V3 Loop Research Articles

Co-Receptor Tropism Determined by Genotypic Assay in HIV-1 Circulating in Cuba

Introduction: R5-tropic viruses predominated at the time of initial HIV-1 infection and a switch to X4-tropic occurs in about 50% of patients in late-stage disease. Objective: To associate different variants of Cuban HIV-1 with the co-receptor use, and the implication for the use of co-receptor inhibitors. Methodology: Viral HIV-1 subtype was determined in 42 Cuban individuals using COMET V.2, Rega subtyping toolV.3 algorithms and phylogenetic analysis. Co-receptor tropism was predicted using the geno2pheno [co-receptor] algorithm. Additionally, all V3 loop HIV-1 Cuban sequences deposited previously at Los Alamos database were also analyzed for comparison of subtype and co-receptor tropism. Results: The most frequent subtypes detected were CRF20–23–24_BG (35.7%), subtypes B (33.3%) and CRF19_ cpx (14.3%) when pol and V3 regions were analyzed. Overall, 61.9% of the samples were R5 viruses and 14.3% were X4. Viruses CRF19_cpx were more often R5X4/X4 (5/6 samples, p=0.009) or X4 strains (3/6 samples, p=0.019). The additional analysis of 359 Cuban env sequences demonstrated that only 29.3% were X4 viruses. Interestingly, 43.6% of the CRF19_cpx were R5X4/X4 viruses, confirming the previous association (p=0.011). Characteristic amino acids in the V3 loop (V/T12, R13, Q18, V19, G22) were identified at higher frequencies in CRF19_cpx viruses than in subtype B (p<0.0001). Conclusion: CRF19_cpx is a genetic form with high proportion of X4-tropic viruses. This supports the increased pathogenicity of CRF19_cpx, potentially leading to rapid disease progression. The high frequency of X4 tropism in CRF19_cpx infected patients would imply that CCR5 antagonists could be ineffective in most of these patients.

HIV coreceptor tropism among patients with newly diagnosed infection

Introduction: Coreceptor tropism is the ability of human immunodeficiency virus (HIV) to use CCR5 or CXCR4 coreceptor to enter the host cell. Tropism depends on the structure of the surface glycoprotein, involved in binding molecules to receptor and coreceptor. Viruses that CCR5use are named R5, viruses that CXCR4 use are X4-tropic, and dual/mixed (DM) viruses use either of the two. During early stages of HIV infection,R5 strains are usually present and with the progression of HIV disease, frequency of X4 occurs. Aim: The aim of the study was to analyze the coreceptor tropism in newly diagnosed HIV infected patients. Material and methods: The study group included 26 patients with newly diagnosed HIV infection, in the hospital care from 2010 to 2012 at the Clinic for Infectious and Tropical diseases of the Clinical Center of Serbia. Upon RNA isolation from plasma, PCR amplification and DNA sequencing of the V3 loop of the env gene were performed. The sequences were used for prediction of the coreceptor tropism, using the Geno2pheno bioinformatics algorithms. Results: R5 and X4 tropism were detected in 21/26 (81%) and 5/26 (19%) patients, respectively. Five out of twenty-six patients (19.2%) were in the C3 clinical stadium of the disease, and all of them were infected with R5-tropic virus. Fourteen out of twenty-five patients (56%) were late presenters of HIV infection, where 12 (86%) of them were infected with R5-tropic virus. Conclusion: Our results imply that the majority of HIV strains in patients, with newly diagnosed infection in Serbia, are characterized by R5 tropism. However, the prevalence of X4 strains is not negligible, indicating late presentation of newly diagnosed patients, but also possibly implying increased virulence of the circulating strains. Considering that CCR5 antagonists would not be effective in 19% of studied patients, the prediction of coreceptor tropism is undoubtedly very important.

