Single amino acid change in gp41 region of HIV-1 alters bystander apoptosis and CD4 decline in humanized mice

<p>Abstract</p> <p>Background</p> <p>The mechanism by which HIV infection leads to a selective depletion of CD4 cells leading to immunodeficiency remains highly debated. Whether the loss of CD4 cells is a direct consequence of virus infection or bystander apoptosis of uninfected cells is also uncertain.</p> <p>Results</p> <p>We have addressed this issue in the humanized mouse model of HIV infection using a HIV variant with a point mutation in the gp41 region of the Env glycoprotein that alters its fusogenic activity. We demonstrate here that a single amino acid change (V38E) altering the cell-to-cell fusion activity of the Env minimizes CD4 loss in humanized mice without altering viral replication. This differential pathogenesis was associated with a lack of bystander apoptosis induction by V38E virus even in the presence of similar levels of infected cells. Interestingly, immune activation was observed with both WT and V38E infection suggesting that the two phenomena are likely not interdependent in the mouse model.</p> <p>Conclusions</p> <p>We conclude that Env fusion activity is one of the determinants of HIV pathogenesis and it may be possible to attenuate HIV by targeting gp41.</p

Asymmetry of Early Endosome Distribution in C. elegans Embryos

development, we examined the distribution and dynamics of early endosomes (EEs) in embryos.EEs are primarily found at the cell periphery with an initially uniform distribution after fertilization. Strikingly, we find that during the first cell cycle, EEA-1 positive EEs become enriched at the anterior cortex. In contrast, the Golgi compartment shows no asymmetry in distribution. Asymmetric enrichment of EEs depends on acto-myosin contractility and embryonic PAR polarity. In addition to their localization at the cortex, EEs are also found around the centrosome. These EEs move rapidly (1.3um/s) from the cortex directly to the centrosome, a speed comparable to that of the minus end directed motor dynein.We speculate that the asymmetry of early endosomes might play a role in cell asymmetries or fate decisions

Viremic HIV Infected Individuals with High CD4 T Cells and Functional Envelope Proteins Show Anti-gp41 Antibodies with Unique Specificity and Function

BACKGROUND: CD4 T-cell decay is variable among HIV-infected individuals. In exceptional cases, CD4 T-cell counts remain stable despite high plasma viremia. HIV envelope glycoprotein (Env) properties, namely tropism, fusion or the ability to induce the NK ligand NKp44L, or host factors that modulate Env cytopathic mechanisms may be modified in such situation. METHODS: We identified untreated HIV-infected individuals showing non-cytopathic replication (VL>10,000 copies/mL and CD4 T-cell decay<50 cells/µL/year, Viremic Non Progressors, VNP) or rapid progression (CD4 T-cells<350 cells/µL within three years post-infection, RP). We isolated full-length Env clones and analyzed their functions (tropism, fusion activity and capacity to induce NKp44L expression on CD4 cells). Anti-Env humoral responses were also analyzed. RESULTS: Env clones isolated from VNP or RP individuals showed no major phenotypic differences. The percentage of functional clones was similar in both groups. All clones tested were CCR5-tropic and showed comparable expression and fusogenic activity. Moreover, no differences were observed in their capacity to induce NKp44L expression on CD4 T cells from healthy donors through the 3S epitope of gp41. In contrast, anti- Env antibodies showed clear functional differences: plasma from VNPs had significantly higher capacity than RPs to block NKp44L induction by autologous viruses. Consistently, CD4 T-cells isolated from VNPs showed undetectable NKp44L expression and specific antibodies against a variable region flanking the highly conserved 3S epitope were identified in plasma samples from these patients. Conversely, despite continuous antigen stimulation, VNPs were unable to mount a broad neutralizing response against HIV. CONCLUSIONS: Env functions (fusion and induction of NKp44L) were similar in viremic patients with slow or rapid progression to AIDS. However, differences in humoral responses against gp41 epitopes nearby 3S sequence may contribute to the lack of CD4 T cell decay in VNPs by blocking the induction of NKp44L by gp41

Escape from Autologous Neutralizing Antibodies in Acute/Early Subtype C HIV-1 Infection Requires Multiple Pathways

