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The adhesion of HIV gp120 antigen to human cells is modulated in part by interactions with heparan sulfate. The HXB2 strain of gp120 has been shown to interact with heparin primarily through the V3 loop, although other domains including the C-terminal domain were also implicated. However, the JR-FL strain (representative of CCR5-interacting strains that make up newest infections) was shown to have a drastically lowered affinity to heparin due to the loss of several basic residues in the V3 loop, and deletion of the V3 loop in JR-FL gp120 was shown to abrogate some, but not all, heparin binding. Here, we use high resolution hydroxyl radical protein footprinting to measure the changes in protein surface oxidation levels that result from the binding of a model heparin fragment (fondaparinux). Protection in both the V3 loop and the N-terminus of JR-FP gp120 is observed. The well-defined composition of fondaparinux allowed us to perform docking simulations, which showed two clusters of fondaparinux binding: the V3 loop, and a domain consisting of the N- and C-termini. Together, the experimental and theoretical results indicate the heparin/heparan sulfate binding sites on JR-FL gp120 and the efficient interaction of fondaparinux, a widely exploited therapeutic carbohydrate, on gp120.

Human immunodeficiency virus type 1 (HIV-1) envelope gp120 is partly an intrinsically disordered (unstructured/disordered) protein as it contains regions that do not fold into well-defined protein structures. These disordered regions play important roles in HIV's life cycle, particularly, V3 loop-dependent cell entry, which determines how the virus uses two coreceptors on immune cells, the chemokine receptors CCR5 (R5), CXCR4 (X4) or both (R5X4 virus). Most infecting HIV-1 variants utilise CCR5, while a switch to CXCR4-use occurs in the majority of infections. Why does this ‘rewiring’ event occur in HIV-1 infected patients? As changes in the charge of the V3 loop are associated with this receptor switch and it has been suggested that charged residues promote structure disorder, we hypothesise that the intrinsic disorder of the V3 loop plays a role in determining cell tropism. To test this we use three independent data sets of gp120 to analyse V3 loop disorder. We find that the V3 loop of X4 virus has significantly higher intrinsic disorder tendency than R5 and R5X4 virus, while R5X4 virus has the lowest. These results indicate that structural disorder plays an important role in determining HIV-1 cell tropism and CXCR4 binding. We speculate that changes in N-linked glycosylation associated with tropism change (from R5 to X4) are required to stabilise the V3 loop with increased disorder tendency during HIV-1 evolution. We discuss the potential evolutionary mechanisms leading to the fixation of disorder promoting mutations and the adaptive potential of protein structural disorder in viral host adaptation.

Objectives Better understanding of the dynamics of HIV reservoirs under ART is a critical step to achieve a functional HIV cure. Our objective was to assess the genetic diversity of archived HIV-1 DNA over 48 weeks in blood cells of individuals starting treatment with a dolutegravir-based regimen. Methods Eighty blood samples were prospectively and longitudinally collected from 20 individuals (NCT02557997) including: acutely (n = 5) and chronically (n = 5) infected treatment-naive individuals, as well as treatment-experienced individuals who switched to a dolutegravir-based regimen and were either virologically suppressed (n = 5) or had experienced treatment failure (n = 5). The integrase and V3 loop regions of HIV-1 DNA isolated from PBMCs were analysed by pyrosequencing at baseline and weeks 4, 24 and 48. HIV-1 genetic diversity was calculated using Shannon entropy. Results All individuals achieved or maintained viral suppression throughout the study. A low and stable genetic diversity of archived HIV quasispecies was observed in individuals starting treatment during acute infection. A dramatic reduction of the genetic diversity was observed at week 4 of treatment in the other individuals. In these patients and despite virological suppression, a recovery of the genetic diversity of the reservoirs was observed up to 48 weeks. Viral variants bearing dolutegravir resistance-associated substitutions at integrase position 50, 124, 230 or 263 were detected in five individuals (n = 5/20, 25%) from all groups except those who were ART-failing at baseline. None of these substitutions led to virological failure. Conclusions These data demonstrate that the genetic diversity of the HIV-1 reservoir is reshaped following the initiation of a dolutegravir-based regimen and strongly suggest that HIV-1 can continue to replicate despite successful treatment.

The human immunodeficiency virus type 1 (HIV-1)/simian immunodeficiency virus (SIV) envelope spike (Env) mediates viral entry into host cells. The V3 loop of the gp120 component of the Env trimer contributes to the coreceptor binding site and is a target for neutralizing antibodies. We used cryo-electron tomography to visualize the binding of CD4 and the V3 loop monoclonal antibody (MAb) 36D5 to gp120 of the SIV Env trimer. Our results show that 36D5 binds gp120 at the base of the V3 loop and suggest that the antibody exerts its neutralization effect by blocking the coreceptor binding site. The antibody does this without altering the dynamics of the spike motion between closed and open states when CD4 is bound. The interaction between 36D5 and SIV gp120 is similar to the interaction between some broadly neutralizing anti-V3 loop antibodies and HIV-1 gp120. Two conformations of gp120 bound with CD4 are revealed, suggesting an intrinsic dynamic nature of the liganded Env trimer. CD4 binding substantially increases the binding of 36D5 to gp120 in the intact Env trimer, consistent with CD4-induced changes in the conformation of gp120 and the antibody binding site. Binding by MAb 36D5 does not substantially alter the proportions of the two CD4-bound conformations. The position of MAb 36D5 at the V3 base changes little between conformations, indicating that the V3 base serves as a pivot point during the transition between these two states. IMPORTANCE Glycoprotein spikes on the surfaces of SIV and HIV are the sole targets available to the immune system for antibody neutralization. Spikes evade the immune system by a combination of a thick layer of polysaccharide on the surface (the glycan shield) and movement between spike domains that masks the epitope conformation. Using SIV virions whose spikes were “decorated” with the primary cellular receptor (CD4) and an antibody (36D5) at part of the coreceptor binding site, we visualized multiple conformations trapped by the rapid freezing step, which were separated using statistical analysis. Our results show that the CD4-induced conformational dynamics of the spike enhances binding of the antibody.

Background Vaccine against HIV-1 is not currently available. In present, Virus like particles (VLPs) as effective strategy was used in several vaccine developing. Two conserved sequences; V3 loop of gp120 and the membrane-proximal external region (MPER) of gp41 are dominant sites for vaccine studies. Objective In this study, we used fusion gene of MPER and V3 to product recombinant VLPs and introduced a novel retroviral VLPs harboring high copy of MPER-V3 for HIV-1 vaccine design. Methods The pEGFP-N1 plasmid harboring MPER-V3 sequence with Vpr linker was constructed. To produce virus-like particles, HEK 293T cells were co-transfected with the recombinant plasmid, pSPAX-2, pMD2-G and pWPXLd plasmids, evaluated by AFM and SEM microscopy and quantified using P24 end-point ELISA assay. Results Time-course quantification of p24 protein as the characteristics of viral production evidenced for the efficient secretion of virus-like structures (up to 120 ng/ml) to the culture supernatant of transfected cells. Examination of the centrifuge-concentrated VLPs by AFM and SEM microscope, also illustrated particles with spherical morphologies and diameters of around 150 nm that had similar sizes to HIV virions. Conclusion These data indicated the production of HIV-1 virus-like particles harboring high copy of MPER-V3 that maintained their antigenic structure. These VLPs represented a good implication as a potential vaccine candidate and this guarantees the further investigations towards the assessment of its immunogenicity.