P-D5 Two conserved tyrosines in the HIV-1 envelope V2 loop play a critical role in gp120/gp41 association

P-D5 Two conserved tyrosines in the HIV-1 envelope V2 loop play a critical role in gp120/gp41 association

Drug resistance and tropism as markers of the dynamics of HIV-1 DNA quasispecies in blood cells of heavily pretreated patients who achieved sustained virological suppression

The objective of this study was to address the dynamics of archived resistant quasispecies in cell-associated HIV-1 DNA over time in heavily ART-experienced patients with currently suppressed plasma HIV-1 RNA. Longitudinal ultra-deep sequencing (UDS) analysis of reverse transcriptase, protease and V3 Env regions was performed on blood-cell-associated HIV-1 DNA samples. Drug-resistance-associated mutations (DRAMs) and tropism were interpreted using the ANRS and Geno2Pheno algorithms. We analysed frozen blood cells from patients enrolled in the INNOVE and ANRS 123 ETOILE studies who achieved sustained viral suppression after salvage optimized ART (SOT). Samples were available at baseline and 6 and ≥12 months after SOT initiation in 10 patients. V3 loop sequences displayed wide intra-individual dynamics over time. Viral variants harbouring DRAMs exhibited three non-exclusive scenarios. First, when SOT exerted the same selective pressure as previous failing regimens, some viral quasispecies still harboured the same DRAMs at the same level as at the time of virological failure. Thus, as DRAMs were mostly associated with the same viral variant, variants with a complete resistance pattern remained archived. Second, some viral variants harbouring DRAMs were no longer detected over time when SOT consisted of new antiretroviral classes or had resistance profiles distinct from those of previous failing regimens. Third, variants with new DRAMs associated with SOT emerged in blood cells during follow-up despite sustained virological control. Using longitudinal UDS analysis and focusing on DRAMs and tropism as markers, we demonstrated that, despite sustained virological control, archived HIV-1 DNA quasispecies continued to evolve.

Roles of glycans in interactions between gp120 and HIV broadly neutralizing antibodies

Many novel broadly neutralizing antibodies against human immunodeficiency virus (HIV) have been identified during the past decade, providing promising templates for the development of an effective HIV-1 vaccine. Structural studies reveal that the epitopes of some of these antibodies involve one or more crucial glycans, without which the binding is completely abolished. In this study, we have investigated the critical roles of glycans in interactions between HIV-1 gp120 and two broadly neutralizing antibodies PG9 (targeting V1/V2) and PGT128 (targeting V3) that are able to neutralize more than 70% of HIV-1 isolates. We have performed molecular dynamics simulations of a number of systems including antibody-gp120 complex with and without glycans, antibody, gp120 with and without glycans, and glycan-only systems. The simulation results show that the complex structures are stabilized by the glycans, and the multivalent interactions between the antibody and gp120 promote cooperativities to further enhance the binding. In the free gp120, the glycans increase the flexibility of the V1/V2 and V3 loops, which likely increases the entropy cost of the antibody recognition. However, the antibodies are able to bind the flexible interface by recognizing the preexisting glycan conformation, and penetrating the glycan shield with flexible complementarity determining region loops that sample the bound conformations occasionally.

Incompatible Natures of the HIV-1 Envelope in Resistance to the CCR5 Antagonist Cenicriviroc and to Neutralizing Antibodies.