One aim for an HIV vaccine is to elicit neutralizing antibodies (Nab) that can limit replication of genetically diverse viruses and prevent establishment of a new infection. Thus, identifying the strengths and weaknesses of Nab during the early stages of natural infection could prove useful in achieving this goal. Here we demonstrate that viral escape readily occurred despite the development of high titer autologous Nab in two subjects with acute/early subtype C infection. To provide a detailed portrayal of the escape pathways, Nab resistant variants identified at multiple time points were used to create a series of envelope (Env) glycoprotein chimeras and mutants within the background of a corresponding newly transmitted Env. In one subject, Nab escape was driven predominantly by changes in the region of gp120 that extends from the beginning of the V3 domain to the end of the V5 domain (V3V5). However, Nab escape pathways in this subject oscillated and at times required cooperation between V1V2 and the gp41 ectodomain. In the second subject, escape was driven by changes in V1V2. This V1V2-dependent escape pathway was retained over time, and its utility was reflected in the virus's ability to escape from two distinct monoclonal antibodies (Mabs) derived from this same patient via introduction of a single potential N-linked glycosylation site in V2. Spatial representation of the sequence changes in gp120 suggested that selective pressure acted upon the same regions of Env in these two subjects, even though the Env domains that drove escape were different. Together the findings argue that a single mutational pathway is not sufficient to confer escape in early subtype C HIV-1 infection, and support a model in which multiple strategies, including potential glycan shifts, direct alteration of an epitope sequence, and cooperative Env domain conformational masking, are used to evade neutralization

R5 Clade C SHIV Strains with Tier 1 or 2 Neutralization Sensitivity: Tools to Dissect Env Evolution and to Develop AIDS Vaccines in Primate Models

Background: HIV-1 clade C (HIV-C) predominates worldwide, and anti-HIV-C vaccines are urgently needed. Neutralizing antibody (nAb) responses are considered important but have proved difficult to elicit. Although some current immunogens elicit antibodies that neutralize highly neutralization-sensitive (tier 1) HIV strains, most circulating HIVs exhibiting a less sensitive (tier 2) phenotype are not neutralized. Thus, both tier 1 and 2 viruses are needed for vaccine discovery in nonhuman primate models. Methodology/Principal Findings: We constructed a tier 1 simian-human immunodeficiency virus, SHIV-1157ipEL, by inserting an “early,” recently transmitted HIV-C env into the SHIV-1157ipd3N4 backbone [1] encoding a “late” form of the same env, which had evolved in a SHIV-infected rhesus monkey (RM) with AIDS. SHIV-1157ipEL was rapidly passaged to yield SHIV-1157ipEL-p, which remained exclusively R5-tropic and had a tier 1 phenotype, in contrast to “late” SHIV-1157ipd3N4 (tier 2). After 5 weekly low-dose intrarectal exposures, SHIV-1157ipEL-p systemically infected 16 out of 17 RM with high peak viral RNA loads and depleted gut CD4$^+$ T cells. SHIV-1157ipEL-p and SHIV-1157ipd3N4 env genes diverge mostly in V1/V2. Molecular modeling revealed a possible mechanism for the increased neutralization resistance of SHIV-1157ipd3N4 Env: V2 loops hindering access to the CD4 binding site, shown experimentally with nAb b12. Similar mutations have been linked to decreased neutralization sensitivity in HIV-C strains isolated from humans over time, indicating parallel HIV-C Env evolution in humans and RM. Conclusions/Significance: SHIV-1157ipEL-p, the first tier 1 R5 clade C SHIV, and SHIV-1157ipd3N4, its tier 2 counterpart, represent biologically relevant tools for anti-HIV-C vaccine development in primates

Increased Sensitivity to Broadly Neutralizing Antibodies of End-Stage Disease R5 HIV-1 Correlates with Evolution in Env Glycosylation and Charge

BACKGROUND: Induction of broadly neutralizing antibodies, such as the monoclonal antibodies IgGb12, 2F5 and 2G12, is the objective of most antibody-based HIV-1 vaccine undertakings. However, despite the relative conserved nature of epitopes targeted by these antibodies, mechanisms underlying the sensitivity of circulating HIV-1 variants to broadly neutralizing antibodies are not fully understood. Here we have studied sensitivity to broadly neutralizing antibodies of HIV-1 variants that emerge during disease progression in relation to molecular alterations in the viral envelope glycoproteins (Env), using a panel of primary R5 HIV-1 isolates sequentially obtained before and after AIDS onset. PRINCIPAL FINDINGS: HIV-1 R5 isolates obtained at end-stage disease, after AIDS onset, were found to be more sensitive to neutralization by TriMab, an equimolar mix of the IgGb12, 2F5 and 2G12 antibodies, than R5 isolates from the chronic phase. The increased sensitivity correlated with low CD4(+) T cell count at time of virus isolation and augmented viral infectivity. Subsequent sequence analysis of multiple env clones derived from the R5 HIV-1 isolates revealed that, concomitant with increased TriMab neutralization sensitivity, end-stage R5 variants displayed envelope glycoproteins (Envs) with reduced numbers of potential N-linked glycosylation sites (PNGS), in addition to increased positive surface charge. These molecular changes in Env also correlated to sensitivity to neutralization by the individual 2G12 monoclonal antibody (mAb). Furthermore, results from molecular modeling suggested that the PNGS lost at end-stage disease locate in the proximity to the 2G12 epitope. CONCLUSIONS: Our study suggests that R5 HIV-1 variants with increased sensitivity to broadly neutralizing antibodies, including the 2G12 mAb, may emerge in an opportunistic manner during severe immunodeficiency as a consequence of adaptive molecular Env changes, including loss of glycosylation and gain of positive charge

