directed against these regions.     

N-linked glycosylation sites adjacent to and within the V1/V2 and the V3 loops of dualtropic human immunodeficiency virus type 1 isolate DH12 gp120 affect coreceptor usage and cellular tropism

The envelope glycoprotein of human immunodeficiency virus type 1 (HIV-1) is extensively glycosylated, containing approximately 23 asparagine (N)-linked glycosylation sites on its gp120 subunit. In this study, specific glycosylation sites on gp120 of a dualtropic primary HIV-1 isolate, DH12, were eliminated by site-directed mutagenesis and the properties of the resulting mutant envelopes were evaluated using a recombinant vaccinia virus-based cell-to-cell fusion assay alone or in the context of viral infections. Of the glycosylation sites that were evaluated, those proximal to the V1/V2 loops (N135, N141, N156, N160) and the V3 loops (N301) of gp120 were functionally critical. The glycosylation site mutations near the V1/V2 loop compromised the use of CCR5 and CXCR4 equally. In contrast, a mutation within the V3 loop preferentially inhibited the usage of CCR5; although this mutant protein completely lost its CCR5-dependent fusion activity, it retained 50% of the wild-type fusion activity with CXCR4. The replication of a virus containing this mutation was severely compromised in peripheral blood mononuclear cells, MT-4 cells, and primary monocyte-derived macrophages. A revertant virus, which acquired second site changes in the V3 loop that resulted in an increase in net positive charge, was isolated. The revertant virus fully recovered the usage of CXCR4 but not of CCR5, thereby altering the tropism of the parental virus from dualtropic to T-tropic. These results suggest that carbohydrate moieties near the V1/V2 and the V3 loops play critical roles in maintaining proper conformation of the variable loops for optimal interaction with receptors. Our results, combined with those of previously reported studies, further demonstrate that the function of individual glycans may be virus isolate dependent.     

Removal of a single N-linked glycan in human immunodeficiency virus type 1 gp120 results in an enhanced ability to induce neutralizing antibody responses

Glycans on human immunodeficiency virus (HIV) envelope protein play an important role in infection and evasion from host immune responses. To examine the role of specific glycans, we introduced single or multiple mutations into potential N-linked glycosylation sites in hypervariable regions (V1 to V3) of the env gene of HIV type 1 (HIV-1) 89.6. Three mutants tested showed enhanced sensitivity to soluble CD4. Mutant N7 (N197Q) in the carboxy-terminal stem of the V2 loop showed the most pronounced increase in sensitivity to broadly neutralizing antibodies (NtAbs), including those targeting the CD4-binding site (IgG1b12) and the V3 loop (447-52D). This mutant is also sensitive to CD4-induced NtAb 17b in the absence of CD4. Unlike the wild-type (WT) Env, mutant N7 mediates CD4-independent infection in U87-CXCR4 cells. To study the immunogenicity of mutant Env, we immunized pig-tailed macaques with recombinant vaccinia viruses, one expressing SIVmac239 Gag-Pol and the other expressing HIV-1 89.6 Env gp160 in WT or mutant forms. Animals were boosted 14 to 16 months later with simian immunodeficiency virus gag DNA and the cognate gp140 protein before intrarectal challenge with SHIV89.6P-MN. Day-of-challenge sera from animals immunized with mutant N7 Env had significantly higher and broader neutralizing activities than sera from WT Env-immunized animals. Neutralizing activity was observed against SHIV89.6, SHIV89.6P-MN, HIV-1 SF162, and a panel of subtype B primary isolates. Compared to control animals, immunized animals showed significant reduction of plasma viral load and increased survival after challenge, which correlated with prechallenge NtAb titers. These results indicate the potential advantages for glycan modification in vaccine design, although the role of specific glycans requires further examination.     

Detailed characterization of antibody responses against HIV-1 group M consensus gp120 in rabbits

Background

We recently reported induction of broadly neutralizing antibodies (bnAbs) against multiple HIV-1 (human immunodeficiency virus type 1) isolates in rabbits, albeit weak against tier 2 viruses, using a monomeric gp120 derived from an M group consensus sequence (MCON6). To better understand the nature of the neutralizing activity, detailed characterization of immunological properties of the protein was performed. Immunogenic linear epitopes were identified during the course of immunization, and spatial distribution of these epitopes was determined. Subdomain antibody target analyses were done using the gp120 outer domain (gp120-OD) and eOD-GT6, a protein based on a heterologous sequence. In addition, refined epitope mapping analyses were done by competition assays using several nAbs with known epitopes.