Cenicriviroc is a CCR5 antagonist which prevents human immunodeficiency virus type 1 (HIV-1) from cellular entry. The CCR5-binding regions of the HIV-1 envelope glycoprotein are important targets for neutralizing antibodies (NAbs), and mutations conferring cenicriviroc resistance may therefore affect sensitivity to NAbs. Here, we used the in vitro induction of HIV-1 variants resistant to cenicriviroc or NAbs to examine the relationship between resistance to cenicriviroc and resistance to NAbs. The cenicriviroc-resistant variant KK652-67 (strain KK passaged 67 times in the presence of increasing concentrations of cenicriviroc) was sensitive to neutralization by NAbs against the V3 loop, the CD4-induced (CD4i) region, and the CD4-binding site (CD4bs), whereas the wild-type (WT) parental HIV-1 strain KKWT from which cenicriviroc-resistant strain KK652-67 was obtained was resistant to these NAbs. The V3 region of KK652-67 was important for cenicriviroc resistance and critical to the high sensitivity of the V3, CD4i, and CD4bs epitopes to NAbs. Moreover, induction of variants resistant to anti-V3 NAb 0.5γ and anti-CD4i NAb 4E9C from cenicriviroc-resistant strain KK652-67 resulted in reversion to the cenicriviroc-sensitive phenotype comparable to that of the parental strain, KKWT. Resistance to 0.5γ and 4E9C was caused by the novel substitutions R315K, G324R, and E381K in the V3 and C3 regions near the substitutions conferring cenicriviroc resistance. Importantly, these amino acid changes in the CCR5-binding region were also responsible for reversion to the cenicriviroc-sensitive phenotype. These results suggest the presence of key amino acid residues where resistance to cenicriviroc is incompatible with resistance to NAbs. This implies that cenicriviroc and neutralizing antibodies may restrict the emergence of variants resistant to each other.

Conserved Role of an N-Linked Glycan on the Surface Antigen of Human Immunodeficiency Virus Type 1 Modulating Virus Sensitivity to Broadly Neutralizing Antibodies against the Receptor and Coreceptor Binding Sites.

HIV-1 establishes persistent infection in part due to its ability to evade host immune responses. Occlusion by glycans contributes to masking conserved sites that are targets for some broadly neutralizing antibodies (bNAbs). Previous work has shown that removal of a highly conserved potential N-linked glycan (PNLG) site at amino acid residue 197 (N7) on the surface antigen gp120 of HIV-1 increases neutralization sensitivity of the mutant virus to CD4 binding site (CD4bs)-directed antibodies compared to its wild-type (WT) counterpart. However, it is not clear if the role of the N7 glycan is conserved among diverse HIV-1 isolates and if other glycans in the conserved regions of HIV-1 Env display similar functions. In this work, we examined the role of PNLGs in the conserved region of HIV-1 Env, particularly the role of the N7 glycan in a panel of HIV-1 strains representing different clades, tissue origins, coreceptor usages, and neutralization sensitivities. We demonstrate that the absence of the N7 glycan increases the sensitivity of diverse HIV-1 isolates to CD4bs- and V3 loop-directed antibodies, indicating that the N7 glycan plays a conserved role masking these conserved epitopes. However, the effect of the N7 glycan on virus sensitivity to neutralizing antibodies directed against the V2 loop epitope is isolate dependent. These findings indicate that the N7 glycan plays an important and conserved role modulating the structure, stability, or accessibility of bNAb epitopes in the CD4bs and coreceptor binding region, thus representing a potential target for the design of immunogens and therapeutics. N-linked glycans on the HIV-1 envelope protein have been postulated to contribute to viral escape from host immune responses. However, the role of specific glycans in the conserved regions of HIV-1 Env in modulating epitope recognition by broadly neutralizing antibodies has not been well defined. We show here that a single N-linked glycan plays a unique and conserved role among conserved glycans on HIV-1 gp120 in modulating the exposure or the stability of the receptor and coreceptor binding site without affecting the integrity of the Env in mediating viral infection or the ability of the mutant gp120 to bind to CD4. The observation that the antigenicity of the receptor and coreceptor binding sites can be modulated by a single glycan indicates that select glycan modification offers a potential strategy for the design of HIV-1 vaccine candidates.

Disulfide Sensitivity in the Env Protein Underlies Lytic Inactivation of HIV-1 by Peptide Triazole Thiols.

