Gp120 V3 Research Articles

The influence of N-linked glycans on the molecular dynamics of the HIV-1 gp120 V3 loop.

N-linked glycans attached to specific amino acids of the gp120 envelope trimer of a HIV virion can modulate the binding affinity of gp120 to CD4, influence coreceptor tropism, and play an important role in neutralising antibody responses. Because of the challenges associated with crystallising fully glycosylated proteins, most structural investigations have focused on describing the features of a non-glycosylated HIV-1 gp120 protein. Here, we use a computational approach to determine the influence of N-linked glycans on the dynamics of the HIV-1 gp120 protein and, in particular, the V3 loop. We compare the conformational dynamics of a non-glycosylated gp120 structure to that of two glycosylated gp120 structures, one with a single, and a second with five, covalently linked high-mannose glycans. Our findings provide a clear illustration of the significant effect that N-linked glycosylation has on the temporal and spatial properties of the underlying protein structure. We find that glycans surrounding the V3 loop modulate its dynamics, conferring to the loop a marked propensity towards a more narrow conformation relative to its non-glycosylated counterpart. The conformational effect on the V3 loop provides further support for the suggestion that N-linked glycosylation plays a role in determining HIV-1 coreceptor tropism.

Discovery of novel anti-HIV-1 agents based on a broadly neutralizing antibody against the envelope gp120 V3 loop: a computational study

A computer-aided search for novel anti-HIV-1 agents able to mimic the pharmacophore properties of broadly neutralizing antibody (bNAb) 3074 was carried out based on the analysis of X-ray complexes of this antibody Fab with the MN, UR29, and VI191 peptides from the V3 loop of the HIV envelope protein gp120. Using these empirical data, peptidomimetic candidates of bNAb 3074 were identified by a public, web-oriented virtual screening platform (pepMMsMIMIC) and models of these candidates bound to the above V3 peptides were generated by molecular docking. The docking calculations identified four molecules exhibiting a high affinity to all of the V3 peptides. These molecules were selected as the most probable peptidomimetics of bNAb 3074. Finally, the stability of the complexes of these molecules with the MN, UR29, and VI191 V3 peptides was estimated by molecular dynamics and free energy simulations. Specific binding to the V3 loop was accomplished primarily by π–π interactions between the aromatic rings of the peptidomimetics and the conserved Phe-20 and/or Tyr-21 of the V3 immunogenic crown. In a mechanism similar to that of bNAb 3074, these compounds were found to block the tip of the V3 loop forming its invariant structural motif that contains residues critical for cell tropism. Based on these findings, the compounds selected are considered as promising basic structures for the rational design of novel, potent, and broad-spectrum anti-HIV-1 therapeutics.

Stability of unfrozen whole blood DNA for remote genotypic analysis of HIV-1 coreceptor tropism

BackgroundMaraviroc is an HIV-1 coreceptor antagonist that has shown good efficacy and tolerability in treatment-naive and treatment-experienced patients harboring CCR5-tropic virus. The use of Maraviroc in treatment simplification in patients with suppressed plasma HIV-1 RNA requires analysis of HIV-1 DNA. Coreceptor tropism testing is often performed remotely at reference laboratories. In this study paired whole blood stored at + 4°C and at−20°C were compared as a source for genotypic coreceptor tropism testing.MethodsTwo hundred paired whole blood samples from different patients were analysed. Each sample was stored in two different conditions: one aliquot was stored at−20°C until spin column DNA extraction (WB20) and one aliquot was stored at +4°C for two weeks and then placed at room temperature (22-24°C) for two days before DNA extraction (WB4). Subsequently, a fragment encompassing the HIV-1 gp120 V3 domain was amplified by a singlicate nested PCR followed by triplicate nested PCR in the negative samples. A randomly selected panel of 20 paired WB4 and WB20 duplicate amplification products were sequenced and coreceptor tropism was inferred by geno2pheno [coreceptor].ResultsWB20 yielded a higher amount of DNA than WB4 (median [IQR] values 332.5 ng/μl [117.5-401] and 107 ng/μl [56.6-318], respectively; P < 0.001). However, the DNA purity was higher for WB4 than for WB20 (median distance from the optimal OD260/280 ratio, 0.14 [0.07-0.79] and 0.96 [0.36-1.10], respectively; P < 0.0001). The number of samples successfully amplified was 152 (76.0%) for WB20 and 155 (77.5%) for WB4 with the first PCR and 179 (89.5%) for WB20 and 181 (90.5%) for WB4 (P = ns) following subsequent triplicate analysis. The inferred coreceptor tropism was concordant in 18 out of 20 paired WB4 and WB20 samples. Two samples yielded discordant results, consistent with the discordance rate within duplicates from the same sample source (2/20 with WB4 and 1/20 with WB20) due to the inherent gp120 V3 variability.ConclusionsStoring whole blood at +4°C for up to two weeks and shipping at room temperature is a convenient method for obtaining HIV-1 gp120 V3 sequence information via testing at a remote laboratory in patients with suppressed viremia.