A phase I/IIA clinical trial with the chimeric mouse-human monoclonal antibody CGP 47,439 to the principal neutralization determinant in the V3 region of human immunodeficiency virus type 1 (HIV-1) strain IIIB envelope protein gp120 is reported. The trial was an uncontrolled single-center, open-label, multidose tolerability, immunogenicity, and pharmacokinetic study in homosexual men with advanced HIV disease. Patient groups were formed on the basis of the reactivity of the antibody with the gp120 of their HIV-1 isolates. Intravenous infusions of 1, 10, and 25 mg of antibody were followed by seven escalated doses of 50, 100, and 200 mg, every 3 weeks. The antibody was well tolerated; no toxicity was observed. Some patients showed a transient but insignificant antibody response to the antibody with no apparent adverse reactions or accelerated elimination of it. Substantial serum levels of the antibody were maintained with a mean t1/2 beta of 8-16 days. A virus burden reduction was observed in some patients.

HIV subtypes not only predominate in different geographical regions but also differ in key phenotypic characteristics. To determine if genotypic and/or phenotypic differences in the Envelope (Env) glycoprotein can explain subtype related differences, we cloned 37 full length Envs from Subtype B and AE HIV infected individuals from Singapore. Our data demonstrates that CRF01_AE Envs have lower Potential N Glycosylation Sites and higher risk of ×4 development. Phenotypically, CRF01_AE were less infectious than subtype B Envs in cells expressing low levels of CCR5. Moreover, the Maraviroc IC50 was higher for subtype B Envs and correlated with infectivity in low CCR5 expressing cells as well as PNGS. Specifically, the glycosylation site N301 in the V3 loop was seen less frequently in AE subtype and CXCR4 topic viruses. CRF01_AE differs from B subtype in terms of CCR5 usage and Maraviroc susceptibility which may have implications for HIV pathogenesis and virus evolution.

Summary CCR5 is the primary chemokine receptor utilized by HIV to infect leukocytes, whereas CCR5 ligands inhibit infection by blocking CCR5 engagement with HIV gp120. To guide the design of improved therapeutics, we solved the structure of CCR5 in complex with chemokine antagonist [5P7]CCL5. Several structural features appeared to contribute to the anti-HIV potency of [5P7]CCL5, including the distinct chemokine orientation relative to the receptor, the near-complete occupancy of the receptor binding pocket, the dense network of intermolecular hydrogen bonds, and the similarity of binding determinants with the FDA-approved HIV inhibitor Maraviroc. Molecular modeling indicated that HIV gp120 mimicked the chemokine interaction with CCR5, providing an explanation for the ability of CCR5 to recognize diverse ligands and gp120 variants. Our findings reveal that structural plasticity facilitates receptor-chemokine specificity and enables exploitation by HIV, and provide insight into the design of small molecule and protein inhibitors for HIV and other CCR5-mediated diseases.

HIV cure research is increasingly focused on anatomical tissues as sites for residual HIV replication during combined antiretroviral therapy (cART). Tissue-based HIV could contribute to low-level immune activation and viral rebound over the course of infection and could also influence the development of diseases, such as atherosclerosis, neurological disorders and cancers. cART-treated subjects have a decreased and irregular presence of HIV among tissues, which has resulted in a paucity of actual evidence concerning how or if HIV persists, replicates and evolves in various anatomical sites during therapy. In this study, we pooled 1806 HIV envelope V3 loop sequences from twenty-six tissue types (seventy-one total tissues) of six pre-cART subjects, four subjects with an unknown cART history who died with profound AIDS, and five subjects who died while on cART with an undetectable plasma viral load. A computational approach was used to assess sequences for their ability to utilize specific cellular coreceptors (R5, R5 and X4, or X4). We found that autopsied tissues obtained from virally suppressed cART + subjects harbored both integrated and expressed viruses with similar coreceptor usage profiles to subjects with no or ineffective cART therapy (i.e., significant plasma viral load at death). The study suggests that tissue microenvironments provide a sanctuary for the continued evolution of HIV despite cART.

HIV-1 entry and fusion with target cells is an important target for antiviral therapy. However, a few currently approved treatments are not effective as monotherapy due to the emergence of drug resistance. This consideration has fueled efforts to develop new bioavailable inhibitors targeting different steps of the HIV-1 entry process. Here, a high-throughput screen was performed of a large library of 100,000 small molecules for HIV-1 entry/fusion inhibitors, using a direct virus-cell fusion assay in a 384 half-well format. Positive hits were validated using a panel of functional assays, including HIV-1 specificity, cytotoxicity, and single-cycle infectivity assays. One compound-4-(2,5-dimethyl-pyrrol-1-yl)-2-hydroxy-benzoic acid (DPHB)-that selectively inhibited HIV-1 fusion was further characterized. Functional experiments revealed that DPHB caused irreversible inactivation of HIV-1 Env on cell-free virions and that this effect was related to binding to the third variable loop (V3) of the gp120 subunit of HIV-1 Env. Moreover, DPHB selectively inhibited HIV-1 strains that use CXCR4 or both CXCR4 and CCR5 co-receptors for entry, but not strains exclusively using CCR5. This selectivity was mapped to the gp120 V3 loop using chimeric Env glycoproteins. However, it was found that pure DPHB was not active against HIV-1 and that its degradation products (most likely polyanions) were responsible for inhibition of viral fusion. These findings highlight the importance of post-screening validation of positive hits and are in line with previous reports of the broad antiviral activity of polyanions.

Cete d׳Ivoire continues to have the highest HIV-1 prevalence rate in West Africa, although the infection number is in constant decline. The external envelope protein of the viruses is a likely site of selection, and responsible for receptor binding and entry into host cells, and therefore constitutes an ideal region with which to investigate the evolutionary processes acting on HIV-1. In this study, we analyse 189 envelope glycoprotein V3 loop region sequences of viruse isolates from 1995 to 2009, from HIV-1 untreated patients living in Cete d׳Ivoire, to decipher the temporal relationship between disease diversity, divergence and selection. Our analyses show that the nonsynonymous and synonymous ratio (dN/dS) was lower than 1 for viral populations analysed within 15 years, which showed the sequences did not undergo adequate immune pressure. The phylogenetic tree of the sequences analysed demonstrated distinctly long internal branches and short external branches, suggesting that only a small number of viruses infected the new host cell at each transmission. In addition to identifying sites under purifying selection, we also identified neutral sites that can cause false positive inference of selection. These sites presented form a resource for future studies of selection pressures acting on HIV-1 enν gene in Cete d׳Ivoire and other West African countries.

The gp120 portion of the envelope spike on human immunodeficiency virus type 1 (HIV-1) plays a critical role in viral entry into host cells and is a key target for the humoral immune response, and yet many structural details remain elusive. We have used cryoelectron tomography to visualize the binding of the broadly neutralizing monoclonal antibody (MAb) 447-52D to intact envelope spikes on virions of HIV-1 MN strain. Antibody 447-52D has previously been shown to bind to the tip of the V3 loop. Our results show antibody arms radiating from the sides of the gp120 protomers at a range of angles and place the antibody-bound V3 loop in an orientation that differs from that predicted by most current models but consistent with the idea that antibody binding dislodges the V3 loop from its location in the Env spike, making it flexible and disordered. These data reveal information on the position of the V3 loop and its relative flexibility and suggest that 447-52D neutralizes HIV-1 MN by capturing the V3 loop, blocking its interaction with the coreceptor and altering the structure of the envelope spike. IMPORTANCE Antibody neutralization is one of the primary ways that the body fights infection with HIV. Because HIV is a highly mutable virus, the body must constantly produce new antibodies to counter new strains of HIV that the body itself is producing. Consequently, antibodies capable of neutralizing multiple HIV strains are comparatively few. An improved understanding of the mechanism of antibody neutralization might advance the development of immunogens. Most neutralizing antibodies target the Env glycoprotein spikes found on the virus surface. The broadly neutralizing antibody 447-52D targets the highly conserved β-turn of variable loop 3 (V3) of gp120. The importance of V3 lies in its contribution to the coreceptor binding site on the target cell. We show here that 447-52D binding to V3 converts the Env conformation from closed to open and makes the V3 loop highly flexible, implying disruption of coreceptor binding and attachment to the target cell.