We investigated the mode of action underlying lytic inactivation of HIV-1 virions by peptide triazole thiol (PTT), in particular the relationship between gp120 disulfides and the C-terminal cysteine-SH required for virolysis. Obligate PTT dimer obtained by PTT SH cross-linking and PTTs with serially truncated linkers between pharmacophore isoleucine-ferrocenyltriazole-proline-tryptophan and cysteine-SH were synthesized. PTT variants showed loss of lytic activity but not binding and infection inhibition upon SH blockade. A disproportionate loss of lysis activity vs binding and infection inhibition was observed upon linker truncation. Molecular docking of PTT onto gp120 argued that, with sufficient linker length, the peptide SH could approach and disrupt several alternative gp120 disulfides. Inhibition of lysis by gp120 mAb 2G12, which binds at the base of the V3 loop, as well as disulfide mutational effects, argued that PTT-induced disruption of the gp120 disulfide cluster at the base of the V3 loop is an important step in lytic inactivation of HIV-1. Further, PTT-induced lysis was enhanced after treating virus with reducing agents dithiothreitol and tris (2-carboxyethyl)phosphine. Overall, the results are consistent with the view that the binding of PTT positions the peptide SH group to interfere with conserved disulfides clustered proximal to the CD4 binding site in gp120, leading to disulfide exchange in gp120 and possibly gp41, rearrangement of the Env spike, and ultimately disruption of the viral membrane. The dependence of lysis activity on thiol-disulfide interaction may be related to intrinsic disulfide exchange susceptibility in gp120 that has been reported previously to play a role in HIV-1 cell infection.

Rationally Targeted Mutations at the V1V2 Domain of the HIV-1 Envelope to Augment Virus Neutralization by Anti-V1V2 Monoclonal Antibodies.

HIV-1 envelope glycoproteins (Env) are the only viral antigens present on the virus surface and serve as the key targets for virus-neutralizing antibodies. However, HIV-1 deploys multiple strategies to shield the vulnerable sites on its Env from neutralizing antibodies. The V1V2 domain located at the apex of the HIV-1 Env spike is known to encompass highly variable loops, but V1V2 also contains immunogenic conserved elements recognized by cross-reactive antibodies. This study evaluates human monoclonal antibodies (mAbs) against V2 epitopes which overlap with the conserved integrin α4β7-binding LDV/I motif, designated as the V2i (integrin) epitopes. We postulate that the V2i Abs have weak or no neutralizing activities because the V2i epitopes are often occluded from antibody recognition. To gain insights into the mechanisms of the V2i occlusion, we evaluated three elements at the distal end of the V1V2 domain shown in the structure of V2i epitope complexed with mAb 830A to be important for antibody recognition of the V2i epitope. Amino-acid substitutions at position 179 that restore the LDV/I motif had minimal effects on virus sensitivity to neutralization by most V2i mAbs. However, a charge change at position 153 in the V1 region significantly increased sensitivity of subtype C virus ZM109 to most V2i mAbs. Separately, a disulfide bond introduced to stabilize the hypervariable region of V2 loop also enhanced virus neutralization by some V2i mAbs, but the effects varied depending on the virus. These data demonstrate that multiple elements within the V1V2 domain act independently and in a virus-dependent fashion to govern the antibody recognition and accessibility of V2i epitopes, suggesting the need for multi-pronged strategies to counter the escape and the shielding mechanisms obstructing the V2i Abs from neutralizing HIV-1.

Structure/Function Studies Involving the V3 Region of the HIV-1 Envelope Delineate Multiple Factors That Affect Neutralization Sensitivity.

