Comparative Immunogenicity of Evolved V1V2-Deleted HIV-1 Envelope Glycoprotein Trimers

Despite almost 30 years of research, no effective vaccine has yet been developed against HIV-1. Probably such a vaccine would need to induce both an effective T cell and antibody response. Any vaccine component focused on inducing humoral immunity requires the HIV-1 envelope (Env) glycoprotein complex as it is the only viral protein exposed on the virion surface. HIV-1 has evolved several mechanisms to evade broadly reactive neutralizing antibodies. One such a mechanism involves variable loop domains, which are highly flexible structures that shield the underlying conserved epitopes. We hypothesized that removal of such loops would increase the exposure and immunogenicity of these conserved regions. Env variable loop deletion however often leads to protein misfolding and aggregation because hydrophobic patches becoming solvent accessible. We have therefore previously used virus evolution to acquire functional Env proteins lacking the V1V2 loop. We then expressed them in soluble (uncleaved) gp140 forms. Three mutants were found to perform optimally in terms of protein expression, stability, trimerization and folding. In this study, we characterized the immune responses to these antigens in rabbits. The V1V2 deletion mutant ΔV1V2.9.VK induced a prominent response directed to epitopes that are not fully available on the other Env proteins tested but that effectively bound and neutralized the ΔV1V2 Env virus. This Env variant also induced more efficient neutralization of the tier 1 virus SF162. The immune refocusing effect was lost after booster immunization with a full-length gp140 protein with intact V1V2 loops. Collectively, this result suggests that deletion of variable domains could alter the specificity of the humoral immune response, but did not result in broad neutralization of neutralization-resistant virus isolates.     

Determinants of Human Immunodeficiency Virus Type 1 Envelope Glycoprotein Activation by Soluble CD4 and Monoclonal Antibodies

Infection by some human immunodeficiency virus type 1 (HIV-1) isolates is enhanced by the binding of subneutralizing concentrations of soluble receptor, soluble CD4 (sCD4), or monoclonal antibodies directed against the viral envelope glycoproteins. In this work, we studied the abilities of different antibodies to mediate activation of the envelope glycoproteins of a primary HIV-1 isolate, YU2, and identified the regions of gp120 envelope glycoprotein contributing to activation. Binding of antibodies to a variety of epitopes on gp120, including the CD4 binding site, the third variable (V3) loop, and CD4-induced epitopes, enhanced the entry of viruses containing YU2 envelope glycoproteins. Fab fragments of antibodies directed against either the CD4 binding site or V3 loop also activated YU2 virus infection. The activation phenotype was conferred on the envelope glycoproteins of a laboratory-adapted HIV-1 isolate (HXBc2) by replacing the gp120 V3 loop or V1/V2 and V3 loops with those of the YU2 virus. Infection by the YU2 virus in the presence of activating antibodies remained inhibitable by macrophage inhibitory protein 1β, indicating dependence on the CCR5 coreceptor on the target cells. Thus, antibody enhancement of YU2 entry involves neither Fc receptor binding nor envelope glycoprotein cross-linking, is determined by the same variable loops that dictate enhancement by sCD4, and probably proceeds by a process fundamentally similar to the receptor-activated virus entry pathway.     

A Twin-Cysteine Motif in the V2 Region of gp120 Is Associated with SIV Envelope Trimer Stabilization

The V1 and V2 variable regions of the primate immunodeficiency viruses contribute to the trimer association domain of the gp120 exterior envelope glycoprotein. A pair of V2 cysteine residues at 183 and 191 (“twin cysteines”) is present in several simian immunodeficiency viruses, human immunodeficiency virus type 2 (HIV-2) and some SIV_cpz lineages, but not in HIV-1. To examine the role of this potentially disulfide-bonded twin-cysteine motif, the cysteine residues in the SIVmac239 envelope glycoproteins were individually and pairwise substituted by alanine residues. All of the twin-cysteine mutants exhibited decreases in gp120 association with the Env trimer, membrane-fusing activity, and ability to support virus entry. Thus, the twin-cysteine motif plays a role in Env trimer stabilization in SIV and may do so in HIV-2 and some SIV_cpz as well. This implies that HIV-1 lost the twin-cysteines, and may have relatively unstable Env trimers compared to SIV and HIV-2.     