Results

Based on linear epitope mapping analyses, the V3 loop was most immunogenic, followed by C1 and C5 regions. The V1/V2 loop was surprisingly non-immunogenic. Many immunogenic epitopes were clustered together even when they were distantly separated in primary sequence, suggesting the presence of immunogenic hotspots on the protein surface. Although substantial antibody responses were directed against the outer domain, only about 0.1% of the antibodies bound eOD-GT6. Albeit weak, antibodies against peptides that corresponded to a part of the bnAb VRC01 binding site were detected. Although gp120-induced antibodies could not block VRC01 binding to eOD-GT6, they were able to inhibit VRC01 binding to both gp120 and trimeric BG505 SOSIP gp140. The immune sera also efficiently competed with CD4-IgG2, as well as nAbs 447-52D, PGT121 and PGT126, in binding to gp120.

Conclusions

The results suggest that some antibodies that bind at or near known bnAb epitopes could be partly responsible for the breadth of neutralizing activity induced by gp120 in our study. Immunization strategies that enhance induction of these antibodies relative to others (e.g. V3 loop), and increase their affinity, could improve protective efficacy of an HIV-1 vaccine.

     

Three amino acid residues in the envelope of human immunodeficiency virus type 1 CRF07_BC regulate viral neutralization susceptibility to the human monoclonal neutralizing antibody IgG1b12

The CD4 binding site(CD4bs) of envelope glycoprotein(Env) is an important conserved target for anti-human immunodeficiency virus type 1(HIV-1) neutralizing antibodies. Neutralizing monoclonal antibodies IgG1 b12(b12) could recognize conformational epitopes that overlap the CD4 bs of Env. Different virus strains, even derived from the same individual, showed distinct neutralization susceptibility to b12. We examined the key amino acid residues affecting b12 neutralization susceptibility using single genome amplification and pseudovirus neutralization assay. Eleven amino acid residues were identified that affect the sensitivity of Env to b12. Through site-directed mutagenesis, an amino acid substitution at position 182 in the V2 region of Env was confirmed to play a key role in regulating the b12 neutralization susceptibility. The introduction of V182 L to a resistant strain enhanced its sensitivity to b12 more than twofold. Correspondingly, the introduction of L182 V to a sensitive strain reduced its sensitivity to b12 more than tenfold. Amino acid substitution at positions 267 and 346 could both enhance the sensitivity to b12 more than twofold. However, no additive effect was observed when the three site mutageneses were introduced into the same strain, and the sensitivity was equivalent to the single V182 L mutation. CRF07_BC is a major circulating recombinant form of HIV-1 prevalent in China. Our data may provide important information for understanding the molecular mechanism regulating the neutralization susceptibility of CRF07_BC viruses to b12 and may be helpful for a vaccine design targeting the CD4 bs epitopes.     

A conserved determinant in the V1 loop of HIV-1 modulates the V3 loop to prime low CD4 use and macrophage infection

The CD4 binding site (CD4bs) on the HIV-1 envelope plays a major role in determining the capacity of R5 viruses to infect primary macrophages. Thus, envelope determinants within or proximal to the CD4bs have been shown to control the use of low CD4 levels on macrophages for infection. These residues affect the affinity for CD4 either directly or indirectly by altering the exposure of CD4 contact residues. Here, we describe a single amino acid determinant in the V1 loop that also modulates macrophage tropism. Thus, we identified an E153G substitution that conferred high levels of macrophage infectivity for several heterologous R5 envelopes, while the reciprocal G153E substitution abrogated infection. Shifts in macrophage tropism were associated with dramatic shifts in sensitivity to the V3 loop monoclonal antibody (MAb), 447-52D and soluble CD4, as well as more modest changes in sensitivity to the CD4bs MAb, b12. These observations are consistent with an altered conformation or exposure of the V3 loop that enables the envelope to use low CD4 levels for infection. The modest shifts in b12 sensitivity suggest that residue 153 impacts on the exposure of the CD4bs. However, the more intense shifts in sCD4 sensitivity suggest additional mechanisms that likely include an increased ability of the envelope to undergo conformational changes following binding to suboptimal levels of cell surface CD4. In summary, we show that a conserved determinant in the V1 loop modulates the V3 loop to prime low CD4 use and macrophage infection.     

