Enhancing exposure of HIV-1 neutralization epitopes through mutations in gp41

Background

The generation of broadly neutralizing antibodies is a priority in the design of vaccines against HIV-1. Unfortunately, most antibodies to HIV-1 are narrow in their specificity, and a basic understanding of how to develop antibodies with broad neutralizing activity is needed. Designing methods to target antibodies to conserved HIV-1 epitopes may allow for the generation of broadly neutralizing antibodies and aid the global fight against AIDS by providing new approaches to block HIV-1 infection. Using a naturally occurring HIV-1 Envelope (Env) variant as a template, we sought to identify features of Env that would enhance exposure of conserved HIV-1 epitopes.

Methods and Findings

Within a cohort study of high-risk women in Mombasa, Kenya, we previously identified a subtype A HIV-1 Env variant in one participant that was unusually sensitive to neutralization. Using site-directed mutagenesis, the unusual neutralization sensitivity of this variant was mapped to two amino acid mutations within conserved sites in the transmembrane subunit (gp41) of the HIV-1 Env protein. These two mutations, when introduced into a neutralization-resistant variant from the same participant, resulted in 3- to >360-fold enhanced neutralization by monoclonal antibodies specific for conserved regions of both gp41 and the Env surface subunit, gp120, >780-fold enhanced neutralization by soluble CD4, and >35-fold enhanced neutralization by the antibodies found within a pool of plasmas from unrelated individuals. Enhanced neutralization sensitivity was not explained by differences in Env infectivity, Env concentration, Env shedding, or apparent differences in fusion kinetics. Furthermore, introduction of these mutations into unrelated viral Env sequences, including those from both another subtype A variant and a subtype B variant, resulted in enhanced neutralization susceptibility to gp41- and gp120-specific antibodies, and to plasma antibodies. This enhanced neutralization sensitivity exceeded 1,000-fold in several cases.

Conclusions

Two amino acid mutations within gp41 were identified that expose multiple discontinuous neutralization epitopes on diverse HIV-1 Env proteins. These exposed epitopes were shielded on the unmodified viral Env proteins, and several of the exposed epitopes encompass desired target regions for protective antibodies. Env proteins containing these modifications could act as a scaffold for presentation of such conserved domains, and may aid in developing methods to target antibodies to such regions.     

Mutation at a Single Position in the V2 Domain of the HIV-1 Envelope Protein Confers Neutralization Sensitivity to a Highly Neutralization-Resistant Virus

Understanding the determinants of neutralization sensitivity and resistance is important for the development of an effective human immunodeficiency virus type 1 (HIV-1) vaccine. In these studies, we have made use of the swarm of closely related envelope protein variants (quasispecies) from an extremely neutralization-resistant clinical isolate in order to identify mutations that conferred neutralization sensitivity to antibodies in sera from HIV-1-infected individuals. Here, we describe a virus with a rare mutation at position 179 in the V2 domain of gp120, where replacement of aspartic acid (D) by asparagine (N) converts a virus that is highly resistant to neutralization by multiple polyclonal and monoclonal antibodies, as well as antiviral entry inhibitors, to one that is sensitive to neutralization. Although the V2 domain sequence is highly variable, D at position 179 is highly conserved in HIV-1 and simian immunodeficiency virus (SIV) and is located within the LDI/V recognition motif of the recently described α4β7 receptor binding site. Our results suggest that the D179N mutation induces a conformational change that exposes epitopes in both the gp120 and the gp41 portions of the envelope protein, such as the CD4 binding site and the MPER, that are normally concealed by conformational masking. Our results suggest that D179 plays a central role in maintaining the conformation and infectivity of HIV-1 as well as mediating binding to α4β7.A major goal in human immunodeficiency virus type 1 (HIV-1) vaccine research is the identification of immunogens able to elicit protective immunity from HIV-1 infection. Results from the recent RV144 clinical trial in Thailand (53) have provided evidence that immunization with vaccines containing the recombinant HIV-1 envelope glycoprotein gp120 (6, 7) can protect humans from HIV infection when incorporated in a prime/boost immunization regimen. Although the level of protection observed in the RV144 trial (31%) was modest, it represents a significant advance in HIV-1 vaccine research and has rekindled the efforts to identify improved subunit vaccine antigens that might achieve even higher levels of protection. In these studies, we have sought to understand the molecular determinants of neutralization sensitivity and resistance in HIV-1 envelope proteins for the purpose of developing improved vaccine antigens.In previous studies (47), we have described a novel method of mutational analysis of the HIV-1 envelope protein, termed swarm analysis, for identification of mutations that confer sensitivity and/or resistance to broadly neutralizing antibodies (bNAbs). This method makes use of the natural amino acid sequence virus variation that occurs in each HIV-infected individual to establish panels of closely related envelope proteins that differ from each other by a limited number of amino acid substitutions. We have previously used this method to identify a novel amino acid substitution in gp41 that conferred sensitivity to neutralization by monoclonal and polyclonal antibodies as well as virus entry inhibitors. In this paper, we describe a mutation in the V2 domain of gp120 that similarly induces a neutralization-sensitive phenotype in an otherwise neutralization-resistant envelope sequence.Previous studies (10, 14, 33, 40, 43, 52, 72, 74) have suggested that sequences in the V2 domain act as the “global regulator of neutralization sensitivity” and confer neutralization resistance by restricting access to epitopes located in the V3 domain, the CD4 binding site, and chemokine receptor binding sites through “conformational masking” of neutralizing epitopes. Deletion of the V2 domain markedly increases neutralization sensitivity (10, 57, 62, 74), and several envelope proteins with V2 domain deletions have been developed as candidate HIV-1 vaccines (5, 42, 61). In this paper, we show that a single substitution of asparagine (N) for aspartic acid (D) at position 179 in the C-terminal portion of the V2 domain (corresponding to position 180 in HXB2 numbering) converts a highly neutralization-resistant virus to a neutralization-sensitive virus with a phenotype similar to that described for V2 domain deletion mutants. Position 179 has recently attracted attention as a critical element of the α4β7 integrin binding site that affects virus tropism to the gut (2). Our results suggest that mutation at position 179 results in a conformational change that increases neutralization sensitivity by exposure of epitopes in both gp120 and gp41 that are normally masked in the trimeric structure of gp160 and thus are unavailable for antibody binding.     