Replication-competent variants of human immunodeficiency virus type 2 lacking the V3 loop exhibit resistance to chemokine receptor antagonists

Entry of human immunodeficiency virus type 1 (HIV-1) and HIV-2 requires interactions between the envelope glycoprotein (Env) on the virus and CD4 and a chemokine receptor, either CCR5 or CXCR4, on the cell surface. The V3 loop of the HIV gp120 glycoprotein plays a critical role in this process, determining tropism for CCR5- or CXCR4-expressing cells, but details of how V3 interacts with these receptors have not been defined. Using an iterative process of deletion mutagenesis and in vitro adaptation of infectious viruses, variants of HIV-2 were derived that could replicate without V3, either with or without a deletion of the V1/V2 variable loops. The generation of these functional but markedly minimized Envs required adaptive changes on the gp120 core and gp41 transmembrane glycoprotein. V3-deleted Envs exhibited tropism for both CCR5- and CXCR4-expressing cells, suggesting that domains on the gp120 core were mediating interactions with determinants shared by both coreceptors. Remarkably, HIV-2 Envs with V3 deletions became resistant to small-molecule inhibitors of CCR5 and CXCR4, suggesting that these drugs inhibit wild-type viruses by disrupting a specific V3 interaction with the coreceptor. This study represents a proof of concept that HIV Envs lacking V3 alone or in combination with V1/V2 that retain functional domains required for viral entry can be derived. Such minimized Envs may be useful in understanding Env function, screening for new inhibitors of gp120 core interactions with chemokine receptors, and designing novel immunogens for vaccines.     

The V4 and V5 Variable Loops of HIV-1 Envelope Glycoprotein Are Tolerant to Insertion of Green Fluorescent Protein and Are Useful Targets for Labeling

The mature human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) comprises the non-covalently associated gp120 and gp41 subunits generated from the gp160 precursor. Recent structural analyses have provided quaternary structural models for gp120/gp41 trimers, including the variable loops (V1–V5) of gp120. In these models, the V3 loop is located under V1/V2 at the apical center of the Env trimer, and the V4 and V5 loops project outward from the trimeric protomers. In addition, the V4 and V5 loops are predicted to have less movement upon receptor binding during membrane fusion events. We performed insertional mutagenesis using a GFP variant, GFP_OPT, placed into the variable loops of HXB2 gp120. This allowed us to evaluate the current structural models and to simultaneously generate a GFP-tagged HIV-1 Env, which was useful for image analyses. All GFP-inserted mutants showed similar levels of whole-cell expression, although certain mutants, particularly V3 mutants, showed lower levels of cell surface expression. Functional evaluation of their fusogenicities in cell-cell and virus-like particle-cell fusion assays revealed that V3 was the most sensitive to the insertion and that the V1/V2 loops were less sensitive than V3. The V4 and V5 loops were the most tolerant to insertion, and certain tag proteins other than GFP_OPT could also be inserted without functional consequences. Our results support the current structural models and provide a GFP_OPT-tagged Env construct for imaging studies.     

An Envelope Modification That Renders a Primary, Neutralization-Resistant Clade B Human Immunodeficiency Virus Type 1 Isolate Highly Susceptible to Neutralization by Sera from Other Clades

SF162 is a primary (PR), non-syncytium-inducing, macrophagetropic human immunodeficiency virus type 1 (HIV-1) clade B isolate which is resistant to antibody-mediated neutralization. Deletion of the first or second hypervariable envelope gp120 region (V1 or V2 loop, respectively) of this virus does not abrogate its ability to replicate in peripheral blood mononuclear cells and primary macrophages, nor does it alter its coreceptor usage profile. The mutant virus with the V1 loop deletion, SF162ΔV1, remains as resistant to antibody-mediated neutralization as the wild-type virus SF162. In contrast, the mutant virus with the V2 loop deletion, SF162ΔV2, exhibits enhanced susceptibility to neutralization by certain monoclonal antibodies whose epitopes are located within the CD4-binding site and conserved regions of gp120. More importantly, SF162ΔV2 is now up to 170-fold more susceptible to neutralization than SF162 by sera collected from patients infected with clade B HIV-1 isolates. In addition, it becomes susceptible to neutralization by sera collected from patients infected with clade A, C, D, E, and F HIV-1 isolates. These findings suggest that the V2, but not the V1, loop of SF162 shields an as yet unidentified region of the HIV envelope rich in neutralization epitopes and that the overall structure of this region appears to be conserved among clade B, C, D, E, and F HIV-1 PR isolates.     