The efficiency of the human immunodeficiency virus type-1 (HIV-1) to enter cells is defined primarily by amino acid exchanges in the external glycoprotein gp120 and in, especially its highly variable V3 loop region. To study entry efficiency of HIV-1 a competitive viral entry assay was developed, to be comprised of infectious virus as well as soluble gp120 (sgp120) as an entry competitor. Entry of viruses using the coreceptor CXCR4 was reduced by adding CXCR4-tropic sgp120 (X4-sgp120) SF2 or LAV expressed in the baculovirus system or by adding X4-sgp120 from NL-952 and NL-V3A virus mutants produced in a HeLa-P4 cell culture expression system. Adding X4-sgp120 into a CCR5-specific infection assay revealed that X4-sgp120 enhanced the infection of CCR5-tropic virus. Furthermore, the role of the V3 loop N-glycan g15 on entry efficiency was studied using virus mutants and sgp120 with different N-glycosylation and different coreceptor usage. These experiments showed that viral entry of R5-tropic viruses lacking the N-glycan g15 within the V3 loop was inhibited by CCR5-tropic sgp120 harboring the g15 N-glycan. Altogether, the data demonstrate that HIV-1 entry efficiency can be studied easily by using sgp120 as an internal control or by using autologous or heterologous sgp120–virus pairs.

One strategy being evaluated for HIV-1 vaccine development is focusing immune responses towards neutralizing epitopes on the gp120 outer domain (OD) by removing the immunodominant, but non-neutralizing, inner domain. Previous OD constructs have not elicited strong neutralizing antibodies (nAbs). We constructed two immunogens, a monomeric gp120-OD and a trimeric gp120-OD×3, based on an M group consensus sequence (MCON6). Their biochemical and immunological properties were compared with intact gp120. Results indicated better preservation of critical neutralizing epitopes on gp120-OD×3. In contrast to previous studies, our immunogens induced potent, cross-reactive nAbs in rabbits. Although nAbs primarily targeted Tier 1 viruses, they exhibited significant breadth. Epitope mapping analyses indicated that nAbs primarily targeted conserved V3 loop elements. Although the potency and breadth of nAbs were similar for all three immunogens, nAb induction kinetics indicated that gp120-OD×3 was superior to gp120-OD, suggesting that gp120-OD×3 is a promising prototype for further gp120 OD-based immunogen development.

The clinical application of CCR5 antagonists involves first determining the coreceptor usage by the infecting viral strain. Bioinformatics programs that predict coreceptor usage could provide an alternative method to screen candidates for treatment with CCR5 antagonists, particularly in countries with limited financial resources. Thus, the present study aims to identify the best approach using bioinformatics tools for determining HIV-1 coreceptor usage in clinical practice. Proviral DNA sequences and Trofile results from 99 HIV-1-infected subjects under clinical monitoring were analyzed in this study. Based on the Trofile results, the viral variants present were 81.1% R5, 21.4% R5X4 and 1.8% X4. Determination of tropism using a Geno2pheno[coreceptor] analysis with a false positive rate of 10% gave the most suitable performance in this sampling: the R5 and X4 strains were found at frequencies of 78.5% and 28.4%, respectively, and there was 78.6% concordance between the phenotypic and genotypic results. Further studies are needed to clarify how genetic diversity amongst virus strains affects bioinformatics-driven approaches for determining tropism. Although this strategy could be useful for screening patients in developing countries, some limitations remain that restrict the wider application of coreceptor usage tests in clinical practice. A aplicação clínica dos antagonistas de CCR5 envolve em primeiro lugar determinar o uso de co-receptor pela cepa viral infectante. Programas de bioinformática que prevêem o uso co-receptor poderiam fornecer um método alternativo para selecionar candidatos para o tratamento com os antagonistas do CCR5, particularmente em países com poucos recursos financeiros. Assim, o presente estudo teve por objetivo identificar a melhor abordagem utilizando ferramentas de bioinformática para determinar qual o tipo de co-receptor do HIV-1 que poderia ser usado na prática clínica. Sequências de DNA proviral e Trofile resultados a partir de 99 pacientes infectados pelo HIV-1 sob monitorização clínica foram avaliadas. Com base nos resultados do Teste Trofile, as variantes virais presentes eram R5 (81,1%), R5X4 (21,4%) e X4 (1,8%). Determinação do tropismo pela análise do Geno2pheno, com taxa de falso positivos de 10% apresentou desempenho mais adequado para esta amostragem: as cepas R5 e X4 foram encontradas em frequências de 78,5% e 28,4%, respectivamente, e foi de 78,6% a concordância entre os resultados fenotípicos e genotípicos. Mais estudos são necessários para esclarecer como a diversidade genética entre as cepas do vírus afeta abordagens baseadas na determinação do tropismo pelas ferramentas de bioinformática. Embora esta estratégia possa ser útil para o rastreio de pacientes em países em desenvolvimento, permanecem algumas limitações que restringem a aplicação mais ampla para utilização de testes de co-receptor na prática clínica.

Novel anti-Human immunodeficiency virus (HIV)-1 agents targeting the V3 loop of envelope protein gp120 were designed by computer modeling based on glycosphingolipid β-galactosylceramide (β-GalCer), which is an alternative receptor allowing HIV-1 entry into CD4-negative cells of neural and colonic origin. Models of these β-GalCer analogs bound to the V3 loops from five various HIV-1 variants were generated by molecular docking and their stability was estimated by molecular dynamics (MDs) and binding free energy simulations. Specific binding to the V3 loop was accomplished primarily by non-conventional XH…π interactions between CH/OH sugar groups of the glycolipids and the conserved V3 residues with π-conjugated side chains. The designed compounds were found to block the tip and/or the base of the V3 loop, which form invariant structural motifs that contain residues critical for cell tropism. With the MDs calculations, the docked models of the complexes of the β-GalCer analogs with V3 are energetically stable in all of the cases of interest and exhibit low values of free energy of their formation. Based on the data obtained, these compounds are considered as promising basic structures for the rational design of novel, potent, and broad-spectrum anti-HIV-1 therapeutics.

Entry of HIV virus into the host cell is initiated by the interaction of its surface exposed gp120 protein with the cell surface CD4 receptor and a co-receptor that can be either CCR5 or CXCR4. The third variable region (V3 loop) of gp120 has an important role in co-receptor selection by gp120 and forms an epitope for neutralizing antibodies. In this work the dynamical behavior of the V3 loop in a trimeric environment has been investigated by generating an atomistic trimer model of gp120-CD4 complex and has been compared with the result of a monomeric gp120-CD4 complex. The main results coming from this work are that the three V3 loops belonging to the three subunits of the trimer display a different dynamical behavior in terms of its flexibility, spatial orientation, motion along the principal modes, conformations, solvent exposure and electrostatic potential distribution. We propose that the ability of the V3 loop to present, in the trimeric environment, simultaneous multiple alternative conformations that increase its capability of co-receptor recognition, is at least in part due to the effect of electrostatic potential generated by two subunits over the third one.

The Pro-Ser-Ala-Pro (PSAP) motif in the p2 domain of feline immunodeficiency virus (FIV) Gag is required for efficient virus release, virus replication, and Gag binding to the ubiquitin-E2-variant (UEV) domain of Tsg101. As a result of this direct interaction, expression of an N-terminal fragment of Tsg101 containing the UEV domain (referred to as TSG-5′) inhibits FIV release. In these respects, the FIV p2 Gag PSAP motif is analogous to the PTAP motif of HIV-1 p6 Gag . To evaluate the feasibility of a late domain-targeted inhibition of virus replication, we created an enriched Crandell-Rees feline kidney (CRFK) cell line (T5′ hi ) that stably expresses high levels of TSG-5′. Here we show that mutations in either the V3 loop or the second heptad repeat (HR2) domain of the FIV envelope glycoprotein (Env) rescue FIV replication in T5′ hi cells without increasing FIV release efficiency. TSG-5′-resistance mutations in Env enhance virion infectivity and the cell–cell spread of FIV when diffusion is limited using a semi-solid growth medium. These findings show that mutations in functional domains of Env confer TSG-5′-resistance, which we propose enhances specific infectivity and the cell–cell transmission of virus to counteract inefficient virus release. This article is part of a Special Issue entitled: Viral Membrane Proteins—Channels for Cellular Networking.