Crystal structure of human antibody 2909 reveals conserved features of quaternary structure-specific antibodies that potently neutralize HIV-1

Monoclonal antibody 2909 belongs to a class of potently neutralizing antibodies that recognize quaternary epitopes on HIV-1. Some members of this class, such as 2909, are strain specific, while others, such as antibody PG16, are broadly neutralizing; all, however, recognize a region on the gp120 envelope glycoprotein that includes two loops (V2 and V3) and forms appropriately only in the oligomeric HIV-1 spike (gp120₃/gp41₃). Here we present the crystal structure of 2909 and report structure-function analysis with antibody chimeras composed of 2909 and other members of this antibody class. The 2909 structure was dominated by a heavy-chain third-complementarity-determining region (CDR H3) of 21 residues, which comprised 36% of the combining surface and formed a β-hairpin club extending ∼20 Å beyond the rest of the antibody. Sequence analysis and mass spectrometry identified sites of tyrosine sulfation at the middle and top of CDR H3; substitutions with phenylalanine either ablated (middle substitution) or substantially diminished (top substitution) neutralization. Chimeric antibodies composed of heavy and light chains, exchanged between 2909 and other members of the class, indicated a substantial lack of complementation. Comparison of 2909 to PG16 (which is tyrosine sulfated and the only other member of the class for which a structure has previously been reported) showed that both utilize protruding, anionic CDR H3s for recognition. Thus, despite some diversity, members of this class share structural and functional similarities, with conserved features of the CDR H3 subdomain likely reflecting prevalent solutions by the human immune system for recognition of a quaternary site of HIV-1 vulnerability.Identification of conserved regions accessible on the HIV-1 envelope and design of immunogens that elicit broadly neutralizing antibodies against these sites continue to be major challenges in the development of an effective HIV-1 vaccine. The HIV-1 viral spike—composed of three exterior gp120 subunits and three transmembrane gp41 subunits—is highly protected, but a limited number of these conserved regions exist on the spike, identified primarily by the broadly neutralizing antibodies that target them. One region is quaternary in nature and appropriately formed only on the assembled viral spike (gp120₃/gp41₃). This region is targeted by a recently discovered (14) and fast expanding class of monoclonal antibodies (36, 40) that recognize epitopes with quaternary structural constraints, which are composed of portions of two gp120-variable loops, V2 and V3 (reviewed in reference 49). These quaternary structure-specific (or quaternary-specific) antibodies (also called quaternary-neutralizing epitope or “QNE” antibodies) are found in the sera of selected HIV-1-infected individuals who have broadly neutralizing serum antibodies (41); individual members of the class, however, vary greatly in their breadth of neutralization.Initial evidence for the existence of quaternary-specific antibodies arose in simian/human immunodeficiency virus-infected rhesus macaques and HIV-1-infected chimpanzees (6, 9, 13). Characterization of polyclonal sera from these infected animals suggested the presence of antibodies targeting a conformational epitope involving the variable loop regions of the gp120 viral envelope.Antibody 2909 was the first human monoclonal antibody against HIV-1 to be characterized as being specific for an epitope dependent on the quaternary interaction of envelope glycoproteins (14). It was identified by direct screening for neutralization activity against a pseudovirus derived from strain SF162 of HIV-1. It recognizes a quaternary epitope on the surface of native virions and infected cells but does not bind soluble gp120/gp140 envelope proteins or cell surface-expressed gp120 monomers (14, 20). Competition analysis and virological assays indicate that the 2909 epitope includes portions of the V2 and V3 loops of gp120 (14, 16), with the V2-V3 elements originating either from within a gp120 monomer or between gp120 protomers in the trimer context. Mapping of 2909 recognition identifies a particular anomaly in its recognition (16); neutralization by 2909 depends on the presence of a rare lysine at position 160 in the V2 loop rather than the conserved N-linked site of glycosylation found at this position in most HIV-1 isolates (providing a residue-specific explanation for the neutralization specificity of 2909 for the SF162 virus, which contains this rare lysine).Other strain-specific monoclonal antibodies like 2909 have been isolated from rhesus macaques infected with a chimeric simian/human immunodeficiency virus that contained an SF162 isolate-derived viral spike (SHIV_SF162P4) (36). These rhesus monoclonal antibodies exhibit properties similar to those of 2909 in their potent neutralization of SF162 and their recognition of V2-V3 only in the context of the functional viral spike (e.g., on virus particles) (36). Details from epitope mapping indicate that these rhesus antibodies and human antibody 2909 recognize overlapping epitopes, with some differences in requirements for V2 N-linked glycosylation (36).The somatically related human monoclonal antibodies, PG9 and PG16, were also identified by a direct screen for neutralization (40). They target a quaternary-specific V2-V3 epitope, but unlike 2909, they neutralize an extraordinary 70 to 80% of circulating primary HIV-1 isolates and appear to have some reactivity for monomeric gp120 (40). Much of their increased breadth of neutralization arises from their ability to recognize an N-linked glycan at position 160 in the V2 loop, a motif which is found in greater than 90% of HIV-1 group M isolates (25).Despite substantial differences in their neutralization breadth, antibodies 2909 and PG9/PG16 may be closely related. Notably, an N160K mutation in the V2 loop of typical primary HIV-1 isolates like YU2 and JR-FL can recover 2909 activity (16). Conversely, isolate SF162 can be converted to a PG9- and PG16-sensitive pseudovirus by the K160N mutation (40). Thus, a single N or K at position 160 appears to control much of the neutralization difference between 2909 and PG16. Together the results suggest that 2909 and PG9/PG16 antibodies recognize distinct immunotypes of a similar quaternary epitope.To gain insight into how antibodies achieve recognition of this epitope, we determined the crystal structure of the antigen-binding fragment (Fab) of 2909 at a 3.3-Å resolution and compared this structure to the previously determined structure of PG16 (31, 33). Mutational analysis was used to confirm structural hot spots, and chimeric analysis of domain swaps between 2909 and other quaternary-specific antibodies was used to refine assessments of functional similarity. By identifying structural features—shared between 2909 and PG16 but otherwise highly uncommon in antibodies—the results provide insight into conserved solutions by human antibodies for recognition of an important vaccine target on HIV-1.     

Cloaked similarity between HIV-1 and SARS-CoV suggests an anti-SARS strategy

Background

Severe acute respiratory syndrome (SARS) is a febrile respiratory illness. The disease has been etiologically linked to a novel coronavirus that has been named the SARS-associated coronavirus (SARS-CoV), whose genome was recently sequenced. Since it is a member of the Coronaviridae, its spike protein (S2) is believed to play a central role in viral entry by facilitating fusion between the viral and host cell membranes. The protein responsible for viral-induced membrane fusion of HIV-1 (gp41) differs in length, and has no sequence homology with S2.

Results

Sequence analysis reveals that the two viral proteins share the sequence motifs that construct their active conformation. These include (1) an N-terminal leucine/isoleucine zipper-like sequence, and (2) a C-terminal heptad repeat located upstream of (3) an aromatic residue-rich region juxtaposed to the (4) transmembrane segment.

Conclusions

This study points to a similar mode of action for the two viral proteins, suggesting that anti-viral strategy that targets the viral-induced membrane fusion step can be adopted from HIV-1 to SARS-CoV. Recently the FDA approved Enfuvirtide, a synthetic peptide corresponding to the C-terminal heptad repeat of HIV-1 gp41, as an anti-AIDS agent. Enfuvirtide and C34, another anti HIV-1 peptide, exert their inhibitory activity by binding to a leucine/isoleucine zipper-like sequence in gp41, thus inhibiting a conformational change of gp41 required for its activation. We suggest that peptides corresponding to the C-terminal heptad repeat of the S2 protein may serve as inhibitors for SARS-CoV entry.     

Selective killing of human immunodeficiency virus infected cells by non-nucleoside reverse transcriptase inhibitor-induced activation of HIV protease

Background

The gp41 subunit of the HIV-1 envelope glycoprotein (Env) has been widely regarded as a type I transmembrane protein with a single membrane-spanning domain (MSD). An alternative topology model suggested multiple MSDs. The major discrepancy between the two models is that the cytoplasmic Kennedy sequence in the single MSD model is assigned as the extracellular loop accessible to neutralizing antibodies in the other model. We examined the membrane topology of the gp41 subunit in both prokaryotic and mammalian systems. We attached topological markers to the C-termini of serially truncated gp41. In the prokaryotic system, we utilized a green fluorescent protein (GFP) that is only active in the cytoplasm. The tag protein (HaloTag) and a membrane-impermeable ligand specific to HaloTag was used in the mammalian system.