Genetic diversity of the highly variable V1 region interferes with Human Immunodeficiency Virus type 1 envelope functionality

BackgroundThe HIV envelope (Env) promotes viral entry in the host cell. During this process, Env undergoes several conformational changes to ensure its function. At the same time, the gp120 component of Env is the protein of the virus presenting the largest genetic diversity. Understanding how the virus maintains the balance between the competing requirements for maintenance of functionality and antigenic variation of this protein is central for the comprehension of its strategies of evolution and can highlight vulnerable aspects of its replication cycle. We focused on the variable domains V1 and V2 of the HIV-1 gp120 that are involved in conformational changes and are critical for viral escape from antibody neutralization.ResultsDespite the extensive sequence diversity found in the epidemic for these regions and their location on the external face of the protein, we observed that replacing V1V2 of one primary isolate with that of another severely interferes with Env functionality in more than half of the cases studied. Similar results were obtained for intra- and intersubtype chimeras. These observations are indicative of an interference of genetic diversity in these regions with Env functionality. Therefore, despite the extensive sequence diversity that characterizes these regions in the epidemic, our results show that functional constraints seem to limit their genetic variation. Defects in the V1V2 chimeras were not relieved by the insertion of the V3 region from the same isolate, suggesting that the decrease in functionality is not due to perturbation of potential coevolution networks between V1V2 and V3. Within the V1V2 domain, the sequence of the hypervariable loop of the V1 domain seems to be crucial for the functionality of the protein.ConclusionsBesides the well-documented role of V1V2 in the interplay with the immune response, this work shows that V1 is also involved in the selection of functional envelopes. By documenting a compromise between the opposing forces of sequence diversification and retention of functionality, these observations improve our understanding of the evolutionary trajectories of the HIV-1 envelope gene.

Plasma IgG to Linear Epitopes in the V2 and V3 Regions of HIV-1 gp120 Correlate with a Reduced Risk of Infection in the RV144 Vaccine Efficacy Trial

Neutralizing and non-neutralizing antibodies to linear epitopes on HIV-1 envelope glycoproteins have potential to mediate antiviral effector functions that could be beneficial to vaccine-induced protection. Here, plasma IgG responses were assessed in three HIV-1 gp120 vaccine efficacy trials (RV144, Vax003, Vax004) and in HIV-1-infected individuals by using arrays of overlapping peptides spanning the entire consensus gp160 of all major genetic subtypes and circulating recombinant forms (CRFs) of the virus. In RV144, where 31.2% efficacy against HIV-1 infection was seen, dominant responses targeted the C1, V2, V3 and C5 regions of gp120. An analysis of RV144 case-control samples showed that IgG to V2 CRF01_AE significantly inversely correlated with infection risk (OR= 0.54, p=0.0042), as did the response to other V2 subtypes (OR=0.60-0.63, p=0.016-0.025). The response to V3 CRF01_AE also inversely correlated with infection risk but only in vaccine recipients who had lower levels of other antibodies, especially Env-specific plasma IgA (OR=0.49, p=0.007) and neutralizing antibodies (OR=0.5, p=0.008). Responses to C1 and C5 showed no significant correlation with infection risk. In Vax003 and Vax004, where no significant protection was seen, serum IgG responses targeted the same epitopes as in RV144 with the exception of an additional C1 reactivity in Vax003 and infrequent V2 reactivity in Vax004. In HIV-1 infected subjects, dominant responses targeted the V3 and C5 regions of gp120, as well as the immunodominant domain, heptad repeat 1 (HR-1) and membrane proximal external region (MPER) of gp41. These results highlight the presence of several dominant linear B cell epitopes on the HIV-1 envelope glycoproteins. They also generate the hypothesis that IgG to linear epitopes in the V2 and V3 regions of gp120 are part of a complex interplay of immune responses that contributed to protection in RV144.