Stabilized HIV-1 Envelope Glycoprotein Trimers Lacking the V1V2 Domain,Obtained by Virus Evolution

The envelope glycoproteins (Env) are the focus of HIV-1 vaccine development strategies based on the induction of humoral immunity, but the mechanisms the virus has evolved to limit the induction and binding of neutralizing antibodies (NAbs) constitute substantial obstacles. Conserved neutralization epitopes are shielded by variable regions and carbohydrates, so one strategy to increase their exposure and, it is hoped, their immunogenicity is to delete the overlying variable loops. However, deleting the variable regions from Env trimers can be problematic, because hydrophobic patches that are normally solvent-inaccessible now become exposed, causing protein misfolding or aggregation, for example. Here, we describe the construction and characterization of recombinant gp140 trimers lacking variable domains 1 and 2 (ΔV1V2). The design of the trimers was guided by HIV-1 evolution studies that identified compensatory changes in V1V2-deleted but functional Env proteins (Bontjer, I., Land, A., Eggink, D., Verkade, E., Tuin, K., Baldwin, C., Pollakis, G., Paxton, W. A., Braakman, I., Berkhout, B., and Sanders, R. W. (2009) J. Virol. 83, 368–383). We now show that specific compensatory changes improved the function of ΔV1V2 Env proteins and hence HIV-1 replication. The changes acted by reducing the exposure of a hydrophobic surface either by replacing a hydrophobic residue with a hydrophilic one or by covering the surface with a glycan. The compensatory changes allowed the efficient expression of well folded, soluble gp140 trimers derived from various HIV-1 isolates. The evolved ΔV1V2 Env viruses were extremely sensitive to NAbs, indicating that neutralization epitopes are well exposed, which was confirmed by studies of NAb binding to the soluble ΔV1V2 gp140 trimers. These evolved ΔV1V2 trimers could be useful reagents for immunogenicity and structural studies.     

Critical Amino Acids within the Human Immunodeficiency Virus Type 1 Envelope Glycoprotein V4 N- and C-Terminals Contribute to Virus Entry

The importance of the fourth variable (V4) region of the human immunodeficiency virus 1 (HIV-1) envelope glycoprotein (Env) in virus infection has not been well clarified, though the polymorphism of this region has been found to be associated with disease progression to acquired immunodeficiency syndrome (AIDS). In the present work, we focused on the correlation between HIV-1 gp120 V4 region polymorphism and the function of the region on virus entry, and the possible mechanisms for how the V4 region contributes to virus infectivity. Therefore, we analyzed the differences in V4 sequences along with coreceptor usage preference from CCR5 to CXCR4 and examined the importance of the amino acids within the V4 region for CCR5- and CXCR4-tropic virus entry. In addition, we determined the influence of the V4 amino acids on Env expression and gp160 processing intracellularly, as well as the amount of Env on the pseudovirus surface. The results indicated that V4 tended to have a shorter length, fewer potential N-linked glycosylation sites (PNGS), greater evolutionary distance, and a lower negative net charge when HIV-1 isolates switched from a coreceptor usage preference for CCR5 to CXCR4. The N- and C-terminals of the HIV-1 V4 region are highly conserved and critical to maintain virus entry ability, but only the mutation at position 417 in the context of ADA (a R5-tropic HIV-1 strain) resulted in the ability to utilize CXCR4. In addition, 390L, 391F, 414I, and 416L are critical to maintain gp160 processing and maturation. It is likely that the hydrophobic properties and the electrostatic surface potential of gp120, rather than the conformational structure, greatly contribute to this V4 functionality. The findings provide information to aid in the understanding of the functions of V4 in HIV-1 entry and offer a potential target to aid in the development of entry inhibitors.     