Results

In the absence of membrane fusion, both the prokaryotic and mammalian systems (293FT cells) supported the single MSD model. In the presence of membrane fusion in mammalian cells (293CD4 cells), the data obtained seem to support the multiple MSD model. However, the region predicted to be a potential MSD is the highly hydrophilic Kennedy sequence and is least likely to become a MSD based on several algorithms. Further analysis revealed the induction of membrane permeability during membrane fusion, allowing the membrane-impermeable ligand and antibodies to cross the membrane. Therefore, we cannot completely rule out the possible artifacts. Addition of membrane fusion inhibitors or alterations of the MSD sequence decreased the induction of membrane permeability.

Conclusions

It is likely that a single MSD model for HIV-1 gp41 holds true even in the presence of membrane fusion. The degree of the augmentation of membrane permeability we observed was dependent on the membrane fusion and sequence of the MSD.     

The role of <Emphasis Type="Italic">N</Emphasis>-glycans of HIV-1 gp41 in virus infectivity and susceptibility to the suppressive effects of carbohydrate-binding agents

Background

Carbohydrate-binding agents (CBAs) are potent antiretroviral compounds that target the N-glycans on the HIV-1 envelope glycoproteins. The development of phenotypic resistance to CBAs by the virus is accompanied by the deletion of multiple N-linked glycans of the surface envelope glycoprotein gp120. Recently, also an N-glycan on the transmembrane envelope glycoprotein gp41 was shown to be deleted during CBA resistance development.

Results

We generated HIV-1 mutants lacking gp41 N-glycans and determined the influence of these glycan deletions on the viral phenotype (infectivity, CD4 binding, envelope glycoprotein incorporation in the viral particle and on the transfected cell, virus capture by DC-SIGN⁺ cells and transmission of DC-SIGN-captured virions to CD4⁺ T-lymphocytes) and on the phenotypic susceptibility of HIV-1 to a selection of CBAs. It was shown that some gp41 N-glycans are crucial for the infectivity of the virus. In particular, lack of an intact N616 glycosylation site was shown to result in the loss of viral infectivity of several (i.e. the X4-tropic III_B and NL4.3 strains, and the X4/R5-tropic HE strain), but not all (i.e. the R5-tropic ADA strain) studied HIV-1 strains. In accordance, we found that the gp120 levels in the envelope of N616Q mutant gp41 strains NL4.3, III_B and HE were severely decreased. In contrast, N616Q gp41 mutant HIV-1_ADA contained gp120 levels similar to the gp120 levels in WT HIV-1_ADA virus. Concomitantly deleting multiple gp41 N-glycans was often highly detrimental for viral infectivity. Using surface plasmon resonance technology we showed that CBAs have a pronounced affinity for both gp120 and gp41. However, the antiviral activity of CBAs is not dependent on the concomitant presence of all gp41 glycans. Single gp41 glycan deletions had no marked effects on CBA susceptibility, whereas some combinations of two to three gp41 glycan-deletions had a minor effect on CBA activity.

Conclusions

We revealed the importance of some gp41 N-linked glycans, in particular the N616 glycan which was shown to be absolutely indispensable for the infectivity potential of several virus strains. In addition, we demonstrated that the deletion of up to three gp41 N-linked glycans only slightly affected CBA susceptibility.

     

Characterization of HIV-1 envelope gp41 genetic diversity and functional domains following perinatal transmission

Background

HIV-1 envelope gp41 is a transmembrane protein that promotes fusion of the virus with the plasma membrane of the host cells required for virus entry. In addition, gp41 is an important target for the immune response and development of antiviral and vaccine strategies, especially when targeting the highly variable envelope gp120 has not met with resounding success. Mutations in gp41 may affect HIV-1 entry, replication, pathogenesis, and transmission. We, therefore, characterized the molecular properties of gp41, including genetic diversity, functional motifs, and evolutionary dynamics from five mother-infant pairs following perinatal transmission.

Results

The gp41 open reading frame (ORF) was maintained with a frequency of 84.17% in five mother-infant pairs' sequences following perinatal transmission. There was a low degree of viral heterogeneity and estimates of genetic diversity in gp41 sequences. Both mother and infant gp41 sequences were under positive selection pressure, as determined by ratios of non-synonymous to synonymous substitutions. Phylogenetic analysis of 157 mother-infant gp41 sequences revealed distinct clusters for each mother-infant pair, suggesting that the epidemiologically linked mother-infant pairs were evolutionarily closer to each other as compared with epidemiologically unlinked sequences. The functional domains of gp41, including fusion peptide, heptad repeats, glycosylation sites and lentiviral lytic peptides were mostly conserved in gp41 sequences analyzed in this study. The CTL recognition epitopes and motifs recognized by fusion inhibitors were also conserved in the five mother-infant pairs.

Conclusion

The maintenance of an intact envelope gp41 ORF with conserved functional domains and a low degree of genetic variability as well as positive selection pressure for adaptive evolution following perinatal transmission is consistent with an indispensable role of envelope gp41 in HIV-1 replication and pathogenesis.     

Identification of broadly neutralizing antibody epitopes in the HIV-1 envelope glycoprotein using evolutionary models

Background

Identification of the epitopes targeted by antibodies that can neutralize diverse HIV-1 strains can provide important clues for the design of a preventative vaccine.

Methods

We have developed a computational approach that can identify key amino acids within the HIV-1 envelope glycoprotein that influence sensitivity to broadly cross-neutralizing antibodies. Given a sequence alignment and neutralization titers for a panel of viruses, the method works by fitting a phylogenetic model that allows the amino acid frequencies at each site to depend on neutralization sensitivities. Sites at which viral evolution influences neutralization sensitivity were identified using Bayes factors (BFs) to compare the fit of this model to that of a null model in which sequences evolved independently of antibody sensitivity. Conformational epitopes were identified with a Metropolis algorithm that searched for a cluster of sites with large Bayes factors on the tertiary structure of the viral envelope.

Results

We applied our method to ID₅₀ neutralization data generated from seven HIV-1 subtype C serum samples with neutralization breadth that had been tested against a multi-clade panel of 225 pseudoviruses for which envelope sequences were also available. For each sample, between two and four sites were identified that were strongly associated with neutralization sensitivity (2ln(BF)?>?6), a subset of which were experimentally confirmed using site-directed mutagenesis.

Conclusions

Our results provide strong support for the use of evolutionary models applied to cross-sectional viral neutralization data to identify the epitopes of serum antibodies that confer neutralization breadth.