Selected HIV-1 Env Trimeric Formulations Act as Potent Immunogens in a Rabbit Vaccination Model

BackgroundTen to 30% of HIV-1 infected subjects develop broadly neutralizing antibodies (bNAbs) during chronic infection. We hypothesized that immunizing rabbits with viral envelope glycoproteins (Envs) from these patients may induce bNAbs, when formulated as a trimeric protein and in the presence of an adjuvant.MethodsBased on in vitro neutralizing activity in serum, patients with bNAbs were selected for cloning of their HIV-1 Env. Seven stable soluble trimeric gp140 proteins were generated from sequences derived from four adults and two children infected with either clade A or B HIV-1. From one of the clade A Envs both the monomeric and trimeric Env were produced for comparison. Rabbits were immunized with soluble gp120 or trimeric gp140 proteins in combination with the adjuvant dimethyl dioctadecyl ammonium/trehalose dibehenate (CAF01). Env binding in rabbit immune serum was determined using ELISAs based on gp120-IIIB protein. Neutralizing activity of IgG purified from rabbit immune sera was measured with the pseudovirus-TZMbl assay and a PBMC-based neutralization assay for selected experiments.ResultsIt was initially established that gp140 trimers induce better antibody responses over gp120 monomers and that the adjuvant CAF01 was necessary for such strong responses. Gp140 trimers, based on HIV-1 variants from patients with bNAbs, were able to elicit both gp120IIIB specific IgG and NAbs to Tier 1 viruses of different subtypes. Potency of NAbs closely correlated with titers, and an gp120-binding IgG titer above a threshold of 100,000 was predictive of neutralization capability. Finally, peptide inhibition experiments showed that a large fraction of the neutralizing IgG was directed against the gp120 V3 region.ConclusionsOur results indicate that the strategy of reverse immunology based on selected Env sequences is promising when immunogens are delivered as stabilized trimers in CAF01 adjuvant and that the rabbit is a valuable model for HIV vaccine studies.

Molecular Recognition of CXCR4 by a Dual Tropic HIV-1 gp120 V3 Loop

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.

Novel directions in HIV-1 vaccines revealed from clinical trials.

Considerable HIV-1 vaccine development efforts have been deployed over the past decade. Put into perspective, the results from efficacy trials and the identification of correlates of risk have opened large and unforeseen avenues for vaccine development. The Thai efficacy trial, RV144, provided the first evidence that HIV-1 vaccine protection against HIV-1 acquisition could be achieved. The correlate of risk analysis showed that IgG antibodies against the gp120 V2 loop inversely correlated with a decreased risk of infection, whereas Env-specific IgA directly correlated with risk. Further clinical trials will focus on testing new envelope subunit proteins formulated with adjuvants capable of inducing higher and more durable functional antibody responses (both binding and broadly neutralizing antibodies). Moreover, vector-based vaccine regimens that can induce cell-mediated immune responses in addition to humoral responses remain a priority. Future efficacy trials will focus on prevention of HIV-1 transmission in heterosexual population in Africa and MSM in Asia. The recent successes leading to novel directions in HIV-1 vaccine development are a result of collaboration and commitment among vaccine manufacturers, funders, scientists and civil society stakeholders. Sustained and broad collaborative efforts are required to advance new vaccine strategies for higher levels of efficacy.