Critical Involvement of the ATM-Dependent DNA Damage Response in the Apoptotic Demise of HIV-1-Elicited Syncytia

DNA damage can activate the oncosuppressor protein ataxia telangiectasia mutated (ATM), which phosphorylates the histone H2AX within characteristic DNA damage foci. Here, we show that ATM undergoes an activating phosphorylation in syncytia elicited by the envelope glycoprotein complex (Env) of human immunodeficiency virus-1 (HIV-1) in vitro. This was accompanied by aggregation of ATM in discrete nuclear foci that also contained phospho-histone H2AX. DNA damage foci containing phosphorylated ATM and H2AX were detectable in syncytia present in the brain or lymph nodes from patients with HIV-1 infection, as well as in a fraction of blood leukocytes, correlating with viral status. Knockdown of ATM or of its obligate activating factor NBS1 (Nijmegen breakage syndrome 1 protein), as well as pharmacological inhibition of ATM with KU-55933, inhibited H2AX phosphorylation and prevented Env-elicited syncytia from undergoing apoptosis. ATM was found indispensable for the activation of MAP kinase p38, which catalyzes the activating phosphorylation of p53 on serine 46, thereby causing p53 dependent apoptosis. Both wild type HIV-1 and an HIV-1 mutant lacking integrase activity induced syncytial apoptosis, which could be suppressed by inhibiting ATM. HIV-1-infected T lymphoblasts from patients with inactivating ATM or NBS1 mutations also exhibited reduced syncytial apoptosis. Altogether these results indicate that apoptosis induced by a fusogenic HIV-1 Env follows a pro-apoptotic pathway involving the sequential activation of ATM, p38MAPK and p53

Structure Activity Relationship of Dendrimer Microbicides with Dual Action Antiviral Activity

Topical microbicides, used by women to prevent the transmission of HIV and other sexually transmitted infections are urgently required. Dendrimers are highly branched nanoparticles being developed as microbicides. However, the anti-HIV and HSV structure-activity relationship of dendrimers comprising benzyhydryl amide cores and lysine branches, and a comprehensive analysis of their broad-spectrum anti-HIV activity and mechanism of action have not been published.Dendrimers with optimized activity against HIV-1 and HSV-2 were identified with respect to the number of lysine branches (generations) and surface groups. Antiviral activity was determined in cell culture assays. Time-of-addition assays were performed to determine dendrimer mechanism of action. In vivo toxicity and HSV-2 inhibitory activity were evaluated in the mouse HSV-2 susceptibility model. Surface groups imparting the most potent inhibitory activity against HIV-1 and HSV-2 were naphthalene disulfonic acid (DNAA) and 3,5-disulfobenzoic acid exhibiting the greatest anionic charge and hydrophobicity of the seven surface groups tested. Their anti-HIV-1 activity did not appreciably increase beyond a second-generation dendrimer while dendrimers larger than two generations were required for potent anti-HSV-2 activity. Second (SPL7115) and fourth generation (SPL7013) DNAA dendrimers demonstrated broad-spectrum anti-HIV activity. However, SPL7013 was more active against HSV and blocking HIV-1 envelope mediated cell-to-cell fusion. SPL7013 and SPL7115 inhibited viral entry with similar potency against CXCR4-(X4) and CCR5-using (R5) HIV-1 strains. SPL7013 was not toxic and provided at least 12 h protection against HSV-2 in the mouse vagina.Dendrimers can be engineered with optimized potency against HIV and HSV representing a unique platform for the controlled synthesis of chemically defined multivalent agents as viral entry inhibitors. SPL7013 is formulated as VivaGel(R) and is currently in clinical development to provide protection against HIV and HSV. SPL7013 could also be combined with other microbicides

G-protein signaling: back to the future

Heterotrimeric G-proteins are intracellular partners of G-protein-coupled receptors (GPCRs). GPCRs act on inactive Gα·GDP/Gβγ heterotrimers to promote GDP release and GTP binding, resulting in liberation of Gα from Gβγ. Gα·GTP and Gβγ target effectors including adenylyl cyclases, phospholipases and ion channels. Signaling is terminated by intrinsic GTPase activity of Gα and heterotrimer reformation — a cycle accelerated by ‘regulators of G-protein signaling’ (RGS proteins). Recent studies have identified several unconventional G-protein signaling pathways that diverge from this standard model. Whereas phospholipase C (PLC) β is activated by Gαq and Gβγ, novel PLC isoforms are regulated by both heterotrimeric and Ras-superfamily G-proteins. An Arabidopsis protein has been discovered containing both GPCR and RGS domains within the same protein. Most surprisingly, a receptor-independent Gα nucleotide cycle that regulates cell division has been delineated in both Caenorhabditis elegans and Drosophila melanogaster. Here, we revisit classical heterotrimeric G-protein signaling and explore these new, non-canonical G-protein signaling pathways