ABSTRACTAntibodies (Abs) specific for the V3 loop of the HIV-1 gp120 envelope neutralize most tier 1 and many tier 2 viruses and are present in essentially all HIV-infected individuals as well as immunized humans and animals. Vaccine-induced V3 Abs are associated with reduced HIV infection rates in humans and affect the nature of transmitted viruses in infected vaccinees, despite the fact that V3 is often occluded in the envelope trimer. Here, we link structural and experimental data showing how conformational alterations of the envelope trimer render viruses exceptionally sensitive to V3 Abs. The experiments interrogated the neutralization sensitivity of pseudoviruses with single amino acid mutations in various regions of gp120 that were predicted to alter packing of the V3 loop in the Env trimer. The results indicate that the V3 loop is metastable in the envelope trimer on the virion surface, flickering between states in which V3 is either occluded or available for binding to chemokine receptors (leading to infection) and to V3 Abs (leading to virus neutralization). The spring-loaded V3 in the envelope trimer is easily released by disruption of the stability of the V3 pocket in the unliganded trimer or disruption of favorable V3/pocket interactions. Formation of the V3 pocket requires appropriate positioning of the V1V2 domain, which is, in turn, dependent on the conformation of the bridging sheet and on the stability of the V1V2 B-C strand-connecting loop.IMPORTANCE The levels of antibodies to the third variable region (V3) of the HIV envelope protein correlate with reduced HIV infection rates. Previous studies showed that V3 is often occluded, as it sits in a pocket of the envelope trimer on the surface of virions; however, the trimer is flexible, allowing occluded portions of the envelope (like V3) to flicker into an exposed position that binds antibodies. Here we provide a systematic interrogation of mechanisms by which single amino acid changes in various regions of gp120 (i) render viruses sensitive to neutralization by V3 antibodies, (ii) result in altered packing of the V3 loop, and (iii) activate an open conformation that exposes V3 to the effects of V3 Abs. Taken together, these and previous studies explain how V3 antibodies can protect against HIV-1 infection and why they should be one of the targets of vaccine-induced antibodies.

Reactivation of Neutralized HIV-1 by Dendritic Cells Is Dependent on the Epitope Bound by the Antibody

Ab-neutralized HIV-1 can be captured by dendritic cells (DCs), which subsequently transfer infectious HIV-1 to susceptible CD4(+) T cells. In this study, we examined the capacity of early Abs, as well as recently identified broadly neutralizing Abs (bNAbs) targeting different envelope glycoprotein (Env) epitopes, to block HIV-1 transmission by immature and mature DCs to HIV-1-sensitive cells. Three bNAbs directed against the gp41 membrane proximal region of Env (2F5, 4E10, and 10E8) and three gp120 bNAbs targeting the CD4 binding site (b12, VRC01, and NIH45-46) were examined. In addition, eight glycan-dependent bNAbs targeting the V1V2 apex (PG9, PG16, and PGT145), the V3 loop (2G12, PGT121, and PGT128), and the gp120-gp41 interface of Env (PGT151 and 35O22) were tested. bNAbs that bound specific glycans showed, depending on the immature or mature state of the DC, diverse efficiencies in HIV-1 trans-infection. All bNAbs that bound the CD4 binding site blocked trans-infection, whereas all bNAbs directed against the membrane proximal region lost neutralizing activity after DC-mediated HIV-1 transmission. To understand how preneutralized HIV-1 can be transferred as infectious virus by DCs, we followed the processing of 2F5-treated HIV-1 by DCs with confocal microscopy. Inhibition of DC-internalization pathways could not reverse the dissociation of 2F5 from HIV-1, suggesting that Ab dissociation occurs directly at the plasma membrane. Collectively, these findings imply that the location of the epitope and the neutralization capacity of these Abs determine the efficiency of DC-mediated HIV-1 transfer.

The Evolving HIV-1 Epidemic in Warao Amerindians Is Dominated by an Extremely High Frequency of CXCR4-Utilizing Strains

We previously reported a high prevalence of HIV-1 infection in Warao Amerindians from Venezuela due to the rapid spread of a single B subtype strain. In this study we evaluated the coreceptor use of the HIV-1 strains infecting this Amerindian community. Sequences of the HIV-1 V3 loop from 56 plasma samples were genotyped for coreceptor use. An extremely high frequency of CXCR4 strains was found among HIV-1-infecting Waraos (47/49, 96%), compared to HIV-1 strains infecting the non-Amerindian Venezuelan population (35/79, 44%, p < 0.00001). Evolutionary analysis showed that a significant number of infections occurred between 1 and 12 months before collection and that a great proportion (50-70%) of HIV-1 transmissions occurred within the very early phase of infection (≤12 months). This is consistent with an initial infection dominated by an X4 strain or a very rapid selection of X4 variants after infection. This Amerindian population also exhibits the highest prevalence of tuberculosis in Venezuela, being synergistically bad prognostic factors for the evolution of morbidity and mortality in this vulnerable population.