Heavy Chain-Only IgG2b Llama Antibody Effects Near-Pan HIV-1 Neutralization by Recognizing a CD4-Induced Epitope That Includes Elements of Coreceptor- and CD4-Binding Sites

The conserved HIV-1 site of coreceptor binding is protected from antibody-directed neutralization by conformational and steric restrictions. While inaccessible to most human antibodies, the coreceptor site has been shown to be accessed by antibody fragments. In this study, we used X-ray crystallography, surface plasmon resonance, and pseudovirus neutralization to characterize the gp120-envelope glycoprotein recognition and HIV-1 neutralization of a heavy chain-only llama antibody, named JM4. We describe full-length IgG2b and IgG3 versions of JM4 that target the coreceptor-binding site and potently neutralize over 95% of circulating HIV-1 isolates. Contrary to established trends that show improved access to the coreceptor-binding region by smaller antibody fragments, the single-domain (VHH) version of JM4 neutralized less well than the full-length IgG2b version of JM4. The crystal structure at 2.1-Å resolution of VHH JM4 bound to HIV-1 YU2 gp120 stabilized in the CD4-bound state by the CD4-mimetic miniprotein, M48U1, revealed a JM4 epitope that combined regions of coreceptor recognition (including the gp120 bridging sheet, V3 loop, and β19 strand) with gp120 structural elements involved in recognition of CD4 such as the CD4-binding loop. The structure of JM4 with gp120 thus defines a novel CD4-induced site of vulnerability involving elements of both coreceptor- and CD4-binding sites. The potently neutralizing JM4 IgG2b antibody that targets this newly defined site of vulnerability adds to the expanding repertoire of broadly neutralizing antibodies that effectively neutralize HIV-1 and thereby potentially provides a new template for vaccine development and target for HIV-1 therapy.

Influences on Trimerization and Aggregation of Soluble, Cleaved HIV-1 SOSIP Envelope Glycoprotein

We describe methods to improve the properties of soluble, cleaved gp140 trimers of the human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins (Env) for use in structural studies and as immunogens. In the absence of nonionic detergents, gp140 of the KNH1144 genotype, terminating at residue 681 in gp41 (SOSIP.681), has a tendency to form higher-order complexes or aggregates, which is particularly undesirable for structure-based research. We found that this aggregation in the absence of detergent does not involve the V1, V2, or V3 variable regions of gp120. Moreover, we observed that detergent forms micelles around the membrane-proximal external region (MPER) of the SOSIP.681 gp140 trimers, whereas deletion of most of the MPER residues by terminating the gp140 at residue 664 (SOSIP.664) prevented the aggregation that otherwise occurs in SOSIP.681 in the absence of detergent. Although the MPER can contribute to trimer formation, truncation of most of it only modestly reduced trimerization and lacked global adverse effects on antigenicity. Thus, the MPER deletion minimally influenced the kinetics of the binding of soluble CD4 and a CD4-binding site antibody to immobilized trimers, as detected by surface plasmon resonance. Furthermore, the MPER deletion did not alter the overall three-dimensional structure of the trimers, as viewed by negative-stain electron microscopy. Homogeneous and aggregate-free MPER-truncated SOSIP Env trimers are therefore useful for immunogenicity and structural studies.

S12.3 Understanding How Broadly Cross-Neutralising Antibodies Develop in HIV Infection - Clues For HIV Vaccine Development?

Neutralizing antibodies are likely to play a crucial role in a preventative HIV-1 vaccine. Although efforts to elicit broadly cross-neutralising (BCN) antibodies by vaccination have been unsuccessful, a minority of...

Structure and dynamics of the gp120 V3 loop that confers noncompetitive resistance in R5 HIV-1(JR-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-1V3-M5 derived from HIV-1JR-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-1JR-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.