HIV-1 Envelope Glycoprotein Biosynthesis, Trafficking, and Incorporation

The HIV-1 envelope (Env) glycoproteins play an essential role in the virus replication cycle by mediating the fusion between viral and cellular membranes during the entry process. The Env glycoproteins are synthesized as a polyprotein precursor (gp160) that is cleaved by cellular proteases to the mature surface glycoprotein gp120 and the transmembrane glycoprotein gp41. During virus assembly, the gp120/gp41 complex is incorporated as heterotrimeric spikes into the lipid bilayer of nascent virions. These gp120/gp41 complexes then initiate the infection process by binding receptor and coreceptor on the surface of target cells. Much is currently known about the HIV-1 Env glycoprotein trafficking pathway and the structure of gp120 and the extracellular domain of gp41. However, the mechanism by which the Env glycoprotein complex is incorporated into virus particles remains incompletely understood. Genetic data support a major role for the cytoplasmic tail of gp41 and the matrix domain of Gag in Env glycoprotein incorporation. Still to be defined are the identities of host cell factors that may promote Env incorporation and the role of specific membrane microdomains in this process. Here, we review our current understanding of HIV-1 Env glycoprotein trafficking and incorporation into virions.     

Alternative coreceptor requirements for efficient CCR5- and CXCR4-mediated HIV-1 entry into macrophages

Macrophage tropism of human immunodeficiency virus type 1 (HIV-1) is distinct from coreceptor specificity of the viral envelope glycoproteins (Env), but the virus-cell interactions that contribute to efficient HIV-1 entry into macrophages, particularly via CXCR4, are not well understood. Here, we characterized a panel of HIV-1 Envs that use CCR5 (n = 14) or CXCR4 (n = 6) to enter monocyte-derived macrophages (MDM) with various degrees of efficiency. Our results show that efficient CCR5-mediated MDM entry by Env-pseudotyped reporter viruses is associated with increased tolerance of several mutations within the CCR5 N terminus. In contrast, efficient CXCR4-mediated MDM entry was associated with reduced tolerance of a large deletion within the CXCR4 N terminus. Env sequence analysis and structural modeling identified amino acid variants at positions 261 and 263 within the gp41-interactive region of gp120 and a variant at position 326 within the gp120 V3 loop that were associated with efficient CXCR4-mediated MDM entry. Mutagenesis studies showed that the gp41 interaction domain variants exert a significant but strain-specific influence on CXCR4-mediated MDM entry, suggesting that the structural integrity of the gp120-gp41 interface is important for efficient CXCR4-mediated MDM entry of certain HIV-1 strains. However, the presence of Ile326 in the gp120 V3 loop stem, which we show by molecular modeling is located at the gp120-coreceptor interface and predicted to interact with the CXCR4 N terminus, was found to be critical for efficient CXCR4-mediated MDM entry of divergent CXCR4-using Envs. Together, the results of our study provide novel insights into alternative mechanisms of Env-coreceptor engagement that are associated with efficient CCR5- and CXCR4-mediated HIV-1 entry into macrophages.     

Regulation of epitope exposure in the gp41 membrane-proximal external region through interactions at the apex of HIV-1 Env