     

Identification of a gp41 Core-Binding Molecule with Homologous Sequence of Human TNNI3K-Like Protein as a Novel Human Immunodeficiency Virus Type 1 Entry Inhibitor

Human immunodeficiency virus type 1 (HIV-1) gp41 plays a critical role in the viral fusion process, and its N- and C-terminal heptad repeat domains serve as important targets for developing anti-HIV-1 drugs, like T-20 (generic name, enfuvirtide; brand name, Fuzeon). Here, we conducted a yeast two-hybrid screening on a human bone marrow cDNA library using the recombinant soluble gp41 ectodomain as the bait and identified a novel gp41 core-binding molecule, designated P20. P20 showed no homology with a current HIV fusion inhibitor, T-20, but had sequence homology to a human protein, troponin I type 3 interacting kinase (TNNI3K)-like protein. While it could bind to the six-helix bundle core structure formed by the N- and C-terminal heptad repeats, P20 did not interrupt the formation of the six-helix bundle. P20 was effective in blocking HIV-1 Env-mediated syncytium formation and inhibiting infection by a broad spectrum of HIV-1 strains with distinct subtypes and coreceptor tropism, while it was ineffective against other enveloped viruses, such as vesicular stomatitis virus and influenza A virus. P20 exhibited no significant cytotoxicity to the CD4⁺ cells that were used for testing antiviral activity. Among the 11 P20 mutants, four analogous peptides with a common motif (WGRLEGRRT) exhibited significantly reduced anti-HIV-1 activity, suggesting that this region is the critical active site of P20. Therefore, this peptide can be used as a lead for developing novel HIV fusion inhibitors and as a probe for studying the membrane-fusogenic mechanism of HIV.Human immunodeficiency virus type 1 (HIV-1) is an enveloped virus, and its envelope protein (Env) complex controls the key processes by which HIV-1 delivers its replicative material into target cells. Specifically, the Env surface subunit, gp120, binds the cellular receptor CD4 and a coreceptor, CCR5 or CXCR4, which triggers conformational changes of the transmembrane subunit, gp41 (8). The N-terminal heptad repeat (NHR) in the gp41 ectodomain interacts with its C-terminal heptad repeat (CHR) to form a trimer of hairpins, or six-helix bundle (6-HB; also known as the gp41 fusion core) (38, 51), which brings the viral and target cell membranes into close proximity and promotes membrane fusion (3, 51). Therefore, the gp41 6-HB core plays an important role in viral fusion and may serve as an attractive target for the development of HIV fusion/entry inhibitors (20).In the early 1990s, a number of peptides derived from the gp41 NHR and CHR regions were discovered to exhibit highly potent anti-HIV-1 activity by binding to the corresponding region of gp41 at the fusion-intermediate state (22, 23, 38, 52, 53) and blocking gp41 6-HB core formation (4, 9, 32, 47). One of the CHR-peptides, T-20 (generic name, enfuvirtide; brand name, Fuzeon), was licensed by the FDA as the first member of a new class of anti-HIV drugs, the HIV fusion inhibitors (33, 53). Although T-20 is very effective in inhibiting infection by a broad spectrum of HIV-1 strains, especially those resistant to current antiretroviral therapies (26), T-20 itself also can easily induce drug resistance in T-20-treated patients, resulting in virologic failure (36, 46, 50, 55). Therefore, it is essential to identify and develop novel HIV-1 fusion inhibitors having a mechanism of action or target different from that for T-20 and with improved drug resistance profiles.Here, we sought to screen a human bone marrow cDNA library in a yeast two-hybrid screening assay using the recombinant soluble gp41 ectodomain (rsgp41e) as the bait in hopes of identifying a novel HIV fusion inhibitor with sequence homology to a human protein and low immunogenicity to humans to avoid its rapid clearance by specific human antibodies (1). We identified a 32-mer peptide, designated P20, with sequence homology to human troponin I type 3 interacting kinase (TNNI3K)-like protein. P20 could specifically bind to the gp41 6-HB core and strongly blocked HIV-1 Env-mediated membrane fusion. It potently inhibited infection by a number of laboratory-adapted HIV-1 strains, including T-20-resistant variants, and a broad spectrum of primary HIV-1 isolates. These results suggest that P20 has the potential to be developed further as a novel anti-HIV-1 therapeutic and can be used as a probe to study the role of the HIV-1 gp41 6-HB core in the membrane fusion process.     

The W100 pocket on HIV-1 gp120 penetrated by b12 is not a target for other CD4bs monoclonal antibodies

Background

The conserved CD4 binding site (CD4bs) on HIV-1 gp120 is a major target for vaccines. It is a priority to determine sites and structures within the CD4bs that are important for inclusion in vaccines. We studied a gp120 pocket penetrated by W100 of the potent CD4bs monoclonal antibody (mab), b12. We compared HIV-1 envelopes and corresponding mutants that carried blocked W100 pockets to evaluate whether other CD4bs mabs target this site.

Findings

All CD4bs mabs tested blocked soluble CD4 binding to gp120 consistent with their designation as CD4bs directed antibodies. All CD4bs mabs tested neutralized pseudovirions carrying NL4.3 wild type (wt) envelope. However, only b12 failed to neutralize pseudoviruses carrying mutant envelopes with a blocked W100 pocket. In addition, for CD4bs mabs that neutralized pseudovirions carrying primary envelopes, mutation of the W100 pocket had little or no effect on neutralization sensitivity.

Conclusions

Our data indicate that the b12 W100 pocket on gp120 is infrequently targeted by CD4bs mabs. This site is therefore not a priority for preservation in vaccines aiming to elicit antibodies targeting the CD4bs.     

Mutations in gp120 Contribute to the Resistance of Human Immunodeficiency Virus Type 1 to Membrane-Anchored C-Peptide maC46

Binding of the human immunodeficiency virus (HIV) envelope glycoprotein (Env) to the cellular CD4 receptor and a chemokine coreceptor initiates a series of conformational changes in the Env subunits gp120 and gp41. Eventually, the trimeric gp41 folds into a six-helix bundle, thereby inducing fusion of the viral and cellular membranes. C peptides derived from the C-terminal heptad repeat (CHR) of gp41 are efficient entry inhibitors as they block the six-helix bundle formation. Previously, we developed a membrane-anchored C peptide (maC46) expressed from a retroviral vector that also shows high activity against virus strains resistant to enfuvirtide (T-20), an antiviral C peptide approved for clinical use. Here, we present a systematic analysis of mutations in Env that confer resistance of HIV type 1 (HIV-1) to maC46. We selected an HIV-1 BaL strain with 10-fold reduced sensitivity to maC46 (BaL_C46) by passaging virus for nearly 200 days in the presence of gradually increasing concentrations of maC46. In comparison to wild-type BaL, BaL_C46 had five mutations at highly conserved positions in Env, three in gp120, one in the N-terminal heptad-repeat (NHR), and one in the CHR of gp41. No mutations were found in the NHR domain around the GIV motif that are known to cause resistance to enfuvirtide. Instead, maC46 resistance was found to depend on complementary mutations in the NHR and CHR that considerably favor binding of the mutated NHR to the mutated CHR over binding to maC46. In addition, resistance was highly dependent on mutations in gp120 that accelerated entry. Taken together, resistance to maC46 did not develop readily and required multiple cooperating mutations at conserved positions of the viral envelope glycoproteins gp120 and gp41.The entry process of the human immunodeficiency virus type 1 (HIV-1) has become a major target for new antiviral drugs. Viral entry is initiated by binding of the HIV-1 envelope glycoprotein subunit gp120 to the CD4 receptor and a chemokine coreceptor, generally CCR5 or CXCR4. Upon coreceptor binding, the viral transmembrane subunit gp41 undergoes conformational changes that eventually lead to the formation of the six-helix bundle (6HB) and membrane fusion. The 6HB is composed of a central trimeric coiled-coil structure formed by the N-terminal heptad repeat (NHR) domains of three gp41 molecules and the corresponding C-terminal heptad repeats (CHRs) that pack into the longitudinal grooves on the surface of the NHR coiled-coil in an antiparallel orientation (23). C-peptide fusion inhibitors (CFI) derived from the CHR of gp41 compete with the viral CHR for binding to the NHR trimer, thus blocking 6HB formation and viral entry (18).T-20 (enfuvirtide) is the first clinically approved CFI with high antiviral activity and a low-toxicity profile. However, as with many anti-HIV-1 drugs, resistance can emerge rapidly (13). The majority of the resistance mutations are found in the NHR of gp41 among the amino acids 544 to 553 (32, 35) (numbering refers to gp160 of the HIV-1 HXB2 strain throughout the article). Most of these mutations cause resistance by reducing the affinity of the NHR target region to inhibitory C peptides (13). Additionally, viral entry kinetics were found to correlate with the baseline susceptibility of different HIV strains to CFI. Determinants for viral entry kinetics are found in gp41 as well as in gp120 (1, 14, 35). Here, the influence of coreceptor affinity on virus entry kinetics and CFI susceptibility has been studied extensively (28, 30, 31). Recently, a statistical approach was used that highlighted positions in gp120 that underwent mutations in patients under enfuvirtide treatment (38). However, to our knowledge, selected CFI resistance mutations outside of gp41 have never been confirmed experimentally.Previously, we developed a retroviral vector expressing a membrane-anchored antiviral C peptide (maC46) that efficiently inhibits a broad range of different HIV-1 isolates. Enfuvirtide-resistant HIV-1 strains with mutations in the GIV motif of NHR were fully susceptible to maC46 (10). In the present study, we selected an HIV-1 variant with reduced sensitivity to maC46 by passaging an enfuvirtide-resistant BaL strain of HIV-1 on cells expressing increasing concentrations of maC46. Mutations in gp120 and gp41 were found to contribute to maC46 resistance.     