The power of mucosal anti-HIV-1 envelope immunoglobulins (Igs) to block virus transmission is underappreciated. We used passive immunization, a classical tool to unequivocally prove whether antibodies are protective. We mucosally instilled recombinant neutralizing monoclonal antibodies (nmAbs) of different Ig classes in rhesus macaques (RMs) followed by mucosal simian–human immunodeficiency virus (SHIV) challenge. We gave anti-HIV-1 IgM, IgG, and dimeric IgA (dIgA) versions of the same human nmAb, HGN194 that targets the conserved V3 loop crown. Surprisingly, dIgA1 with its wide-open, flat hinge protected 83% of the RMs against intrarectal R5-tropic SHIV-1157ipEL-p challenge, whereas dIgA2, with its narrow hinge, only protected 17% of the animals—despite identical epitope specificities and in vitro neutralization curves of the two dIgA isotypes (Watkins et al., AIDS 2013 27(9):F13-20). These data imply that factors in addition to neutralization determine in vivo protection. We propose that this underlying protective mechanism is immune exclusion, which involves large nmAb/virion aggregates that prevent virus penetration of mucosal barriers. Future studies need to find biomarkers that predict effective immune exclusion in vivo. Vaccine development strategies against HIV-1 and/or other mucosally transmissible pathogens should include induction of strong mucosal Abs of different Ig classes to defend epithelial barriers against pathogen invasion.

BackgroundTriple class drug-resistant HIV-1 infection remains a global challenge in individuals with extensive antiretroviral treatment (ART) experience, in terms of high mortality and probability of onward transmission. New therapeutic options within old and new drug classes are therefore essential. We determined if patients failing salvage therapy in Botswana are eligible for maraviroc (MVC) and enfuvirtide (T20) viral entry inhibitors based on the coreceptor usage and drug-resistant mutations in envelope gp120 and gp41.MethodsA total of 38 deep salvage patients were included in the analysis. We amplified and sequenced gp41 and V3 regions of HIV-1 envelope. Drug resistance mutations were analysed according to the IAS-USA 2017 reference mutation lists. Coreceptor usage was determined using PSSM and Geno2Pheno using a false-positive rate (FPR) of 10%.ResultsAmong 38 participants, 34 (89%) were successfully sequenced and amplified gp41 and 26 (68%) gp120 V3 loop sequences were obtained. Major T20 mutation G36S was obtained in 1/34 samples (5.8%) within the study population. Polymorphisms I169V(97%), I135L(100%), E151A(70.6%) and N42S(70.6%) were detected in HR1 and HR2 of gp41. CXCR4 coreceptor associated use, mutation L34M in gp41 HR1 was detected in 2 samples (5%). Analysis of coreceptor usage showed (17/26) 65.4% use of CCR5, and a (9/26) 34.6% use of the CXCR4 coreceptor.ConclusionA moderately high proportion of treatment-experienced (deep salvage) participants had CXCR4 coreceptor using strains. The use of maraviroc in Botswana would require coreceptor tropism testing. Non-T20 treatment experience in Botswana reduces the prevalence of the major mutations that confer resistance to the drug. T20 is therefore a potential alternative drug for patients failing salvage therapy in Botswana.

Although HIV-1 can be successfully eradicated from the circulating blood of HIV-1-infected individuals using anti-retroviral therapy (ART), HIV-1 virions emerge immediately after the interruption of ART. This study was aimed to investigate the origin of the emerged HIV-1. After obtaining informed consent, blood samples from nine HIV-1-infected individuals and endoscopic ileum samples from five of the individuals were obtained. Purified peripheral mononuclear cells (PBMCs)and ileum cells were analyzed by flow-cytometry, and the V3 loop sequences of the HIV-1 envelope protein were determined. By comparing the V3 loop sequences of the samples, we confirmed that the provirus hidden in the CD4(+) PBMCs was not the source of the HIV-1 that emerged after the interruption of ART. Although free virus and HIV-1-p24 antigen (p24)-positive cells were not seen in the blood of patients receiving ART, proviral DNA and p24 could be detected in the ileum from the same patient. Among the HIV-1-infected CD4(+) cells in the ileum samples, Vα24(+) natural killer T (NKT) cells were the major p24-positive cells. These results suggest that the innate NKTcells in the mucosal compartment are the most likely candidates for the origin of the HIV-1 that emerged after ART was interrupted.

A subset of broadly neutralizing anti-HIV antibodies inhibits cell to cell transmission of the virus., The neutralizing activity of anti–HIV-1 antibodies is typically measured in assays where cell-free virions enter reporter cell lines. However, HIV-1 cell to cell transmission is a major mechanism of viral spread, and the effect of the recently described broadly neutralizing antibodies (bNAbs) on this mode of transmission remains unknown. Here we identify a subset of bNAbs that inhibit both cell-free and cell-mediated infection in primary CD4+ lymphocytes. These antibodies target either the CD4-binding site (NIH45-46 and 3BNC60) or the glycan/V3 loop (10-1074 and PGT121) on HIV-1 gp120 and act at low concentrations by inhibiting multiple steps of viral cell to cell transmission. These antibodies accumulate at virological synapses and impair the clustering and fusion of infected and target cells and the transfer of viral material to uninfected T cells. In addition, they block viral cell to cell transmission to plasmacytoid DCs and thereby interfere with type-I IFN production. Thus, only a subset of bNAbs can efficiently prevent HIV-1 cell to cell transmission, and this property should be considered an important characteristic defining antibody potency for therapeutic or prophylactic antiviral strategies.

HIV-1 envelope gp120 is the target for neutralizing antibodies (NAbs) against the virus. Various approaches have been explored to improve immunogenicity of broadly neutralizing epitopes on this antigen with limited success. We previously demonstrated that immunogenicity of gp120 and especially its V3 epitopes was enhanced when gp120 was co-administered as immune-complex vaccines with monoclonal antibodies (mAb) to the CD4-binding site (CD4bs). To define the mechanisms by which immune complexes influence V3 immunogenicity, we compared gp120 complexed with mAbs specific for the C2 region (1006-30), the V2 loop (2158), or the CD4bs (654), and found that the gp120/654 and gp120/2158 complexes elicited anti-V3 NAbs, but the gp120/654 complex was the most effective. gp120 complexed with 654 F(ab')2 was as potent, indicating that V3 immunogenicity is determined by the specificity of the mAb's Fab fragment used to form the complexes. Importantly, the gp120/654 complex not only induced anti-gp120 antibodies (Abs) to higher titers, but also of greater avidity. The Abs were cross-reactive with V3 peptides from most subtype B and some subtype C isolates. Neutralization was detected only against Tier-1 HIV-1 pseudoviruses, while Tier-2 viruses, including the homologous JRFL strain, were not neutralized. However, JRFL produced in the presence of a mannosidase inhibitor was sensitive to anti-V3 NAbs in the immune sera. These results demonstrate that the gp120/654 complex is a potent immunogen for eliciting cross-reactive functional NAbs against V3 epitopes, of which exposure is determined by the specific compositions of glycans shrouding the HIV-1 envelope glycoproteins.