Paradox of Protection: Preferential Recognition of CD4-induced Epitopes by Anti-HIV-1 ADCC Antibodies

Paradox of Protection: Preferential Recognition of CD4-induced Epitopes by Anti-HIV-1 ADCC Antibodies

Design and structure of two HIV-1 clade C SOSIP.664 trimers that increase the arsenal of native-like Env immunogens

A key challenge in the quest toward an HIV-1 vaccine is design of immunogens that can generate a broadly neutralizing antibody (bnAb) response against the enormous sequence diversity of the HIV-1 envelope glycoprotein (Env). We previously demonstrated that a recombinant, soluble, fully cleaved SOSIP.664 trimer based on the clade A BG505 sequence is a faithful antigenic and structural mimic of the native trimer in its prefusion conformation. Here, we sought clade C native-like trimers with comparable properties. We identified DU422 and ZM197M SOSIP.664 trimers as being appropriately thermostable (Tm of 63.4 °C and 62.7 °C, respectively) and predominantly native-like, as determined by negative-stain electron microscopy (EM). Size exclusion chromatography, ELISA, and surface plasmon resonance further showed that these trimers properly display epitopes for all of the major bnAb classes, including quaternary-dependent, trimer-apex (e.g., PGT145) and gp120/gp41 interface (e.g., PGT151) epitopes. A cryo-EM reconstruction of the ZM197M SOSIP.664 trimer complexed with VRC01 Fab against the CD4 binding site at subnanometer resolution revealed a striking overall similarity to its BG505 counterpart with expected local conformational differences in the gp120 V1, V2, and V4 loops. These stable clade C trimers contribute additional diversity to the pool of native-like Env immunogens as key components of strategies to induce bnAbs to HIV-1.

Characterizing the Diverse Mutational Pathways Associated with R5-Tropic Maraviroc Resistance: HIV-1 That Uses the Drug-Bound CCR5 Coreceptor.

ABSTRACTEntry inhibitors represent a potent class of antiretroviral drugs that target a host cell protein, CCR5, an HIV-1 entry coreceptor, and not viral protein. Lack of sensitivity can occur due to preexisting virus that uses the CXCR4 coreceptor, while true resistance occurs through viral adaptation to use a drug-bound CCR5 coreceptor. To understand this R5 resistance pathway, we analyzed >500 envelope protein sequences and phenotypes from viruses of 20 patients from the clinical trials MOTIVATE 1 and 2, in which treatment-experienced patients received maraviroc plus optimized background therapy. The resistant viral population was phylogenetically distinct and associated with a genetic bottleneck in each patient, consistent with de novo emergence of resistance. Recombination analysis showed that the C2-V3-C3 region tends to genotypically correspond to the recombinant's phenotype, indicating its primary importance in conferring resistance. Between patients, there was a notable lack of commonality in the specific sites conferring resistance, confirming the unusual nature of R5-tropic resistance. We used coevolutionary and positive-selection analyses to characterize the genotypic determinants of resistance and found that (i) there are complicated covariation networks, indicating frequent coevolutionary/compensatory changes in the context of protein structure; (ii) covarying sites under positive selection are enriched in resistant viruses; (iii) CD4 binding sites form part of a unique covariation network independent of the V3 loop; and (iv) the covariation network formed between the V3 loop and other regions of gp120 and gp41 intersects sites involved in glycosylation and protein secretion. These results demonstrate that while envelope sequence mutations are the key to conferring maraviroc resistance, the specific changes involved are context dependent and thus inherently unpredictable.IMPORTANCE The entry inhibitor drug maraviroc makes the cell coreceptor CCR5 unavailable for use by HIV-1 and is now used in combination antiretroviral therapy. Treatment failure with drug-resistant virus is particularly interesting because it tends to be rare, with lack of sensitivity usually associated with the presence of CXCR4-using virus (CXCR4 is the main alternative coreceptor HIV-1 uses, in addition to CD4). We analyzed envelope sequences from HIV-1, obtained from 20 patients who enrolled in maraviroc clinical trials and experienced treatment failure, without detection of CXCR4-using virus. Evolutionary analysis was employed to identify molecular changes that confer maraviroc resistance. We found that in these individuals, resistant viruses form a distinct population that evolved once and was successful as a result of drug pressure. Further evolutionary analysis placed the complex network of interdependent mutational changes into functional groups that help explain the impediments to the emergence of maraviroc-associated R5 drug resistance.