Truncation of the Membrane-Spanning Domain of Human Immunodeficiency Virus Type 1 Envelope Glycoprotein Defines Elements Required for Fusion,Incorporation, and Infectivity

The membrane-spanning domain (MSD) of the envelope (Env) glycoprotein from human (HIV) and simian immunodeficiency viruses plays a key role in anchoring the Env complex into the viral membrane but also contributes to its biological function in fusion and virus entry. In HIV type 1 (HIV-1), it has been predicted to span 27 amino acids, from lysine residue 681 to arginine 707, and encompasses an internal arginine at residue 694. By examining a series of C-terminal-truncation mutants of the HIV-1 gp41 glycoprotein that substituted termination codons for amino acids 682 to 708, we show that this entire region is required for efficient viral infection of target cells. Truncation to the arginine at residue 694 resulted in an Env complex that was secreted from the cells. In contrast, a region from residues 681 to 698, which contains highly conserved hydrophobic residues and glycine motifs and extends 4 amino acids beyond 694R, can effectively anchor the protein in the membrane, allow efficient transport to the plasma membrane, and mediate wild-type levels of cell-cell fusion. However, these fusogenic truncated Env mutants are inefficiently incorporated into budding virions. Based on the analysis of these mutants, a “snorkeling” model, in which the flanking charged amino acid residues at 681 and 694 are buried in the lipid while their side chains interact with polar head groups, is proposed for the HIV-1 MSD.Human immunodeficiency virus type 1 (HIV-1) infection is initiated by fusion of the viral membrane with that of the target cell and is mediated by the viral envelope glycoprotein (Env). HIV-1 Env, a type 1 membrane-spanning glycoprotein, is a trimeric complex composed of three noncovalently linked heterodimers of gp120, the receptor-binding surface (SU) component, and gp41, the membrane-spanning, transmembrane (TM) component (12, 26, 44, 45). The gp120 and gp41 glycoproteins are synthesized as a precursor gp160 glycoprotein, which is encoded by the env gene. The gp160 precursor is cotranslationally glycosylated and, following transport to the trans-Golgi network, is cleaved into the mature products by a member of the furin family of endoproteases (45). Mature Env proteins are transported to the plasma membrane, where they are rapidly endocytosed or incorporated into virions (5, 33, 43). Recent evidence suggests that endocytosis and intracellular trafficking of Env is required for its interaction with Gag precursors and for efficient assembly into virions (20).HIV-1 Env molecules function as quasistable “spring-loaded” fusion machines. Recent studies have suggested that several regions of gp120 are reoriented following CD4 binding so that a planar “bridging sheet,” which forms the binding site for the coreceptor (CCR5 or CXCR4), can form (6, 7). Coreceptor binding is necessary for additional conformational changes in gp41 and for complete fusion (3). The gp41 monomer has three subdomains, an ectodomain, a membrane-spanning domain (MSD), and a cytoplasmic domain (39). The ectodomain of gp41, which mediates membrane fusion, is composed of a fusion peptide, two heptad repeats, and a tryptophan-rich membrane-proximal external region. Following the binding of gp120 to the CD4 receptor and the CCR5/CXCR4 coreceptor, conformational changes are induced in Env that result in the exposure of the gp41 fusion peptide (32). This peptide inserts into the target cell membrane, allowing gp41 to form a bridge between the viral and cellular membranes. Interaction of the heptad repeats to form a six-helix bundle then brings the target and viral membranes together, allowing membrane fusion to occur (24).While heptad repeat regions 1 and 2 in the N-terminal ectodomain play key roles in Env-mediated fusion by bringing the viral and cell membranes into close proximity, an important function of gp41 is to anchor the glycoprotein complex within the host-derived viral membrane (18). The precise boundaries of the HIV-1 MSD have not been clearly defined; however, the MSD is one of the most conserved regions in the gp41 sequence. Based on the initial functional studies of HIV-1, the MSD of Env was defined as a stretch of 25 predominantly hydrophobic amino acids that span residues K681 to R705 in the NL4-3 sequence (14, 16, 18). These residues were suggested to cross the viral membrane in the form of an alpha helix, the length of which is approximately equal to the theoretical depth of a membrane bilayer. A major caveat of this model is that it places a basic amino acid residue (R694) into the hydrophobic center of the lipid bilayer. While some transmembrane proteins do contain charged amino acid residues in their MSDs, it is normally considered to be energetically unfavorable without some mechanism to neutralize the charge (8, 13). Point mutation studies have yielded varying results, but in general, substitution of K681 is detrimental to fusion and infectivity while mutation of R694 or R705 has only a limited effect on these activities (16, 29). On the other hand, accumulating data argue for a different intramembrane structure of the HIV-1 MSD. Serial small deletions (3 amino acid residues) in the region between R694 and R705 showed normal cell-cell fusion, although larger deletions were detrimental (29), suggesting that, with respect to the biological functions of the Env glycoprotein, the length of this region is more important than its amino acid conservation.Previous C-terminal-truncation studies of simian immunodeficiency virus (SIV) Env (19, 41) suggested that the entire 27-amino-acid region is not required for the biological function of the protein. In the case of SIV, only the 15 apolar amino acids flanked by K689 and R705 (equivalent to K681 and R694 in HIV) and 6 additional amino acids (for a total of 23 amino acids) were required for near-wild-type (WT) fusion (19, 41). Two subsequent residues were required (total, 25 amino acids) for virus-cell entry and infectivity, while a length of 21 amino acid residues was sufficient for SIV Env to be incorporated into viral particles. These results led to a basic amino acid “snorkeling” model for the SIV MSD (41). In this model, the lysine and arginine (NL4-3 equivalents of K681 and R694) are buried in the lipid bilayer, while their long side chains are proposed to extend outward to the membrane surface and present the positively charged amino groups to the negatively charged head groups of the lipid bilayers. Applied to HIV-1 MSD, this model predicts a hydrophobic intramembrane core of only 12 amino acid residues (compared to 15 amino acid residues in the SIV MSD) between K681 and R694. The hydrophobic region C-terminal to K681 is not sufficient to effectively anchor the protein, since mutation of R694 to a stop codon yielded a nonfunctional protein that appeared to be retained in the endoplasmic reticulum (11). This contrasts with truncation experiments with the vesicular stomatitis virus (VSV) G glycoprotein, which have shown that a region of 12 hydrophobic amino acids flanked by basic residues is sufficient to anchor the protein in the membrane (1).In order to understand if the “snorkeling” model is applicable to the HIV-1 MSD, we constructed a series of nonsense mutants with HIV-1 gp41 truncated in single-amino-acid steps at the C terminus from residue R707 to residue R694. For each mutant Env, we determined the membrane stability, fusogenicity, and ability to mediate infectivity. The results of these studies suggest that the 12-residue “core” (36) plus three subsequent hydrophobic amino acids is the minimal anchor domain for HIV-1 Env, as well as the minimal sequence to mediate cell-cell fusion. In contrast to SIV Env, HIV-1 Env requires the entire 25-amino-acid region from K681 to R707 to mediate near-WT incorporation and infectivity.     