Glycoprotein Env of human immunodeficiency virus type 1 (HIV-1) mediates viral entry through membrane fusion. Composed of gp120 and gp41 subunits arranged as a trimer-of-heterodimers, Env adopts a metastable, highly dynamic conformation on the virion surface. This structural plasticity limits the temporospatial exposure of many highly conserved, neutralizing epitopes, contributing to the difficulty in developing effective HIV-1 vaccines. Here, we employed antibody neutralization of HIV-1 infectivity to investigate how inter- and intra-gp120 interactions mediated by variable loops V1/V2 and V3 at the Env apex regulate accessibility of the gp41 membrane-proximal external region (MPER) at the Env base. Swapping the V3 loop from Env_SF162 into the Env_HXB2 background shifted MPER exposure from the prefusogenic state to a functional intermediate conformation that was distinct from the prehairpin-intermediate state sensitive to gp41-targeted fusion inhibitors. The V3-loop swap had a profound impact on global protein dynamics, biasing the equilibrium to a closed conformation resistant to most anti-gp120 antibodies, stabilizing the protein to both cold- and soluble CD4-induced Env inactivation, and increasing the CD4 requirements for viral entry. Further dissection of the Env_HXB2 V3 loop revealed that residue 306 uniquely modulated epitope exposure and trimer stability. The R306S substitution substantially decreased sensitivity to antibodies targeting the gp41 MPER and, surprisingly, the gp120 V3-loop crown (residues 312–315), but had only modest effects on exposure of intervening gp120 epitopes. Furthermore, the point mutation reduced soluble CD4-induced inactivation, but had no impact on cold inactivation. The residue appeared to exert its effects by electrostatically modifying the strength of intra-subunit interactions between the V1/V2 and V3 loops. The distinct patterns of neutralization and stability pointed to a novel prefusogenic Env conformation along the receptor activation pathway and suggested that apical Env-regulation of gp41 MPER exposure can be decoupled from much of the dynamics of gp120 subunits.     

Coevolution Analysis of HIV-1 Envelope Glycoprotein Complex

The HIV-1 Env spike is the main protein complex that facilitates HIV-1 entry into CD4⁺ host cells. HIV-1 entry is a multistep process that is not yet completely understood. This process involves several protein-protein interactions between HIV-1 Env and a variety of host cell receptors along with many conformational changes within the spike. HIV-1 Env developed due to high mutation rates and plasticity escape strategies from immense immune pressure and entry inhibitors. We applied a coevolution and residue-residue contact detecting method to identify coevolution patterns within HIV-1 Env protein sequences representing all group M subtypes. We identified 424 coevolving residue pairs within HIV-1 Env. The majority of predicted pairs are residue-residue contacts and are proximal in 3D structure. Furthermore, many of the detected pairs have functional implications due to contributions in either CD4 or coreceptor binding, or variable loop, gp120-gp41, and interdomain interactions. This study provides a new dimension of information in HIV research. The identified residue couplings may not only be important in assisting gp120 and gp41 coordinate structure prediction, but also in designing new and effective entry inhibitors that incorporate mutation patterns of HIV-1 Env.     

Molecular architectures of trimeric SIV and HIV-1 envelope glycoproteins on intact viruses: strain-dependent variation in quaternary structure

The initial step in target cell infection by human, and the closely related simian immunodeficiency viruses (HIV and SIV, respectively) occurs with the binding of trimeric envelope glycoproteins (Env), composed of heterodimers of the viral transmembrane glycoprotein (gp41) and surface glycoprotein (gp120) to target T-cells. Knowledge of the molecular structure of trimeric Env on intact viruses is important both for understanding the molecular mechanisms underlying virus-cell interactions and for the design of effective immunogen-based vaccines to combat HIV/AIDS. Previous analyses of intact HIV-1 BaL virions have already resulted in structures of trimeric Env in unliganded and CD4-liganded states at ∼20 Å resolution. Here, we show that the molecular architectures of trimeric Env from SIVmneE11S, SIVmac239 and HIV-1 R3A strains are closely comparable to that previously determined for HIV-1 BaL, with the V1 and V2 variable loops located at the apex of the spike, close to the contact zone between virus and cell. The location of the V1/V2 loops in trimeric Env was definitively confirmed by structural analysis of HIV-1 R3A virions engineered to express Env with deletion of these loops. Strikingly, in SIV CP-MAC, a CD4-independent strain, trimeric Env is in a constitutively “open” conformation with gp120 trimers splayed out in a conformation similar to that seen for HIV-1 BaL Env when it is complexed with sCD4 and the CD4i antibody 17b. Our findings suggest a structural explanation for the molecular mechanism of CD4-independent viral entry and further establish that cryo-electron tomography can be used to discover distinct, functionally relevant quaternary structures of Env displayed on intact viruses.     