Achieving an AIDS-free generation will require elimination of postnatal transmission of HIV-1 while maintaining the nutritional and immunologic benefits of breastfeeding for infants in developing regions. Maternal/infant antiretroviral prophylaxis can reduce postnatal HIV-1 transmission, yet toxicities and the development of drug-resistant viral strains may limit the effectiveness of this strategy. Interestingly, in the absence of antiretroviral prophylaxis, greater than 90% of infants exposed to HIV-1 via breastfeeding remain uninfected, despite daily mucosal exposure to the virus for up to 2 y. Moreover, milk of uninfected women inherently neutralizes HIV-1 and prevents virus transmission in animal models, yet the factor(s) responsible for this anti-HIV activity is not well-defined. In this report, we identify a primary HIV-1-neutralizing protein in breast milk, Tenascin-C (TNC). TNC is an extracellular matrix protein important in fetal development and wound healing, yet its antimicrobial properties have not previously been established. Purified TNC captured and neutralized multiclade chronic and transmitted/founder HIV-1 variants, and depletion of TNC abolished the HIV-1-neutralizing activity of milk. TNC bound the HIV-1 Envelope protein at a site that is induced upon engagement of its primary receptor, CD4, and is blocked by V3 loop- (19B and F39F) and chemokine coreceptor binding site-directed (17B) monoclonal antibodies. Our results demonstrate the ability of an innate mucosal host protein found in milk to neutralize HIV-1 via binding to the chemokine coreceptor site, potentially explaining why the majority of HIV-1-exposed breastfed infants are protected against mucosal HIV-1 transmission.

Despite its sequence variability and structural flexibility, the V3 loop of the HIV-1 envelope glycoprotein gp120 is capable of recognizing cell-bound coreceptors CCR5 and CXCR4 and infecting cells. Viral selection of CCR5 is associated with the early stages of infection, and transition to selection of CXCR4 indicates disease progression. We have developed a predictive statistical model for coreceptor selectivity that uses the discrete property of net charge and the binary coreceptor preference markers of the N(6)X(7)[T/S](8)X(9) glycosylation motif and 11/24/25 positive amino acid rule. The model is based on analysis of 2,054 V3 loop sequences from patient data and allows us to infer the most likely state of the disease from physicochemical characteristics of the sequences. The performance of the model is comparable to established sequence-based predictive methods, and may be used in combination with other methods as a supportive diagnostic for coreceptor selection. This model may be used for personalized medical decisions in administering coreceptor-specific therapies.

Genotypic drug resistance testing is routine practice in HIV-1 clinical care. The visual interpretation of sequencing electropherograms is labour-intensive and subject to intra- and inter-assay variability because decisions are based on operators' judgments. In this study the performance of the automatic editing tool RECall was compared to the current standard of editing manually and editing using the tool ViroSeq. Using RECall a consensus sequence could be generated for 97% of the V3 loop and for 79% of the pol experiments. By comparison, using manual editing a consensus sequence could be reached for 87% of the V3 and 87% of the pol experiments. Using ViroSeq, a consensus sequence was generated for 68% of the pol experiments. On a predefined dataset, manual editing displayed the highest probability to accurately assign mixtures (0.91 vs. 0.88 by ViroSeq vs. 0.76 by RECall) and the lowest probability to inaccurately assign a mixture to a pure base call (0.002 vs. 0.019 by ViroSeq vs. 0.002 by RECall). As differences in base calling have little impact on drug resistance interpretation and hands-on-time could be substantially reduced, RECall could be a valuable tool for the standardization and acceleration of the editing process.

Objective: To study the complex formed between Tat protein and Env soluble trimeric immunogen, and compare with previously determined structures of Env native trimers and Env–CD4m complexes. Design: The soluble Env trimer was used to mimic the spike glycoprotein on the virus surface for the study. To overcome limitations of other structural determination methods, cryoelectron microscopy was employed to image the complex, and single particle reconstruction was utilized to reconstruct the structure of the complex from collected micrographs. Molecular modeling of gp120–Tat was performed to provide atomic coordinates for docking. Methods: Images were preprocessed by multivariate statistical analysis to identify principal components of variation then submitted for reconstruction. Reconstructed structures were docked with modeled gp120–Tat atomic coordinates to study the positions of crucial epitopes. Results: Analysis of the Env–Tat complex demonstrated an intermediate structure between Env native trimers and Env–CD4m structures. Docking results indicate that the CD4-binding site and the V3 loop are exposed in the Env–Tat complex. The integrin-binding sequence in Tat was also exposed in Env–Tat docking. Conclusion: The intermediate structure induced by Tat-interaction with Env could potentially provide an explanation for increased virus infection in the presence of Tat protein. Consequently, exposure of CD4-binding sites and a putative integrin-binding sequence on Tat in the complex may provide a new avenue for rational design of an effective HIV vaccine.

HIV-1 cell entry is initiated by the interaction of the viral envelope glycoprotein gp120 with CD4, and chemokine coreceptors CXCR4 and CCR5. The molecular recognition of CXCR4 or CCR5 by the HIV-1 gp120 is mediated through the V3 loop, a fragment of gp120. The binding of the V3 loop to CXCR4 or CCR5 determines the cell tropism of HIV-1 and constitutes a key step before HIV-1 cell entry. Thus, elucidating the molecular recognition of CXCR4 by the V3 loop is important for understanding HIV-1 viral infectivity and tropism, and for the design of HIV-1 inhibitors. We employed a comprehensive set of computational tools, predominantly based on free energy calculations and molecular-dynamics simulations, to investigate the molecular recognition of CXCR4 by a dual tropic V3 loop. We report what is, to our knowledge, the first HIV-1 gp120 V3 loop:CXCR4 complex structure. The computationally derived structure reveals an abundance of polar and nonpolar intermolecular interactions contributing to the HIV-1 gp120:CXCR4 binding. Our results are in remarkable agreement with previous experimental findings. Therefore, this work sheds light on the functional role of HIV-1 gp120 V3 loop and CXCR4 residues associated with HIV-1 coreceptor activity.

OBJECTIVES R5-tropic viruses are associated with HIV-1 transmission and predominate during the early stages of infection. X4-tropic populations have been detected in ~50% of patients with late-stage disease infected with subtype B viruses. In this study, we compared the frequency of X4 tropism in individuals infected with HIV-1 CRF14_BG viruses, which have a V3 loop of subtype B, with a control group of individuals infected with subtype B viruses. METHODS Sixty-three individuals infected with HIV-1 CRF14_BG (n = 31) or subtype B (n = 32) were studied. Similar proportions of newly diagnosed and chronically infected individuals were included in the subtype B and CRF14_BG groups. V3 sequences were obtained and coreceptor tropism was predicted using the Geno2pheno[coreceptor] algorithm. V3 net charge and 11/25 rules were also used for coreceptor prediction. RESULTS Overall, X4 tropism was more frequent among individuals infected with CRF14_BG viruses (87.1%) than subtype B viruses (34.3%), a difference that was statistically highly significant (P = 0.00001). Importantly, the frequencies among newly diagnosed individuals were 90% and 13.3%, respectively (P = 0.0007). Characteristic amino acids in the V3 loop (T13, M14, V19 and W20) were identified at higher frequencies in CRF14_BG viruses (54%) than subtype B viruses (0%; P < 0.000001). CONCLUSIONS CRF14_BG is the genetic form with the highest proportion of X4-tropic viruses reported to date in newly diagnosed and chronic infections. This suggests high pathogenicity for CRF14_BG viruses, potentially leading to rapid disease progression. CCR5 antagonists will be ineffective in most CRF14_BG-infected patients, even at early stages of infection.