Longitudinal Analysis of CCR5 and CXCR4 Usage in a Cohort of Antiretroviral Therapy-Naïve Subjects with Progressive HIV-1 Subtype C Infection

HIV-1 subtype C (C-HIV) is responsible for most HIV-1 cases worldwide. Although the pathogenesis of C-HIV is thought to predominantly involve CCR5-restricted (R5) strains, we do not have a firm understanding of how frequently CXCR4-using (X4 and R5X4) variants emerge in subjects with progressive C-HIV infection. Nor do we completely understand the molecular determinants of coreceptor switching by C-HIV variants. Here, we characterized a panel of HIV-1 envelope glycoproteins (Envs) (n = 300) cloned sequentially from plasma of 21 antiretroviral therapy (ART)-naïve subjects who experienced progression from chronic to advanced stages of C-HIV infection, and show that CXCR4-using C-HIV variants emerged in only one individual. Mutagenesis studies and structural models suggest that the evolution of R5 to X4 variants in this subject principally involved acquisition of an “Ile-Gly” insertion in the gp120 V3 loop and replacement of the V3 “Gly-Pro-Gly” crown with a “Gly-Arg-Gly” motif, but that the accumulation of additional gp120 “scaffold” mutations was required for these V3 loop changes to confer functional effects. In this context, either of the V3 loop changes could confer possible transitional R5X4 phenotypes, but when present together they completely abolished CCR5 usage and conferred the X4 phenotype. Our results show that the emergence of CXCR4-using strains is rare in this cohort of untreated individuals with advanced C-HIV infection. In the subject where X4 variants did emerge, alterations in the gp120 V3 loop were necessary but not sufficient to confer CXCR4 usage.

Emergence of gp120 V3 Variants Confers Neutralization Resistance in an R5 Simian-Human Immunodeficiency Virus-Infected Macaque Elite Neutralizer That Targets the N332 Glycan of the Human Immunodeficiency Virus Type 1 Envelope Glycoprotein

Neutralization-resistant simian-human immunodeficiency virus AD8 (SHIVAD8) variants that emerged in an infected macaque elite neutralizer targeting the human immunodeficiency virus type 1 (HIV-1) gp120 N332 glycan acquired substitutions of critical amino acids in the V3 region rather than losing the N332 glycosylation site. One of these resistant variants, carrying the full complement of gp120 V3 changes, was also resistant to the potent anti-HIV-1 monoclonal neutralizing antibodies PGT121 and 10-1074, both of which are also dependent on the presence of the gp120 N332 glycan.

Curcumin ameliorates hippocampal neuron damage induced by human immunodeficiency virus-1.

Our previous studies have shown that infection with the gp120 V3 loop can cause human immunodeficiency virus-1 associated neurocognitive disorders. Curcumin has been shown to improve these effects to some degree, but the precise mechanisms remain unknown. The present study analyzed the neuroprotective effect and mechanism of curcumin in relation to hippocampal neurons. Results showed that 1 nmol/L gp120 V3 loop suppressed the growth of synapses. After administration of 1 μmol/L curcumin, synaptic growth improved. Curcumin is neuroprotective against gp120 V3 loop-induced neuronal damage by inhibiting the activation of L-type calcium currents, relieving intracellular Ca2+ overload, promoting Bcl-2 expression, and inhibiting Bax activation. The effect of curcumin was identical to nimodipine, suggesting that curcumin has the same neuroprotective effects against gp120 V3 loop-induced neuronal damage.