Pre-Clinical Development of a Recombinant,Replication-Competent Adenovirus Serotype 4 Vector Vaccine Expressing HIV-1 Envelope 1086 Clade C

Background

There is a well-acknowledged need for an effective AIDS vaccine that protects against HIV-1 infection or limits in vivo viral replication. The objective of these studies is to develop a replication-competent, vaccine vector based on the adenovirus serotype 4 (Ad4) virus expressing HIV-1 envelope (Env) 1086 clade C glycoprotein. Ad4 recombinant vectors expressing Env gp160 (Ad4Env160), Env gp140 (Ad4Env140), and Env gp120 (Ad4Env120) were evaluated.

Methods

The recombinant Ad4 vectors were generated with a full deletion of the E3 region of Ad4 to accommodate the env gene sequences. The vaccine candidates were assessed in vitro following infection of A549 cells for Env-specific protein expression and for posttranslational transport to the cell surface as monitored by the binding of broadly neutralizing antibodies (bNAbs). The capacity of the Ad4Env vaccines to induce humoral immunity was evaluated in rabbits for Env gp140 and V1V2-specific binding antibodies, and HIV-1 pseudovirus neutralization. Mice immunized with the Ad4Env160 vaccine were assessed for IFNγ T cell responses specific for overlapping Env peptide sets.

Results

Robust Env protein expression was confirmed by western blot analysis and recognition of cell surface Env gp160 by multiple bNAbs. Ad4Env vaccines induced humoral immune responses in rabbits that recognized Env 1086 gp140 and V1V2 polypeptide sequences derived from 1086 clade C, A244 clade AE, and gp70 V1V2 CASE A2 clade B fusion protein. The immune sera efficiently neutralized tier 1 clade C pseudovirus MW965.26 and neutralized the homologous and heterologous tier 2 pseudoviruses to a lesser extent. Env-specific T cell responses were also induced in mice following Ad4Env160 vector immunization.

Conclusions

The Ad4Env vaccine vectors express high levels of Env glycoprotein and induce both Env-specific humoral and cellular immunity thus supporting further development of this new Ad4 HIV-1 Env vaccine platform in Phase 1 clinical trials.     

Covariance of Charged Amino Acids at Positions 322 and 440 of HIV-1 Env Contributes to Coreceptor Specificity of Subtype B Viruses,and Can Be Used to Improve the Performance of V3 Sequence-Based Coreceptor Usage Prediction Algorithms

The ability to determine coreceptor usage of patient-derived human immunodeficiency virus type 1 (HIV-1) strains is clinically important, particularly for the administration of the CCR5 antagonist maraviroc. The envelope glycoprotein (Env) determinants of coreceptor specificity lie primarily within the gp120 V3 loop region, although other Env determinants have been shown to influence gp120-coreceptor interactions. Here, we determined whether conserved amino acid alterations outside the V3 loop that contribute to coreceptor usage exist, and whether these alterations improve the performance of V3 sequence-based coreceptor usage prediction algorithms. We demonstrate a significant covariant association between charged amino acids at position 322 in V3 and position 440 in the C4 Env region that contributes to the specificity of HIV-1 subtype B strains for CCR5 or CXCR4. Specifically, positively charged Lys/Arg at position 322 and negatively charged Asp/Glu at position 440 occurred more frequently in CXCR4-using viruses, whereas negatively charged Asp/Glu at position 322 and positively charged Arg at position 440 occurred more frequently in R5 strains. In the context of CD4-bound gp120, structural models suggest that covariation of amino acids at Env positions 322 and 440 has the potential to alter electrostatic interactions that are formed between gp120 and charged amino acids in the CCR5 N-terminus. We further demonstrate that inclusion of a “440 rule” can improve the sensitivity of several V3 sequence-based genotypic algorithms for predicting coreceptor usage of subtype B HIV-1 strains, without compromising specificity, and significantly improves the AUROC of the geno2pheno algorithm when set to its recommended false positive rate of 5.75%. Together, our results provide further mechanistic insights into the intra-molecular interactions within Env that contribute to coreceptor specificity of subtype B HIV-1 strains, and demonstrate that incorporation of Env determinants outside V3 can improve the reliability of coreceptor usage prediction algorithms.     