The purpose of this investigation was to determine the impact on human immunodeficiency virus (HIV) tropism of uncontrolled virus exposure during 2 years of intermittent highly active antiretroviral therapy (HAART). The Istituto Superiore di Sanita-Pulsed Antiretroviral Therapy (ISS-PART) randomized study compared the outcome of 2 years of structured treatment interruptions (STIs) versus standard continuous treatment in first-line HAART responder subjects. The STI schedule consisted of five STIs of 1, 1, 2, 2, and 3 months, respectively, separated by four periods of 3-month therapy. In the present study, coreceptor tropism was assessed in 12 patients of the STI arm at different time points over a period of 2 years. Tropism was determined on DNA and RNA by V3 loop region sequencing. The Geno2pheno algorithm (false-positive rate, FPR: 20 %) was used for data interpretation. At baseline, 9/12 subjects (75.0 %) had CCR5-tropic viruses in their HIV. Three had a CXCR4-tropic virus. Ten patients maintained the same coreceptor in DNA after 2 years, whereas in two patients, a shift occurred (one R5–X4, one X4–R5). In a patient with an R5 virus at baseline, a transient change to X4 tropism was seen in the rebounding virus during STI. Changes in tropism were not associated with the amplitude and duration of virus exposure during STIs, residual viremia at baseline, or the development of resistance mutations in the RT region. Our preliminary results suggest that viral replication, observed after short periods of treatment interruption, is not enough to drive the evolution of HIV tropism.

Insight concerning the switch in HIV-1 coreceptor use will lead to a better understanding of HIV-1 pathogenesis and host-virus dynamics. Predicting CXCR4 utilization by analyzing HIV-1 envelope consensus sequences is highly specific, but minority variants in the viral population are often missed resulting in low sensitivity. Commercial phenotypic assays are costly, and the development of sensitive in-house phenotypic assays to detect CXCR4-using HIV may not be feasible for some laboratories. A sensitive, inexpensive genotyping assay was developed to detect viral sequences associated with CXCR4-utilizing virus (X4). Codon-specific primer pairs were used to detect X4-associated codons at five positions in the HIV-1 envelope V3 loop (11, 13, 24, 25, and 32). Sixty plasma samples from HIV-1-infected individuals were analyzed by consensus sequencing and codon-specific PCR (CS-PCR). Forty-six of these were also phenotyped by Trofile or Enhanced Sensitivity Trofile (ESTA). CS-PCR detected X4 variants 17% more often than 11/24/25 consensus sequencing alone (n=60), 30% more often than Trofile (n=27), and in a limited data set, 16% more often than ESTA (n=19). CS-PCR combined with consensus sequencing had approximately 80% concordance with ESTA.

We previously showed that expression of human FcγRI on TZM-bl cells potentiates neutralization by gp41 membrane-proximal external region (MPER)-specific antibodies. Here we show that lysosomotropic reagents known to block phagocytosis do not diminish this effect. We also show that FcγRI occasionally potentiates neutralization by antibodies against the V3 loop of gp120 and cluster I of gp41. We conclude that FcγRI provides a kinetic advantage for neutralizing antibodies against partially cryptic epitopes independent of phagocytosis.

The production of human monoclonal antibodies (mAbs) has been improved recently using the single B cell and PCR technology. A number of new anti-HIV-1 mAbs directed to various epitopes were produced by selecting single B cells from HIV positive individuals using the HIV-1 envelope (Env) proteins, and we tested whether the peptide can select B cells specific to a particular Env epitope. Using the fluorescently-labeled peptide tetramer representative of the V3 loop of HIV-1 Env gp120 for staining the B cells derived from one HIV-1 infected donor, four clonal human mAbs were produced with specificity to the V3 region. The clonality of the four V3 mAbs was based on the usage of the same immunoglobulin genes and almost identical sequence of CDRs. The amino acid changes were present only in the framework and, possibly, they could be related to the differences observed in the relative affinity binding of these four mAbs to V3 antigen. One representative V3 mAb displayed very potent neutralizing activity to one of two viruses tested. This study shows the feasibility of utilizing a peptide tetramer to select epitope-specific B cells and produce mAbs.

Human immunodeficiency virus type 1 (HIV-1) coreceptor usage and tropism can be modulated by the V3 loop sequence of the gp120 exterior envelope glycoprotein. For coreceptors, R5 viruses use CCR5, X4 viruses use CXCR4, and dual-tropic (R5X4) viruses use either CCR5 or CXCR4. To understand the requirements for dual tropism, we derived and analyzed a dual-tropic variant of an X4 virus. Changes in the V3 base, which allow gp120 to interact with the tyrosine-sulfated CCR5 N-terminus, and deletion of residues 310/311 in the V3 tip were necessary for efficient CCR5 binding and utilization. Thus, both sets of V3 changes allowed CCR5 utilization with retention of the ability to use CXCR4. We also found that the stable association of gp120 with the trimeric envelope glycoprotein complex in R5X4 viruses, as in X4 viruses, is less sensitive to V3 loop changes than gp120-trimer association in R5 viruses.

Human immunodeficiency virus type 1 (HIV-1) V3 loop sequence can be used to infer viral coreceptor use. The effect of input copy number on population-based sequencing of the V3 loop of HIV-1 was examined through replicate deep and population-based sequencing of samples with known tropism, a heterogeneous clinical sample (624 population-based sequences and 47 deep-sequencing replicates), and a large cohort of clinical samples from phase III clinical trials of maraviroc including the MOTIVATE/A4001029 studies ( n = 1,521). Proviral DNA from two independent samples from each of 101 patients from the MOTIVATE/A4001029 studies was also analyzed. Cumulative technical error occurred at a rate of 3 × 10 −4 mismatches/bp, without observed effect on inferred tropism. Increasing PCR replication increased minority species detection with an ∼10% minority population detected in 18% of cases using a single replicate at a viral load of 1,072 copies/ml and in 44% of cases using three replicates. The nucleotide prevalence detected by population-based and deep sequencing were highly correlated (Spearman's ρ, 0.73), and the accuracy increased with increasing input copy number ( P < 0.001). Triplicate sequencing was able to predict tropism changes in the MOTIVATE/A4001029 studies for both low ( P = 0.05) and high ( P = 0.02) viral loads. Sequences derived from independently extracted and processed samples of proviral DNA for the same patient were equivalent to replicates from the same extraction ( P = 0.45) and had correlated position-specific scoring matrix scores (Spearman's ρ, 0.75; P ≪ 0.001); however, concordance in tropism inference was only 83%. Input copy number and PCR replication are important factors in minority species detection in samples with significant heterogeneity.

HIV-specific antibody-dependent cell cytotoxicity (ADCC) is likely to be important in governing protection from human immunodeficiency virus (HIV) and slowing disease progression. Little is known about the ADCC responses to HIV-1 subtype C. We characterized ADCC responses in HIV-1 subtype C-infected Indian subjects with slow disease progression and identified the dominant antigenic regions recognized by these antibodies. ADCC responses were measured in plasma from 34 long-term non-progressors (LTNPs), who were asymptomatic and maintained CD4 count above 500 cells/mm3 for the last 7 years in the absence of antiretroviral therapy (ART), and 58 ART naïve progressors with CD4 count

The human immunodeficiency virus type 1 (HIV-1) envelope protein provides the primary contact between the virus and host, and is the main target of the adaptive humoral immune response. The length of gp120 variable loops and the number of N-linked glycosylation events are key determinants for virus infectivity and immune escape, while the V3 loop overall positive charge is known to affect co-receptor tropism. We selected two families in which both parents and two children had been infected with HIV-1 for nearly 10 years, but who demonstrated variable parameters of disease progression. We analysed the gp120 envelope sequence and compared individuals that progressed to those that did not in order to decipher evolutionary alterations that are associated with disease progression when individuals are infected with genetically related virus strains. The analysis of the V3-positive charge demonstrated an association between higher V3-positive charges with disease progression. The ratio between the amino acid length and the number of potential N-linked glycosylation sites was also shown to be associated with disease progression with the healthier family members having a lower ratio. In conclusion in individuals initially infected with genetically linked virus strains the V3-positive charges and N-linked glycosylation are associated with HIV-1 disease progression and follow varied evolutionary paths for individuals with varied disease progression.