Rapid screening of anti-HIV ingredients in Artemisia rupestris L. extracts interacting with V3 loop region of HIV-1 gp120 and reverse transcriptase by affinity capillary electrophoresis and capillary zone electrophoresis

Rapid screening of anti-HIV ingredients in Artemisia rupestris L. extracts interacting with V3 loop region of HIV-1 gp120 and reverse transcriptase by affinity capillary electrophoresis and capillary zone electrophoresis

O17.2 Characterising clade-specific virus-host interactions in hiv infected clinically asymptomatic and aids presenting subjects

<h3>Introduction</h3> The utilised co-receptor is indicative of the clinical progression in HIV infected subjects. Differences in clades are known to impact the outcome of HIV infection. In this study, we investigated the utilised co-receptor and N-glycosylation sites in clinically asymptomatic and AIDS presenting subjects. <h3>Materials and methods</h3> A total of 1,538 nucleotide sequences encompassing the hyper-variable V3 loop of HIV-1, from clinically asymptomatic and AIDS presenting subjects were downloaded from the Los Alamaos Database, which belonged to clades A, B, C and D of HIV-1. Co-receptor prediction was performed using web-based tools <i>PSSM</i> and <i>(ds) Kernel</i>. Numbers of N-glycosylation sites were also calculated using the ‘<i>N-glycosite</i>’ tool. <h3>Results</h3> CCR5 was the utilised co-receptor in 97% (n = 200) of asymptomatic individuals of clade A and 96.5% (n = 199) of AIDS presenting subjects. In B-clade, 98.9% (n = 194) subjects in asymptomatic group were CCR5 utilising, and 83.5% of AIDS presenting subjects were CCR5 utilising (n = 163, CXCR4 were 22.3%, n = 47). In C-clade the CCR5 was utilised in 193 subjects (asymptomatic, n = 200), and 142 (AIDS presenting, n = 148) utilised both co-receptors (dual co-tropic), and in D clade the co-receptor utilised in 55% subjects was CCR5, n = 154 (CXCR4 in 45% subjects, n = 126), and 81% (n = 198) AIDS presenting subjects utilised CCR5, and 19% utilised CXCR4. Percentage of subjects exhibiting N-glycosylation sites also varied among clades with decrease in number of sites in some and increase in others, when compared between the two clinical categories. <h3>Conclusions</h3> Co-receptor switching and addition of N-glycosylation sites does not seem to occur universally in all clades studied. The number of N-Glycosylation sites is also not increased from clinically asymptomatic to AIDS presenting subjects. In conclusion, co-receptor switching (from CCR5 to CXCR4) and increase in number of N-glycosylation sites, which are predictive of disease progression, does to occur in all clades universally, thus indicating clade specific responses.

MiR-133b-5p regulates the expression of the heat shock protein 70 during rat neuronal cell apoptosis induced by the gp120 V3 loop peptide.