Functional and immunologic characterization of human immunodeficiency virus type 1 envelope glycoproteins containing deletions of the major variable regions.

Deletions of the major variable regions (V1/V2, V3, and V4) of the human immunodeficiency virus type 1 (HIV-1) gp120 exterior envelope glycoprotein were created to study the role of these regions in function and antigenicity. Deletion of the V4 region disrupted processing of the envelope glycoprotein precursor. In contrast, the deletion of the V1/V2 and/or V3 regions yielded processed exterior envelope glycoproteins that retained the ability to interact with the gp41 transmembrane glycoprotein and the CD4 receptor. Shedding of the gp120 exterior glycoprotein by soluble CD4 was observed for the mutant with the V3 deletion but did not occur for the V1/V2-deleted mutant. None of the deletion mutants formed syncytia or supported virus entry. Importantly, the affinity of neutralizing antibodies directed against the CD4-binding region for the multimeric envelope glycoprotein complex was increased dramatically by the removal of both the V1/V2 and V3 structures. These results indicate that, in addition to playing essential roles in the induction of membrane fusion, the major variable regions mask conserved neutralization epitopes of the HIV-1 gp120 glycoprotein from antibodies. These results explain the temporal pattern associated with generation of HIV-1-neutralizing antibodies following infection and suggest stratagems for eliciting improved immune responses to conserved gp120 epitopes.     

Effects of the I559P gp41 Change on the Conformation and Function of the Human Immunodeficiency Virus (HIV-1) Membrane Envelope Glycoprotein Trimer

The mature human immunodeficiency virus (HIV-1) envelope glycoprotein (Env) trimer is produced by proteolytic cleavage of a precursor and consists of three gp120 exterior and three gp41 transmembrane subunits. The metastable Env complex is induced to undergo conformational changes required for virus entry by the binding of gp120 to the receptors, CD4 and CCR5/CXCR4. An isoleucine-to-proline change (I559P) in the gp41 ectodomain has been used to stabilize soluble forms of HIV-1 Env trimers for structural characterization and for use as immunogens. In the native membrane-anchored HIV-1_BG505 Env, the I559P change modestly decreased proteolytic maturation, increased the non-covalent association of gp120 with the Env trimer, and resulted in an Env conformation distinctly different from that of the wild-type HIV-1_BG505 Env. Compared with the wild-type Env, the I559P Env was recognized inefficiently by polyclonal sera from HIV-1-infected individuals, by several gp41-directed antibodies, by some antibodies against the CD4-binding site of gp120, and by antibodies that preferentially recognize the CD4-bound Env. Some of the gp120-associated antigenic differences between the wild-type HIV-1_BG505 Env and the I559P mutant were compensated by the SOS disulfide bond between gp120 and gp41, which has been used to stabilize cleaved soluble Env trimers. Nonetheless, regardless of the presence of the SOS changes, Envs with proline 559 were recognized less efficiently than Envs with isoleucine 559 by the VRC01 neutralizing antibody, which binds the CD4-binding site of gp120, and the PGT151 neutralizing antibody, which binds a hybrid gp120-gp41 epitope. The I559P change completely eliminated the ability of the HIV-1_BG505 Env to mediate cell-cell fusion and virus entry, and abolished the capacity of the SOS Env to support virus infection in the presence of a reducing agent. These results suggest that differences exist between the quaternary structures of functional Env spikes and I559P Envs.     

HIV-1 escape from the CCR5 antagonist maraviroc associated with an altered and less-efficient mechanism of gp120-CCR5 engagement that attenuates macrophage tropism