Broadly Neutralizing Antibody PGT121 Allosterically Modulates CD4 Binding via Recognition of the HIV-1 gp120 V3 Base and Multiple Surrounding Glycans

New broad and potent neutralizing HIV-1 antibodies have recently been described that are largely dependent on the gp120 N332 glycan for Env recognition. Members of the PGT121 family of antibodies, isolated from an African donor, neutralize ∼70% of circulating isolates with a median IC50 less than 0.05 µg ml−1. Here, we show that three family members, PGT121, PGT122 and PGT123, have very similar crystal structures. A long 24-residue HCDR3 divides the antibody binding site into two functional surfaces, consisting of an open face, formed by the heavy chain CDRs, and an elongated face, formed by LCDR1, LCDR3 and the tip of the HCDR3. Alanine scanning mutagenesis of the antibody paratope reveals a crucial role in neutralization for residues on the elongated face, whereas the open face, which accommodates a complex biantennary glycan in the PGT121 structure, appears to play a more secondary role. Negative-stain EM reconstructions of an engineered recombinant Env gp140 trimer (SOSIP.664) reveal that PGT122 interacts with the gp120 outer domain at a more vertical angle with respect to the top surface of the spike than the previously characterized antibody PGT128, which is also dependent on the N332 glycan. We then used ITC and FACS to demonstrate that the PGT121 antibodies inhibit CD4 binding to gp120 despite the epitope being distal from the CD4 binding site. Together, these structural, functional and biophysical results suggest that the PGT121 antibodies may interfere with Env receptor engagement by an allosteric mechanism in which key structural elements, such as the V3 base, the N332 oligomannose glycan and surrounding glycans, including a putative V1/V2 complex biantennary glycan, are conformationally constrained.

A common mechanism of clinical HIV-1 resistance to the CCR5 antagonist maraviroc despite divergent resistance levels and lack of common gp120 resistance mutations

BackgroundThe CCR5 antagonist maraviroc (MVC) inhibits human immunodeficiency virus type 1 (HIV-1) entry by altering the CCR5 extracellular loops (ECL), such that the gp120 envelope glycoproteins (Env) no longer recognize CCR5. The mechanisms of HIV-1 resistance to MVC, the only CCR5 antagonist licensed for clinical use are poorly understood, with insights into MVC resistance almost exclusively limited to knowledge obtained from in vitro studies or from studies of resistance to other CCR5 antagonists. To more precisely understand mechanisms of resistance to MVC in vivo, we characterized Envs isolated from 2 subjects who experienced virologic failure on MVC.ResultsEnvs were cloned from subjects 17 and 24 before commencement of MVC (17-Sens and 24-Sens) and after virologic failure (17-Res and 24-Res). The Envs cloned during virologic failure showed broad divergence in resistance levels, with 17-Res Env exhibiting a relatively high maximal percent inhibition (MPI) of ~90% in NP2-CD4/CCR5 cells and peripheral blood mononuclear cells (PBMC), and 24-Res Env exhibiting a very low MPI of ~0 to 12% in both cell types, indicating relatively “weak” and “strong” resistance, respectively. Resistance mutations were strain-specific and mapped to the gp120 V3 loop. Affinity profiling by the 293-Affinofile assay and mathematical modeling using VERSA (Viral Entry Receptor Sensitivity Analysis) metrics revealed that 17-Res and 24-Res Envs engaged MVC-bound CCR5 inefficiently or very efficiently, respectively. Despite highly divergent phenotypes, and a lack of common gp120 resistance mutations, both resistant Envs exhibited an almost superimposable pattern of dramatically increased reliance on sulfated tyrosine residues in the CCR5 N-terminus, and on histidine residues in the CCR5 ECLs. This altered mechanism of CCR5 engagement rendered both the resistant Envs susceptible to neutralization by a sulfated peptide fragment of the CCR5 N-terminus.ConclusionsClinical resistance to MVC may involve divergent Env phenotypes and different genetic alterations in gp120, but the molecular mechanism of resistance of the Envs studied here appears to be related. The increased reliance on sulfated CCR5 N-terminus residues suggests a new avenue to block HIV-1 entry by CCR5 N-terminus sulfopeptidomimetic drugs.