Various HIV-1 env genetic and biochemical features impact the elicitation of cross-reactive neutralizing antibodies in natural infections. Thus, we aimed to investigate cross-neutralizing antibodies in individuals infected with HIV-1 env subtypes B, F1, C or the B/Bbr variant as well as env characteristics. Therefore, plasma samples from Brazilian chronically HIV-1 infected individuals were submitted to the TZM-bl neutralization assay. We also analyzed putative N-glycosylation sites (PNGLs) and the size of gp120 variable domains in the context of HIV-1 subtypes prevalent in Brazil. We observed a greater breadth and potency of the anti-Env neutralizing response in individuals infected with the F1 or B HIV-1 subtypes compared with the C subtype and the variant B/Bbr. We observed greater V1 B/Bbr and smaller V4 F1 than those of other subtypes (p

The conformation of HIV-1 envelope (Env) glycoprotein trimers is key in ensuring protection against waves of neutralizing antibodies generated during infection, while maintaining sufficient exposure of the CD4 binding site (CD4bs) for viral entry. The CD4 binding loop on Env is an early contact site for CD4 while penetration of a proximal cavity by CD4 triggers Env conformational changes for entry. The role of residues in the CD4 binding loop in regulating the conformation of the trimer and trimer association domain (TAD) was investigated using a novel saturation mutagenesis approach. Single mutations identified, resulted in distinct trimer conformations affecting CD4bs exposure, the glycan shield and the TAD across diverse HIV-1 clades. Importantly, mutations that improve access to the CD4bs without exposing the immunodominant V3 loop were identified. The different trimer conformations identified will affect the specificity and breadth of nabs elicited in vivo and are important to consider in design of Env immunogens for vaccines., Author Summary Spike proteins on the surface of HIV virus particles bind to CD4 receptors on the surface of immune cells and trigger infection. The immune system in an infected person attacks the virus spikes by producing antibodies that bind and neutralize them. To combat this immune attack, HIV continually alters the structure of the spike and thus escapes host antibodies. However, this process must still preserve sites on the spike that bind CD4 receptors for infection. Here, we investigated how the spike regulates its structure. We used a systematic approach to investigate every possible mutation covering a region of the spike critical for binding the CD4 receptor and controlling overall structure. We identified different sites and mechanisms that control the spike structure for diverse HIV-1 strains and impact the exposure of the binding site for CD4 along with targets for neutralizing antibodies. Our observations will help guide the design of spike structures for vaccines that induce neutralizing antibodies effective against different HIV-1 strains across the globe.

Background HIV-1 typically develops resistance to any single antiretroviral agent. Combined anti-retroviral therapy to reduce drug-resistance development is necessary to control HIV-1 infection. Here, to assess the utility of a combination of antibody and fusion inhibitor treatments, we investigated the potency of monoclonal antibodies at neutralizing HIV-1 variants that are resistant to fusion inhibitors. Results Mutations that confer resistance to four fusion inhibitors, enfuvirtide, C34, SC34, and SC34EK, were introduced into the envelope of HIV-1JR-FL, a CCR5-tropic tier 2 strain. Pseudoviruses with these mutations were prepared and used for the assessment of neutralization sensitivity to an array of antibodies. The resulting neutralization data indicate that the potencies of some antibodies, especially of those against the CD4 binding site, V3 loop, and membrane-proximal external region epitopes, were increased by the mutations in gp41 that conferred resistance to the fusion inhibitors. C34-, SC34-, and SC34EK-resistant mutants showed more sensitivity to monoclonal antibodies than enfuvirtide-resistant mutants. An analysis of C34-resistant mutations revealed that the I37K mutation in gp41 HR1 is a key mutation for C34 resistance, low infectivity, neutralization sensitivity, epitope exposure, and slow fusion kinetics. The N126K mutation in the gp41 HR2 domain contributed to C34 resistance and neutralization sensitivity to anti-CD4 binding site antibodies. In the absence of L204I, the effect of N126K was antagonistic to that of I37K. The results of a molecular dynamic simulation of the envelope trimer confirmation suggest that an I37K mutation induces the augmentation of structural fluctuations prominently in the interface between gp41 and gp120. Our observations indicate that the “conformational unmasking” of envelope glycoprotein by an I37K mutation is one of the mechanisms of neutralization sensitivity enhancement. Furthermore, the enhanced neutralization of C34-resistant mutants in vivo was shown by its high rate of neutralization by IgG from HIV patient samples. Conclusions Mutations in gp41 that confer fusion inhibitor resistance exert enhanced sensitivity to broad neutralizing antibodies (e.g., VRC01 and 10E8) and other conventional antibodies developed in HIV-1 infected patients. Therefore, next-generation fusion inhibitors and monoclonal antibodies could be a potential combination for future regimens of combined antiretroviral therapy. Electronic supplementary material The online version of this article (doi:10.1186/s12977-016-0304-7) contains supplementary material, which is available to authorized users.

Human immunodeficiency virus subtype 1B (HIV-1B) binds to the CD4 receptor and co-receptor CCR5 or CXCR4 to enter T lymphocytes. The amino acid sequence of the HIV envelope glycoprotein V3 region determines the co-receptor tropism, thereby influencing the infectivity of the virus. Our research group previously found that the amino acid at position 22 of the V3 region may affect the infectivity of the virus, and in this study, we tested this hypothesis. We constructed pseudoviruses by changing the amino acids at position 22 of the V3 region in CCR5-tropic and CXCR4-tropic viruses and tested their infectivity. When the amino acid at V3 position 22 was altered in the CCR5- and CXCR4-tropic viruses, their ability to infect cells decreased to 20.6% and 17.14%, respectively. Therefore, we propose that residue 22 in the V3 region of subtype HIV-1B significantly influences the infectivity of the virus.

HIV-1 enters immune cells via binding the viral envelope to a host cell CD4 receptor, and then a secondary co-receptor, usually CCR5 (R5) or CXCR4 (X4), and some HIV can utilize both co-receptors (R5X4). Although a small set of amino-acid properties such as charge and sequence length applied to HIV-1 envelope V3 loop sequence data can be used to predict co-receptor usage, we sought to expand the fundamental understanding of the physiochemical basis of tropism by analyzing many, perhaps less obvious, amino-acid properties over a diverse array of HIV sequences. We examined 74 amino-acid physicochemical scales over 1,559 V3 loop sequences with biologically tested tropisms downloaded from the Los Alamos HIV sequence database. Linear regressions were then calculated for each feature relative to three tropism transitions (R5→X4; R5→R5X4; R5X4→X4). Independent correlations were rank ordered to determine informative features. A structural analysis of the V3 loop was performed to better interpret these findings relative to HIV tropism states. Similar structural changes are required for R5 and R5X4 to transition to X4, thus suggesting that R5 and R5X4 types are more similar than either phenotype is to X4. Overall, the analysis suggests a continuum of viral tropism that is only partially related to charge; in fact, the analysis suggests that charge modification may be primarily attributed to decreased R5 usage, and further structural changes, particularly those associated with β-sheet structure, are likely required for full X4 usage.

We have probed the structure of the C4 and V3 domains of human immunodeficiency virus type 1 gp120 by immunochemical techniques. Monoclonal antibodies (MAbs) recognizing an exposed gp120 sequence, (E/K)VGKAMYAPP, in C4 were differentially sensitive to denaturation of gp120, implying a conformational component to some of the epitopes. The MAbs recognizing conformation-sensitive C4 structures failed to bind to a gp120 mutant with an alteration in the sequence of the V3 loop, and their binding to gp120 was inhibited by both V3 and C4 MAbs. This implies an interaction between the V3 and C4 regions of gp120, which is supported by the observation that the binding of some MAbs to the V3 loop was often enhanced by amino acid changes in an around the C4 region.