Maraviroc (MVC) inhibits the entry of human immunodeficiency virus type 1 (HIV-1) by binding to and modifying the conformation of the CCR5 extracellular loops (ECLs). Resistance to MVC results from alterations in the HIV-1 gp120 envelope glycoproteins (Env) enabling recognition of the drug-bound conformation of CCR5. To better understand the mechanisms underlying MVC resistance, we characterized the virus-cell interactions of gp120 from in vitro-generated MVC-resistant HIV-1 (MVC-Res Env), comparing them with those of gp120 from the sensitive parental virus (MVC-Sens Env). In the absence of the drug, MVC-Res Env maintains a highly efficient interaction with CCR5, similar to that of MVC-Sens Env, and displays a relatively modest increase in dependence on the CCR5 N terminus. However, in the presence of the drug, MVC-Res Env interacts much less efficiently with CCR5 and becomes critically dependent on the CCR5 N terminus and on positively charged elements of the drug-modified CCR5 ECL1 and ECL2 regions (His88 and His181, respectively). Structural analysis suggests that the Val323 resistance mutation in the gp120 V3 loop alters the secondary structure of the V3 loop and the buried surface area of the V3 loop-CCR5 N terminus interface. This altered mechanism of gp120-CCR5 engagement dramatically attenuates the entry of HIV-1 into monocyte-derived macrophages (MDM), cell-cell fusion activity in MDM, and viral replication capacity in MDM. In addition to confirming that HIV-1 escapes MVC by becoming heavily dependent on the CCR5 N terminus, our results reveal novel interactions with the drug-modified ECLs that are critical for the utilization of CCR5 by MVC-Res Env and provide additional insights into virus-cell interactions that modulate macrophage tropism.     

Structure and Dynamics of the gp120 V3 Loop That Confers Noncompetitive Resistance in R5 HIV-1JR-FL to Maraviroc

Maraviroc, an (HIV-1) entry inhibitor, binds to CCR5 and efficiently prevents R5 human immunodeficiency virus type 1 (HIV-1) from using CCR5 as a coreceptor for entry into CD4⁺ cells. However, HIV-1 can elude maraviroc by using the drug-bound form of CCR5 as a coreceptor. This property is known as noncompetitive resistance. HIV-1_V3-M5 derived from HIV-1_JR-FLan is a noncompetitive-resistant virus that contains five mutations (I304V/F312W/T314A/E317D/I318V) in the gp120 V3 loop alone. To obtain genetic and structural insights into maraviroc resistance in HIV-1, we performed here mutagenesis and computer-assisted structural study. A series of site-directed mutagenesis experiments demonstrated that combinations of V3 mutations are required for HIV-1_JR-FLan to replicate in the presence of 1 µM maraviroc, and that a T199K mutation in the C2 region increases viral fitness in combination with V3 mutations. Molecular dynamic (MD) simulations of the gp120 outer domain V3 loop with or without the five mutations showed that the V3 mutations induced (i) changes in V3 configuration on the gp120 outer domain, (ii) reduction of an anti-parallel β-sheet in the V3 stem region, (iii) reduction in fluctuations of the V3 tip and stem regions, and (iv) a shift of the fluctuation site at the V3 base region. These results suggest that the HIV-1 gp120 V3 mutations that confer maraviroc resistance alter structure and dynamics of the V3 loop on the gp120 outer domain, and enable interactions between gp120 and the drug-bound form of CCR5.     

Structural Characterization of Cleaved,Soluble HIV-1 Envelope Glycoprotein Trimers

Human immunodeficiency virus type 1 (HIV-1) infection is a significant global public health problem for which development of an effective prophylactic vaccine remains a high scientific priority. Many concepts for a vaccine are focused on induction of appropriate titers of broadly neutralizing antibodies (bNAbs) against the viral envelope (Env) glycoproteins gp120 and gp41, but no immunogen has yet accomplished this goal in animals or humans. One approach to induction of bNAbs is to design soluble, trimeric mimics of the native viral Env trimer. Here, we describe structural studies by negative-stain electron microscopy of several variants of soluble Env trimers based on the KNH1144 subtype A sequence. These Env trimers are fully cleaved between the gp120 and gp41 components and stabilized by specific amino acid substitutions. We also illustrate the structural consequences of deletion of the V1/V2 and V3 variable loops from gp120 and the membrane-proximal external region (MPER) from gp41. All of these variants adopt a trimeric configuration that appropriately mimics native Env spikes, including the CD4 receptor-binding site and the epitope for the VRC PG04 bNAb. These cleaved, soluble trimer designs can be adapted for use with multiple different env genes for both vaccine and structural studies.