HIV-1 Specific IgA Detected in Vaginal Secretions of HIV Uninfected Women Participating in a Microbicide Trial in Southern Africa Are Primarily Directed Toward gp120 and gp140 Specificities

Background

Many participants in microbicide trials remain uninfected despite ongoing exposure to HIV-1. Determining the emergence and nature of mucosal HIV-specific immune responses in such women is important, since these responses may contribute to protection and could provide insight for the rational design of HIV-1 vaccines.

Methods and Findings

We first conducted a pilot study to compare three sampling devices (Dacron swabs, flocked nylon swabs and Merocel sponges) for detection of HIV-1-specific IgG and IgA antibodies in vaginal secretions. IgG antibodies from HIV-1-positive women reacted broadly across the full panel of eight HIV-1 envelope (Env) antigens tested, whereas IgA antibodies only reacted to the gp41 subunit. No Env-reactive antibodies were detected in the HIV-negative women. The three sampling devices yielded equal HIV-1-specific antibody titers, as well as total IgG and IgA concentrations. We then tested vaginal Dacron swabs archived from 57 HIV seronegative women who participated in a microbicide efficacy trial in Southern Africa (HPTN 035). We detected vaginal IgA antibodies directed at HIV-1 Env gp120/gp140 in six of these women, and at gp41 in another three women, but did not detect Env-specific IgG antibodies in any women.

Conclusion

Vaginal secretions of HIV-1 infected women contained IgG reactivity to a broad range of Env antigens and IgA reactivity to gp41. In contrast, Env-binding antibodies in the vaginal secretions of HIV-1 uninfected women participating in the microbicide trial were restricted to the IgA subtype and were mostly directed at HIV-1 gp120/gp140.     

Transitions to and from the CD4-Bound Conformation Are Modulated by a Single-Residue Change in the Human Immunodeficiency Virus Type 1 gp120 Inner Domain

Binding to the primary receptor CD4 induces conformational changes in the human immunodeficiency virus type 1 (HIV-1) gp120 envelope glycoprotein that allow binding to the coreceptor (CCR5 or CXCR4) and ultimately trigger viral membrane-cell membrane fusion mediated by the gp41 transmembrane envelope glycoprotein. Here we report the derivation of an HIV-1 gp120 variant, H66N, that confers envelope glycoprotein resistance to temperature extremes. The H66N change decreases the spontaneous sampling of the CD4-bound conformation by the HIV-1 envelope glycoproteins, thus diminishing CD4-independent infection. The H66N change also stabilizes the HIV-1 envelope glycoprotein complex once the CD4-bound state is achieved, decreasing the probability of CD4-induced inactivation and revealing the enhancing effects of soluble CD4 binding on HIV-1 infection. In the CD4-bound conformation, the highly conserved histidine 66 is located between the receptor-binding and gp41-interactive surfaces of gp120. Thus, a single amino acid change in this strategically positioned gp120 inner domain residue influences the propensity of the HIV-1 envelope glycoproteins to negotiate conformational transitions to and from the CD4-bound state.Human immunodeficiency virus type 1 (HIV-1), the cause of AIDS (6, 29, 66), infects target cells by direct fusion of the viral and target cell membranes. The viral fusion complex is composed of gp120 and gp41 envelope glycoproteins, which are organized into trimeric spikes on the surface of the virus (10, 51, 89). Membrane fusion is initiated by direct binding of gp120 to the CD4 receptor on target cells (17, 41, 53). CD4 binding creates a second binding site on gp120 for the chemokine receptors CCR5 and CXCR4, which serve as coreceptors (3, 12, 19, 23, 25). Coreceptor binding is thought to lead to further conformational changes in the HIV-1 envelope glycoproteins that facilitate the fusion of viral and cell membranes. The formation of an energetically stable six-helix bundle by the gp41 ectodomain contributes to the membrane fusion event (9, 10, 79, 89, 90).The energy required for viral membrane-cell membrane fusion derives from the sequential transitions that the HIV-1 envelope glycoproteins undergo, from the high-energy unliganded state to the low-energy six-helix bundle. The graded transitions down this energetic slope are initially triggered by CD4 binding (17). The interaction of HIV-1 gp120 with CD4 is accompanied by an unusually large change in entropy, which is thought to indicate the introduction of order into the conformationally flexible unliganded gp120 glycoprotein (61). In the CD4-bound state, gp120 is capable of binding CCR5 with high affinity; moreover, CD4 binding alters the quaternary structure of the envelope glycoprotein complex, resulting in the exposure of gp41 ectodomain segments (27, 45, 77, 92). The stability of the intermediate state induced by CD4 binding depends upon several variables, including the virus (HIV-1 versus HIV-2/simian immunodeficiency virus [SIV]), the temperature, and the nature of the CD4 ligand (CD4 on a target cell membrane versus soluble forms of CD4 [sCD4]) (30, 73). For HIV-1 exposed to sCD4, if CCR5 binding occurs within a given period of time, progression along the entry pathway continues. If CCR5 binding is impeded or delayed, the CD4-bound envelope glycoprotein complex decays into inactive states (30). In extreme cases, the binding of sCD4 to the HIV-1 envelope glycoproteins induces the shedding of gp120 from the envelope glycoprotein trimer (31, 56, 58). Thus, sCD4 generally inhibits HIV-1 infection by triggering inactivation events, in addition to competing with CD4 anchored in the target cell membrane (63).HIV-1 isolates vary in sensitivity to sCD4, due in some cases to a low affinity of the envelope glycoprotein trimer for CD4 and in other cases to differences in propensity to undergo inactivating conformational transitions following CD4 binding (30). HIV-1 isolates that have been passaged extensively in T-cell lines (the tissue culture laboratory-adapted [TCLA] isolates) exhibit lower requirements for CD4 than primary HIV-1 isolates (16, 63, 82). TCLA viruses bind sCD4 efficiently and are generally sensitive to neutralization compared with primary HIV-1 isolates. Differences in sCD4 sensitivity between primary and TCLA HIV-1 strains have been mapped to the major variable loops (V1/V2 and V3) of the gp120 glycoprotein (34, 42, 62, 81). Sensitivity to sCD4 has been shown to be independent of envelope glycoprotein spike density or the intrinsic stability of the envelope glycoprotein complex (30, 35).In general, HIV-1 isolates are more sensitive to sCD4 neutralization than HIV-2 or SIV isolates (4, 14, 73). The relative resistance of SIV to sCD4 neutralization can in some cases be explained by a reduced affinity of the envelope glycoprotein trimer for sCD4 (57); however, at least some SIV isolates exhibit sCD4-induced activation of entry into CD4-negative, CCR5-expressing target cells that lasts for several hours after exposure to sCD4 (73). Thus, for some primate immunodeficiency virus envelope glycoproteins, activated intermediates in the CD4-bound conformation can be quite stable.The HIV-1 envelope glycoprotein elements important for receptor binding, subunit interaction, and membrane fusion are well conserved among different viral strains (71, 91). Thus, these elements represent potential targets for inhibitors of HIV-1 entry. Understanding the structure and longevity of the envelope glycoprotein intermediates along the virus entry pathway is relevant to attempts at inhibition. For example, peptides that target the heptad repeat 1 region of gp41 exhibit major differences in potency against HIV-1 strains related to efficiency of chemokine receptor binding (20, 21), which is thought to promote the conformational transition to the next step in the virus entry cascade. The determinants of the duration of exposure of targetable HIV-1 envelope glycoprotein elements during the entry process are undefined.To study envelope glycoprotein determinants of the movement among the distinct conformational states along the HIV-1 entry pathway, we attempted to generate HIV-1 variants that exhibit improved stability. Historically, labile viral elements have been stabilized by selecting virus to replicate under conditions, such as high temperature, that typically weaken protein-protein interactions (38, 39, 76, 102). Thus, we subjected HIV-1 to repeated incubations at temperatures between 42°C and 56°C, followed by expansion and analysis of the remaining replication-competent virus fraction. In this manner, we identified an envelope glycoprotein variant, H66N, in which histidine 66 in the gp120 N-terminal segment was altered to asparagine. The resistance of HIV-1 bearing the H66N envelope glycoproteins to changes in temperature has been reported elsewhere (37). Here, we examine the effect of the H66N change on the ability of the HIV-1 envelope glycoproteins to negotiate conformational transitions, either spontaneously or in the presence of sCD4. The H66N phenotype was studied in the context of both CD4-dependent and CD4-independent HIV-1 variants.     