NMR mapping of RANTES surfaces interacting with CCR5 using linked extracellular domains

Chemokines constitute a large family of small proteins that regulate leukocyte trafficking to the site of inflammation by binding to specific cell-surface receptors belonging to the G-protein-coupled receptor (GPCR) superfamily. The interactions between N-terminal (Nt-) peptides of these GPCRs and chemokines have been studied extensively using NMR spectroscopy. However, because of the lower affinities of peptides representing the three extracellular loops (ECLs) of chemokine receptors to their respective chemokine ligands, information concerning these interactions is scarce. To overcome the low affinity of ECL peptides to chemokines, we linked two or three CC chemokine receptor 5 (CCR5) extracellular domains using either biosynthesis in Escherichia coli or chemical synthesis. Using such chimeras, CCR5 binding to RANTES was followed using (1)H-(15)N-HSQC spectra to monitor titration of the chemokine with peptides corresponding to the extracellular surface of the receptor. Nt-CCR5 and ECL2 were found to be the major contributors to CCR5 binding to RANTES, creating an almost closed ring around this protein by interacting with opposing faces of the chemokine. A RANTES positively charged surface involved in Nt-CCR5 binding resembles the positively charged surface in HIV-1 gp120 formed by the C4 and the base of the third variable loop of gp120 (V3). The opposing surface on RANTES, composed primarily of β2-β3 hairpin residues, binds ECL2 and was found to be analogous to a surface in the crown of the gp120 V3. The chemical and biosynthetic approaches for linking GPCR surface regions discussed herein should be widely applicable to the investigation of interactions of extracellular segments of chemokine receptors with their respective ligands.

CoRSeqV3-C: a novel HIV-1 subtype C specific V3 sequence based coreceptor usage prediction algorithm

BackgroundThe majority of HIV-1 subjects worldwide are infected with HIV-1 subtype C (C-HIV). Although C-HIV predominates in developing regions of the world such as Southern Africa and Central Asia, C-HIV is also spreading rapidly in countries with more developed economies and health care systems, whose populations are more likely to have access to wider treatment options, including the CCR5 antagonist maraviroc (MVC). The ability to reliably determine C-HIV coreceptor usage is therefore becoming increasingly more important. In silico V3 sequence based coreceptor usage prediction algorithms are a relatively rapid and cost effective method for determining HIV-1 coreceptor specificity. In this study, we elucidated the V3 sequence determinants of C-HIV coreceptor usage, and used this knowledge to develop and validate a novel, user friendly, and highly sensitive C-HIV specific coreceptor usage prediction algorithm.ResultsWe characterized every phenotypically-verified C-HIV gp120 V3 sequence available in the Los Alamos HIV Database. Sequence analyses revealed that compared to R5 C-HIV V3 sequences, CXCR4-using C-HIV V3 sequences have significantly greater amino acid variability, increased net charge, increased amino acid length, increased frequency of insertions and substitutions within the GPGQ crown motif, and reduced frequency of glycosylation sites. Based on these findings, we developed a novel C-HIV specific coreceptor usage prediction algorithm (CoRSeqV3-C), which we show has superior sensitivity for determining CXCR4 usage by C-HIV strains compared to all other available algorithms and prediction rules, including Geno2pheno[coreceptor] and WebPSSMSINSI-C, which has been designed specifically for C-HIV.ConclusionsCoRSeqV3-C is now openly available for public use at http://www.burnet.edu.au/coreceptor. Our results show that CoRSeqV3-C is the most sensitive V3 sequence based algorithm presently available for predicting CXCR4 usage of C-HIV strains, without compromising specificity. CoRSeqV3-C may be potentially useful for assisting clinicians to decide the best treatment options for patients with C-HIV infection, and will be helpful for basic studies of C-HIV pathogenesis.

The HIV-1 gp120 Major Variable Regions Modulate Cold Inactivation

HIV-1 entry involves the viral envelope glycoproteins (Env gps) and receptors on the target cell. Receptor binding channels the intrinsic high potential energy of Env into the force required to fuse the membranes of virus and target cell. For some HIV-1 strains, prolonged incubation on ice decreases Env potential energy and results in functional inactivation. By characterizing chimeras between two primary clade C HIV-1 strains that differ in sensitivities to cold, soluble CD4, and neutralizing antibodies, we found that these properties were largely determined by discrete elements within the gp120 variable regions V1V2 and V3.