V2 loop glycosylation of the human immunodeficiency virus type 1 SF162 envelope facilitates interaction of this protein with CD4 and CCR5 receptors and protects the virus from neutralization by anti-V3 loop and anti-CD4 binding site antibodies

We examined the role of asparagine-linked glycosylation of the V2 loop of the human immunodeficiency virus (HIV) SF162 envelope on viral replication potential and neutralization susceptibility. We report that the asparagines located at the amino- and carboxy-terminal sites (at positions 154 and 195, respectively), as well as within the V2 loop of the SF162 envelope (at position 186), are glycosylated during in vitro replication of this virus in human peripheral blood mononuclear cells. Our studies indicate that glycosylation of the V2 loop, in particular at its base, facilitates the interaction of the HIV envelope with the CD4 and CCR5 receptor molecules present on the surface of target cells and affects viral replication kinetics in a cell type-dependent manner. In cells expressing high numbers of receptor molecules on their surfaces, the SF162-derived V2 loop-deglycosylated mutant viruses replicate as efficiently as the parental SF162 virus, while in cells expressing small numbers of receptor molecules, the mutant viruses replicate with markedly reduced efficiency. In addition to expanding the viral tropism, V2 loop glycosylation at the three sites examined prevents neutralization by anti-CD4 binding site antibodies. In contrast, glycosylation at the amino- and carboxy-terminal sites of the V2 loop but not within the loop itself offers protection against anti-V3 loop antibodies. Thus, the epitopes masked by the sugar molecules present on the three glycosylation sites examined are not identical but overlap.     

UCLA1, a synthetic derivative of a gp120 RNA aptamer, inhibits entry of human immunodeficiency virus type 1 subtype C

Entry of human immunodeficiency virus type 1 (HIV-1) into cells is mediated by the virion surface envelope (Env) glycoproteins, making it a desirable target for antiretroviral entry inhibitors. We previously isolated a family of gp120 binding RNA aptamers and showed that they neutralized the infectivity of HIV-1. In this study, we assessed the activity of a shortened synthetic derivative of the B40 aptamer, called UCLA1, against a large panel of HIV-1 subtype C viruses. UCLA1 tightly bound to a consensus HIV-1 subtype C gp120 and neutralized isolates of the same subtype with 50% inhibitory concentrations (IC(50)s) in the nanomolar range. The aptamer had little toxicity in tests with cell lines and primary cells. Furthermore, it exhibited high therapeutic indices, suggesting that it may be effective at very low doses. Mapping of UCLA1 binding sites on gp120 revealed eight amino acid residues that modulated neutralization resistance. This included residues within the coreceptor binding site, at the base of the V3 loop, and in the bridging sheet within the conserved V1/V2 stem-loop of gp120. The aptamer was also shown to have synergistic effects with T20, a gp41 fusion inhibitor, and IgG1b12 (b12), an anti-CD4 binding site monoclonal antibody. These results suggest that UCLA1 may be suitable for development as a potent HIV-1 entry inhibitor.     

N-linked glycosylation of the V3 loop and the immunologically silent face of gp120 protects human immunodeficiency virus type 1 SF162 from neutralization by anti-gp120 and anti-gp41 antibodies