Relationship between Antibody 2F5 Neutralization of HIV-1 and Hydrophobicity of Its Heavy Chain Third Complementarity-Determining Region

The membrane-proximal external region (MPER) of the HIV-1 gp41 transmembrane glycoprotein is the target of the broadly neutralizing antibody 2F5. Prior studies have suggested a two-component mechanism for 2F5-mediated neutralization involving both structure-specific recognition of a gp41 protein epitope and nonspecific interaction with the viral lipid membrane. Here, we mutationally alter a hydrophobic patch on the third complementarity-determining region of the heavy chain (CDR H3) of the 2F5 antibody and assess the abilities of altered 2F5 variants to bind gp41 and to neutralize diverse strains of HIV-1. CDR H3 alterations had little effect on the affinity of 2F5 variants for a peptide corresponding to its gp41 epitope. In contrast, strong effects and a high degree of correlation (P < 0.0001) were found between virus neutralization and CDR H3 hydrophobicity, as defined by predicted free energies of transfer from water to a lipid bilayer interface or to octanol. The effect of CDR H3 hydrophobicity on neutralization was independent of isolate sensitivity to 2F5, and CDR H3 variants with tryptophan substitutions were able to neutralize HIV-1 ∼10-fold more potently than unmodified 2F5. A threshold was observed for increased hydrophobicity of the 2F5 CDR H3 loop beyond which effects on 2F5-mediated neutralization leveled off. Together, the results provide a more complete understanding of the 2F5 mechanism of HIV-1 neutralization and indicate ways to enhance the potency of MPER-directed antibodies.The membrane-proximal external region (MPER) of the human immunodeficiency virus type 1 (HIV-1) gp41 transmembrane glycoprotein is the target of three broadly neutralizing anti-HIV-1 antibodies, 2F5, Z13e, and 4E10, and is thus a potential site of HIV-1 vulnerability to the humoral immune response (21, 24, 27, 48). The MPER encompasses ∼25 residues at the carboxyl-terminal end of the predicted gp41 ectodomain, just before the transmembrane region, and is rich in aromatic residues, typical of bilayer-interfacial regions of membrane proteins (26, 36, 40). Mutation of selected MPER tryptophans abrogates gp41-mediated fusion of the viral and target cell membranes, indicating that this region is crucial for HIV-1 infectivity (23, 28). Structural studies of unbound forms of the gp41 MPER both in solution and in lipid contexts have demonstrated that it adopts a number of conformations, many of which are α-helical, and electron-paramagnetic resonance measurements have indicated lipid bilayer immersion depths for MPER residues that range from acyl to phospholipid headgroup regions (4, 7, 8, 19, 32, 37). The binding of neutralizing antibodies, such as 2F5, to the MPER must therefore account for the membrane milieu in which the epitope is found.The 2F5 antibody has been shown to exhibit ∼100-fold-enhanced binding to its epitope on uncleaved gp140s when presented in the context of lipid proteoliposomes (11, 25), and other studies have shown that 2F5 can contact phospholipids directly in the absence of gp41 (1, 3, 12, 22, 29, 30). The latter finding has led to the suggestion that 2F5 might be autoreactive (12), although passive transfusion of 2F5 does not appear to have deleterious effects (38) and 2F5 failed to react in some clinically based assays for autoreactive lipid antibodies (31, 39). The crystal structures of the 2F5 antibody in complex with its gp41 MPER epitope revealed that, despite the 22-residue length of the 2F5 heavy chain third complementarity-determining region (CDR H3) loop, contacts with the gp41 MPER peptide are made predominantly at the loop base. In some crystal structures, the tip of the loop protrudes away from gp41, while in others, it is disordered (9, 14, 25). A unique feature of the tip of the CDR H3 loop is that it contains a patch of hydrophobic residues, including residues L100_A, F100_B, V100_D, and I100_F (Kabat numbering), which, with the exception of I100_F, do not contact gp41 (9, 10, 14, 25) (Fig. ​(Fig.1).1). While a prior study revealed the importance of residue F100_B of the CDR H3 loop in 2F5-neutralizing activity, nonconservative residue substitutions at this position also appeared to diminish 2F5 binding to the immobilized MPER peptide and gp41 in enzyme-linked immunosorbent assay (ELISA) formats (47). Conversely, a more recent study has shown that alanine mutations in the 2F5 CDR H3 loop can affect neutralization without affecting gp41 binding (2).Open in a separate windowFIG. 1.2F5 CDR H3 loop mutagenesis. (A) Structure of 2F5 Fab (blue and gray) in complex with a gp41 peptide (red). The 2F5 CDR H3 (purple) contacts gp41 only at its base, while the tip extends away from the peptide. (B) Close-up view of the 2F5 CDR H3 loop, with hydrophobic residues at the loop tip shown in stick representation and colored green. (C) Mutations introduced into the tip of the 2F5 CDR H3 (100_A to 100_F) are defined, along with a plot of the Wimley-White predicted free energies of transfer to a lipid bilayer interface (black) or to octanol (gray) for each of the mutations.In this study, we sought to examine the role of the chemical nature of residues at the tip of the 2F5 CDR H3 loop in neutralization of HIV-1. Mutations were introduced into the 2F5 CDR H3 loop that altered its hydrophobicity, and the resulting 2F5 mutants were tested both for binding to a gp41 epitope peptide and for neutralization of HIV-1. The results showed that the tip of the 2F5 CDR H3 loop, and specifically its hydrophobic nature, is required for 2F5-mediated neutralization of HIV-1 by means that appear to be independent both of gp41 affinity and of isolate-specific sensitivity to neutralization by 2F5.     