Neuronal cell dysfunction and apoptosis, the main causes of HIV-associated dementia, and its underlying mechanism are important unsolved health problems. Many research reports suggest that miRNAs regulate HIV-1-induced apoptosis. We used the HIV-1 gp120 V3 Loop peptide to induce primary rat cortical neurons apoptosis. Next, we used a microRNA microarray to identify the significant changes of miRNA in the rat cortical neurons treated with the gp120 V3 loop peptide. We used western blot and real-time PCR to measure the regulation of heat shock protein 70 by rno-miR-133b-5p. In response to the gp120 V3 loop peptide treatment, rat cortical neurons exhibited 11 up-regulated and 21 down-regulated miRNAs. We further examined miR-133b-5p, a microRNA that was up-regulated more than 118-fold. In addition, both HSP70 mRNA and protein expression were dose-dependent in rats cortical neurons treated with gp120 V3 loop peptide for 48 hr. MiR-133b-5p could regulate heat shock protein 70 (HSP70) at both transcription and translation levels. Rno-miR-133b-5p might be less significant for the gp120 V3 loop peptide induced neuron apoptosis. Thus, we discovered a potential new target for the regulation of HIV-1 gp120- induced apoptosis.

Curcumin Increases HSP70 Expression in Primary Rat Cortical Neuronal Apoptosis Induced by gp120 V3 Loop Peptide

Neuronal cell dysfunction and apoptosis are the main causes of the invasion of the central nervous system by human immunodeficiency virus type 1 (HIV-1), although the underlying mechanism has not been well understood. Recent research has shown that curcumin might play an important role in regulating HIV-1 development. Heat shock protein 70 (HSP70), a protein induced by heat, was reported to inhibit apoptosis through various cell signaling pathways in brain. Overexpression of HSP70 could effectively protected neurons in many animal and cellular models of dementia. In the present study, the expression of HSP70 in the gp120 V3 loop peptide-induced neuronal apoptosis was investigated. Our results demonstrated that gp120 V3 loop peptide could induce primary rat cortical neuronal apoptosis. We also found that curcumin could increase HSP70 expression. In addition, the expression level of both HSP70 mRNA and HSP70 protein were dependent on the curcumin dose in the rat cortical neurons. Curcumin could improve HSP70 expression in gp120 V3 loop peptide-induced primary rat cortical neuronal apoptosis. In general, our results indicated that curcumin played an important role in the gp120 V3 loop peptide induced neuronal apoptosis by regulating HSP70.

Short Communication: HIV-1 Variants That Use Mouse CCR5 Reveal Critical Interactions of gp120's V3 Crown with CCR5 Extracellular Loop 1.

The CCR5 coreceptor amino terminus and extracellular (ECL) loops 1 and 2 have been implicated in HIV-1 infections, with species differences in these regions inhibiting zoonoses. Interactions of gp120 with CD4 and CCR5 reduce constraints on metastable envelope subunit gp41, enabling gp41 conformational changes needed for infection. We previously selected HIV-1JRCSF variants that efficiently use CCR5(Δ18) with a deleted amino terminus or CCR5(HHMH) with ECL2 from an NIH/Swiss mouse. Unexpectedly, the adaptive gp120 mutations were nearly identical, suggesting that they function by weakening gp120's grip on gp41 and/or by increasing interactions with ECL1. To analyze this and further wean HIV-1 from human CCR5, we selected variants using CCR5(HMMH) with murine ECL1 and 2 sequences. HIV-1JRCSF mutations adaptive for CCR5(Δ18) and CCR5(HHMH) were generally maladaptive for CCR5(HMMH), whereas the converse was true for CCR5(HMMH) adaptations. The HIV-1JRCSF variant adapted to CCR5(HMMH) also weakly used intact NIH/Swiss mouse CCR5. Our results strongly suggest that HIV-1JRCSF makes functionally critical contacts with human ECL1 and that adaptation to murine ECL1 requires multiple mutations in the crown of gp120's V3 loop.