We examined how asparagine-linked glycans within and adjacent to the V3 loop (C2 and C3 regions) and within the immunologically silent face (V4, C4, and V5 regions) of the human immunodeficiency virus (HIV) SF612 envelope affect the viral phenotype. Five of seven potential glycosylation sites are utilized when the virus is grown in human peripheral blood mononuclear cells, with the nonutilized sites lying within the V4 loop. Elimination of glycans within and adjacent to the V3 loop renders SF162 more susceptible to neutralization by polyclonal HIV(+)-positive and simian/human immunodeficiency virus-positive sera and by monoclonal antibodies (MAbs) recognizing the V3 loop, the CD4- and CCR5-binding sites, and the extracellular region of gp41. Importantly, our studies also indicate that glycans located within the immunologically silent face of gp120, specifically the C4 and V5 regions, also conferred on SF162 resistance to neutralization by anti-V3 loop, anti-CD4 binding site, and anti-gp41 MAbs but not by antibodies targeting the coreceptor binding site. We also observed that the amino acid composition of the V4 region contributes to the neutralization phenotype of SF162 by anti-V3 loop and anti-CD4 binding site MAbs. Collectively, our data support the proposal that the glycosylation and structure of the immunologically silent face of the HIV envelope plays an important role in defining the neutralization phenotype of HIV type 1.     

The synthetic peptide derived from the NH2-terminal extracellular region of an orphan G protein-coupled receptor, GPR1, preferentially inhibits infection of X4 HIV-1

Several G protein-coupled receptors (GPCRs) serve as co-receptors for entry of human immunodeficiency virus type 1 (HIV-1) into target cells. Here we report that a synthetic peptide derived from the NH2-terminal extracellular region of an orphan GPCR, GPR1 (GPR1ntP-(1-27); MEDLEETLFEEFENYSYDLDYYSLESC), inhibited infection of not only an HIV-1 variant that uses GPR1 as a co-receptor, but also X4, R5, and R5X4 viruses. Among these HIV-1 strains tested, viruses that can utilize CXCR4 as their co-receptors were preferentially inhibited. Inhibition of early steps in X4 virus replication was also detected in the primary human peripheral blood lymphocytes. GPR1ntP-(1-27) directly interacted with recombinant X4 envelope glycoprotein (rgp120). This interaction was neither inhibited nor enhanced by the soluble CD4 (sCD4) but inhibited by the anti-third variable (V3) loop-specific monoclonal antibody and heparin known to bind to the V3 loop. Although the conformational changes in gp120, including the V3 loop, have been reported to be required for its interaction with a co-receptor after binding of gp120 to CD4, it has also been reported that the V3 loop is already exposed on the surface of virions before interaction with CD4. We found that GPR1ntP-(1-27) blocked binding of virus to the cells, and this peptide equally bound to rgp120 in the presence or absence of sCD4. Because we detected the binding of GPR1ntP-(1-27) to the highly purified virions even in the absence of sCD4, GPR1ntP-(1-27) probably recognized the V3 loop exposed on the virions, and this interaction was responsible for the anti-HIV-1 activity of GPR1ntP-(1-27).     

Mechanism of neutralization by the broadly neutralizing HIV-1 monoclonal antibody VRC01

The structure of VRC01 in complex with the HIV-1 gp120 core reveals that this broadly neutralizing CD4 binding site (CD4bs) antibody partially mimics the interaction of the primary virus receptor, CD4, with gp120. Here, we extended the investigation of the VRC01-gp120 core interaction to the biologically relevant viral spike to better understand the mechanism of VRC01-mediated neutralization and to define viral elements associated with neutralization resistance. In contrast to the interaction of CD4 or the CD4bs monoclonal antibody (MAb) b12 with the HIV-1 envelope glycoprotein (Env), occlusion of the VRC01 epitope by quaternary constraints was not a major factor limiting neutralization. Mutagenesis studies indicated that VRC01 contacts within the gp120 loop D, the CD4 binding loop, and the V5 region were necessary for optimal VRC01 neutralization, as suggested by the crystal structure. In contrast to interactions with the soluble gp120 monomer, VRC01 interaction with the native viral spike did not occur in a CD4-like manner; VRC01 did not induce gp120 shedding from the Env spike or enhance gp41 membrane proximal external region (MPER)-directed antibody binding to the Env spike. Finally, VRC01 did not display significant reactivity with human antigens, boding well for potential in vivo applications. The data indicate that VRC01 interacts with gp120 in the context of the functional spike in a manner distinct from that of CD4. It achieves potent neutralization by precisely targeting the CD4bs without requiring alterations of Env spike configuration and by avoiding steric constraints imposed by the quaternary structure of the functional Env spike.