Membrane bound modified form of clade B Env,JRCSF is suitable for immunogen design as it is efficiently cleaved and displays all the broadly neutralizing epitopes including V2 and C2 domain-dependent conformational epitopes

Background

Antigenicity of HIV-1 envelope proteins (Envs) of both lab-adapted and primary isolates expressed on the cell surface rarely match with in vitro neutralization of viruses, pseudo-typed with corresponding Envs. Often, both neutralizing and non-neutralizing antibodies bind to Envs expressed on the cell membrane. This could be due to the lack of efficient cleavage of Env expressed on the cell surface. Naturally occurring, efficiently cleaved Envs with appropriate antigenic properties are relatively rare. Given viral diversity it is essential to increase the pool of candidate Envs suitable for immunogen design. Previously, it has been reported that JRFL Env is the only clade B Env, which is efficiently cleaved on the cell surface and retains desirable antigenic properties. JRCSF is a clade B Env isolated from the same patient as JRFL. JRCSF Env has not been explored aggressively for designing immunogen as the binding characteristics of JRCSF Env to broadly neutralizing antibodies on the cell surface and its cleavage status are unknown.

Results

Although JRCSF preferentially binds to most of the other gp120-directed neutralizing antibodies and cleavage dependent antibody, PGT151 efficiently, it binds poorly to CD4-binding-site-directed (CD4-bs-directed) neutralizing antibodies on cell surface. Membrane bound form of modified JRCSF Env containing the N197D mutation binds to CD4-bs-directed neutralizing antibodies better than JRFL, without debilitating its ability to bind quaternary epitope-directed neutralizing antibodies or exposing the CD4i antibody epitopes. In comparison to JRFL (E168K), JRCSF Env binds more efficiently to PG9/PGT145 class of V1/V2-directed conformational antibodies. Biochemical, cell surface staining and gp120 shedding experiments suggest that JRCSF is efficiently cleaved on the cell surface.

Conclusions

Binding of JRCSF Env expressed on cell surface to the various HIV-1 Env-directed antibodies has not been reported earlier. Here, for the first time, we report that compared to JRFL, JRCSF displays epitopes for a larger number of broadly neutralizing antibodies and is also efficiently cleaved when expressed on the cell surface. Thus, considering the diversity of viral Envs and the discovery of conformation dependent glycan-directed antibodies in HIV-1 infected individuals, an innately cleaved JRCSF Env as present on the viral membrane and displaying those distinct epitopes may be an important candidate for immunogen design.

     

Resistance profiles of novel electrostatically constrained HIV-1 fusion inhibitors

Human immunodeficiency virus (HIV) gp41 plays a key role in viral fusion; the N- and C-terminal heptad repeats (N-HR and C-HR) of gp41 form a stable 6-helical conformation for fusion. Therefore, HR-derived peptides, such as enfuvirtide (T-20), inhibit HIV-1 fusion by acting as decoys, and have been used for the treatment of HIV-1 infection. However, the efficacy of T-20 is attenuated by resistance mutations in gp41, including V38A and N43D. To suppress the resistant variants, we previously developed electrostatically constrained peptides, SC34 and SC34EK, and showed that both exhibited potent anti-HIV-1 activity against wild-type and T-20-resistant variants. In this study, to clarify the resistance mechanism to this next generation of fusion inhibitors, we selected variants with resistance to SC34 and SC34EK in vitro. The resistant variants had multiple mutations in gp41. All of these mutations individually caused less than 6-fold resistance to SC34 and SC34EK, indicating that there is a significant genetic barrier for high-level resistance. Cross-resistance to SC34 and SC34EK was reduced by a simple difference in the polarity of two intramolecular electrostatic pairs. Furthermore, the selected mutations enhanced the physicochemical interactions with N-HR variants and restored activities of the parental peptide, C34, even to resistant variants. These results demonstrate that our approach of designing gp41-binding inhibitors using electrostatic constraints and information derived from resistance studies produces inhibitors with enhanced activity, high genetic barrier, and distinct resistance profile from T-20 and other inhibitors. Hence, this is a promising approach for the design of future generation peptide fusion inhibitors.     

Effect of Mutations in the Human Immunodeficiency Virus Type 1 Protease on Cleavage of the gp41 Cytoplasmic Tail

We previously reported that human immunodeficiency virus type 1 (HIV-1) develops resistance to the cholesterol-binding compound amphotericin B methyl ester (AME) by acquiring mutations (P203L and S205L) in the cytoplasmic tail of the transmembrane envelope glycoprotein gp41 that create cleavage sites for the viral protease (PR). In the present study, we observed that a PR inhibitor-resistant (PIR) HIV-1 mutant is unable to efficiently cleave the gp41 cytoplasmic tail in P203L and S205L virions, resulting in loss of AME resistance. To define the pathway to AME resistance in the context of the PIR PR, we selected for resistance with an HIV-1 isolate expressing the mutant enzyme. We identified a new gp41 mutation, R236L, that results in cleavage of the gp41 tail by the PIR PR. These results highlight the central role of gp41 cleavage as the primary mechanism of AME resistance.Cholesterol-enriched membrane microdomains, often referred to as lipid rafts (4, 18, 24), play an important role in the replication of many enveloped viruses, including human immunodeficiency virus type 1 (HIV-1) (22, 30). Lipid rafts are involved in both HIV-1 entry and egress (reviewed in references 6, 22, and 30), and the lipid bilayer of HIV-1 virions is significantly enriched in cholesterol and highly saturated lipids characteristic of lipid rafts (3, 5, 8). We recently demonstrated that the cholesterol-binding polyene fungal antibiotic amphotericin B methyl ester (AME) potently inhibits HIV-1 replication. The antiviral activity of AME is due to a profound inhibition of viral entry (27, 28) and impairment of virus particle production (29).In our previous studies, we showed that the propagation of HIV-1 in the presence of AME leads to viral escape from this compound. The mutations that confer resistance map to the cytoplasmic tail (CT) of the gp41 transmembrane envelope (Env) glycoprotein (27, 28). AME-resistant mutants (P203L and S205L) overcome the defect in viral entry imposed by AME by a novel mechanism of resistance whereby the gp41 CT is cleaved by the viral protease (PR) after incorporation of Env into virions (28). The introduction of stop codons into the gp41-coding region that prematurely truncate the CT also renders virions AME resistant. In the present study, we evaluated the interplay between protease inhibitor resistance (PIR) mutations and AME resistance.