The primary cellular receptor for the human immunodeficiency viruses type 1 (HIV-1) and type 2 (HIV-2) is the CD4 antigen. HIV infection of CD4+ cells is initiated by binding of the virus to the cell surface, via a high affinity interaction between CD4 and the HIV outer envelope glycoprotein, gp120. The development of model systems using soluble recombinant forms of CD4 (sCD4) has allowed kinetic and thermodynamic analyses of CD4 binding to gp120, and study of the post-binding events leading to virus-cell membrane fusion. It has thus been demonstrated that the affinity of sCD4 for gp120 on virions or HIV-infected cells depends on both the primary sequence and the tertiary structure of gp120 in the membrane. With cell-line adapted isolates of HIV-1, sCD4 binding induces conformational changes in gp120, leading to the complete dissociation of gp120 from the transmembrane glycoprotein, gp41, and exposing cryptic epitopes of gp41. Similar observations have been made with cell-anchored CD4; exposure of cryptic gp41 epitopes occurs at the fusion interface between clusters of CD4-expressing and HIV-infected cells. Thus, for HIV-1, CD4 induces exposure of fusogenic components of gp41 which triggers virus-cell membrane coalescence. This is termed receptor-mediated activation of fusion. With primary isolates of HIV-1 and the related lentiviruses, HIV-2 and simian immunodeficiency virus (SIV), the CD4-induced molecular rearrangements in gp120 are more subtle, implying that there is a spectrum of responses to sCD4 binding. The high-affinity binding site on CD4 for gp120 is necessary and probably sufficient for activation of HIV fusion, although other regions of CD4 may indirectly influence viral entry. There are two regions on the envelope glycoproteins which are recognized as playing a role in HIV entry: the N-terminus of gp41 and the gp120 V3 loop. The roles of these domains are discussed.

Genetically engineered herpesviruses have been successfully used for the development of vaccines against important animal diseases, including Aujesky's disease (pseudorabies virus), infectious bovine rhinotracheitis, and swine fever (hog cholera virus) (21, 56, 57). In addition, attenuated herpesviruses have been used for human vaccination (including the Towne strain of human cytomegalovirus and the Oka strain of varicella-zoster virus) (5, 24, 38, 52). Both herpes simplex virus type 1 (HSV-1) and varicella-zoster virus have been used for the expression of foreign genes, since these viruses can accommodate large segments of exogenous DNA with little effect on virus replication (15, 43). Replication-competent and replication-defective gene replacement vectors based on both viruses are being explored as possible human immunodeficiency virus (HIV) vaccine delivery systems (32, 45). The appeal of this approach lies in part in the ability of herpesviruses to (i) elicit strong cytotoxic-T-lymphocyte (CTL) responses; (ii) infect mucosal surfaces; (iii) infect a broad range of cell types, including dendritic cells (1, 23, 30, 40); and (iv) establish a state of persistence in the infected cell. The latter property may conceivably result in more durable immune responses to herpesvirus-based vaccines compared to many other vector approaches. The HSV-1 amplicon is an alternative HSV-1-based gene transfer vector which differs significantly from standard gene replacement vector systems. Most notably, the HSV-1 amplicon is a highly flexible, replication-defective, high-capacity plasmid-based gene transfer vector that contains only the lytic-phase origin of DNA replication and the cleavage and packaging sequences from HSV-1 (13, 46). This plasmid can be replicated and packaged into HSV-1 virions in the presence of either a suitable helper virus or a helper virus genome (13, 46). In this context, ca. 150 kb of concatemeric, replicated amplicon DNA becomes packaged into each virus particle (Fig. ​(Fig.1).1). Thus, the amplicon particle can contain as many as 15 to 20 copies of the packaged amplicon plasmid. This results in the delivery of multiple gene copies to each individual cell that becomes infected by the amplicon particle. FIG. 1. HSV-1 amplicon vector system. The amplicon plasmid contains the HSV-1 origin of DNA replication, the viral cleavage and packaging site, and a mammalian expression cassette encoding the protein of interest (in this case, gp120). This plasmid is cotransfected ... The amplicon vector system has recently been improved through the development of defective helper-virus genomes which lack viral cleavage and packaging sites of their own (12, 53). These defective helper-virus genomes allow amplicon plasmids to become packaged into HSV-1 particles while failing to become packaged themselves. As a consequence, helper-free amplicon stocks can be readily derived (9, 12, 39, 53). These helper-free amplicon particles express only the exogenous inserted sequence of interest and do not express any open reading frame from HSV-1. Thus, helper-free amplicon particles can be considered to be highly safe and devoid of the ability to express any of the immunomodulatory gene products of HSV-1 itself (e.g., ICP47, which downregulates major histocompatibility complex class I [17, 59]). This may in part explain why helper-free amplicon vectors can infect dendritic cells without inhibiting dendritic-cell maturation or immunostimulatory function (58), unlike either wild-type HSV-1 or recombinant HSV-1 vectors (23, 30, 40). With the pressing need for an effective HIV-1 vaccine, it seems prudent to explore a wide array of viral delivery systems. In the present study, we constructed an HSV-1 amplicon plasmid that encodes a codon-optimized HIV-1 gp120 insert (4), under the transcriptional control of a strong viral promoter (HSV:gp120). We then packaged this plasmid into HSV-1 particles, which were inoculated into mice to evaluate their ability to elicit immune responses against the encoded gp120. Since CD8+ CTL activity is considered by many to be critical for controlling both HIV infection and infection with the closely related agent simian immunodeficiency virus (SIV) (6-8, 19, 35, 42), we concentrated our analyses on the ability of the HSV-1 amplicon vector to elicit strong CTL responses to gp120. Among the numerous CD8+-CTL epitopes that potentially could be derived from the HIV-1 virion, few are known to be recognized in mice. Among these, an immunodominant H-2Dd-restricted epitope from the V3 loop of HIV-1 gp120 (RGPGRAFVTI) has been described in BALB/c mice (2, 3, 25, 33, 37, 44). Our analyses of Env-specific cellular immune responses to HSV:gp120 amplicon particles therefore focused on this particular epitope. Overall, the findings reported here show that the HSV:gp120 amplicon vector is capable of eliciting strong Env-specific immune responses after a single inoculation of amplicon particles. The intradermal (i.d.) route of immunization appears to result in the generation of maximal cellular immune responses to gp120, and there is a clear dose effect in terms of the strength of response elicited versus the dose of amplicon delivered. However, even the lowest dose of amplicon that was tested (10,000 infectious particles) was able to generate a strong gp120-specific cellular response. The amplicon-induced immune response was highly durable in mice (lasting >5 months), and strong cellular responses occurred even in animals that had been previously infected with wild-type HSV-1. Overall, these data provide support for further evaluation of this novel approach to HIV-1 vaccine development.

Human immunodeficiency virus (HIV) attachment to host cells is a multi-step process that involves interaction of the viral envelope gp120 with the primary receptor CD4 and coreceptors. HIV gp120 also binds to other cell surface components, including heparan sulfate (HS), a sulfated polysaccharide whose wide interactive properties are exploited by many pathogens for attachment and concentration at the cell surface. To analyze the structural features of gp120 binding to HS, we used soluble CD4 to constrain gp120 in a specific conformation. We first found that CD4 induced conformational change of gp120, dramatically increasing binding to HS. We then showed that HS binding interface on gp120 comprised, in addition to the well characterized V3 loop, a CD4-induced epitope. This epitope is efficiently targeted by nanomolar concentrations of size-defined heparin/HS-derived oligosaccharides. Because this domain of the protein also constitutes the binding site for the viral coreceptors, these results support an implication of HS at late stages of the virus-cell attachment process and suggest potential therapeutic applications.