Direct interaction of HIV gp120 with neuronal CXCR4 and CCR5 receptors induces cofilin-actin rod pathology via a cellular prion protein- and NOX-dependent mechanism

Nearly 50% of individuals with long-term HIV infection are affected by the onset of progressive HIV-associated neurocognitive disorders (HAND). HIV infiltrates the central nervous system (CNS) early during primary infection where it establishes persistent infection in microglia (resident macrophages) and astrocytes that in turn release inflammatory cytokines, small neurotoxic mediators, and viral proteins. While the molecular mechanisms underlying pathology in HAND remain poorly understood, synaptodendritic damage has emerged as a hallmark of HIV infection of the CNS. Here, we report that the HIV viral envelope glycoprotein gp120 induces the formation of aberrant, rod-shaped cofilin-actin inclusions (rods) in cultured mouse hippocampal neurons via a signaling pathway common to other neurodegenerative stimuli including oligomeric, soluble amyloid-β and proinflammatory cytokines. Previous studies showed that synaptic function is impaired preferentially in the distal proximity of rods within dendrites. Our studies demonstrate gp120 binding to either chemokine co-receptor CCR5 or CXCR4 is capable of inducing rod formation, and signaling through this pathway requires active NADPH oxidase presumably through the formation of superoxide (O2-) and the expression of cellular prion protein (PrPC). These findings link gp120-mediated oxidative stress to the generation of rods, which may underlie early synaptic dysfunction observed in HAND.

SERINC5 Inhibits HIV-1 Infectivity by Altering the Conformation of gp120 on HIV-1 Particles

ABSTRACT SERINC5 is a 10-transmembrane-domain cellular protein that is incorporated into budding HIV-1 particles and reduces HIV-1 infectivity by inhibiting virus-cell fusion. HIV-1 susceptibility to SERINC5 is determined by sequences in the viral Env glycoprotein gp120, and the antiviral effect of SERINC5 is counteracted by the viral accessory protein Nef. While the precise mechanism by which SERINC5 inhibits HIV-1 infectivity is unclear, previous studies have suggested that SERINC5 affects Env conformation. To define the effects of SERINC5 on Env conformation, we quantified the binding of HIV-1 particles to immobilized Env-specific monoclonal antibodies. We observed that SERINC5 reduced the binding of HIV-1 particles bearing a SERINC5-susceptible Env to antibodies that recognize the V3 loop, a soluble CD4 (sCD4)-induced epitope, and an N-linked glycan. In contrast, SERINC5 did not alter the capture of HIV-1 particles bearing the SERINC5-resistant Env protein. Moreover, the effect of SERINC5 on antibody-dependent virus capture was abrogated by Nef expression. Our results indicate that SERINC5 inhibits HIV-1 infectivity by altering the conformation of gp120 on virions and/or physical masking of specific HIV-1 Env epitopes. IMPORTANCE SERINC5 is a host cell protein that inhibits the infectivity of HIV-1 by a novel and poorly understood mechanism. Here, we provide evidence that the SERINC5 protein alters the conformation of the HIV-1 Env proteins and that this action is correlated with SERINC5’s ability to inhibit HIV-1 infectivity. Defining the specific effects of SERINC5 on the HIV-1 glycoprotein conformation may be useful for designing new antiviral strategies targeting Env.

In Silico Selection of Gp120 ssDNA Aptamer to HIV-1

Nucleic acid aptamers that specifically bind to other molecules are mostly obtained through the systematic evolution of ligands by exponential enrichment (SELEX). Because SELEX is a time-consuming procedure, the in silico design of specific aptamers has recently become a progressive approach. HIV-1 surface glycoprotein gp120, which is involved in the early stages of HIV-1 infection, is an attractive target for RNA and DNA aptamer selection. In this study, four single-stranded DNA aptamers, referred to as HD2, HD3, HD4, and HD5, that had the ability of HIV-1 inhibition were designed in silico. In a proposed non-SELEX approach, some parts of the B40 aptamer sequence, which interacted with gp120, were isolated and considered as a separate aptamer sequence. Then, to obtain the best docking scores of the HDOCK server and Hex software, some modifications, insertions, and deletions were applied to each selected sequence. Finally, the cytotoxicity and HIV inhibition of the selected aptamers were evaluated experimentally. Results demonstrated that the selected aptamers could inhibit HIV-1 infection by up to 80%, without any cytotoxicity. Therefore, this new non-SELEX approach could be considered a simple, fast, and efficient method for aptamer selection.

A versatile high throughput strategy for cloning the env gene of HIV-1

The trimeric envelope glycoprotein (gp120/gp41)3 of human immunodeficiency virus-1 (HIV-1) mediates viral and host cell membrane fusion, initiated by binding of viral envelope gp120 protein to the CD4 receptor on host immune cells. Functional env genes from infected individuals have been widely used as templates for vaccine design, for setting up viral neutralization assays and to study the viral evolution and pathogenesis. Traditional topoisomerase or T4 DNA polymerase mediated approaches for cloning single genome amplified (SGA) env genes are labor-intensive, cost-ineffective with low-throughput, thereby enabling functional analysis of only a limited number of env genes from the diverse circulating quasispecies in infected individuals. Herein, we report an efficient, easy to optimize and high-throughput approach for cloning diverse HIV-1 env genes. Multiple env/rev gene cassettes, derived from infected infants, were subjected to SGA using Phusion polymerase and utilized as megaprimers in overlap extension PCR mediated cloning (OEC), circumventing the requirement for novel enzymes. Furthermore, utilization of Phusion polymerase for both the amplification of env/rev cassettes and OEC allows convenient monitoring and optimization, thereby providing much greater flexibility and versatility for analysis of env genes from HIV-1 infected individuals.

Probing intrinsic dynamics and conformational transition of HIV gp120 by molecular dynamics simulation

The HIV envelope glycoprotein gp120 has evolved two distinct conformational states to balance viral infection and immune escape.

Serine substitutions are linked to codon usage and differ for variable and conserved protein regions

Serine is the only amino acid that is encoded by two disjoint codon sets (TCN & AGY) so that a tandem substitution of two nucleotides is required to switch between the two sets. We show that these codon sets underlie distinct substitution patterns at positions subject to purifying and diversifying selections. We found that in humans, positions that are conserved among ~100 vertebrates, and thus subjected to purifying selection, are enriched for substitutions involving serine (TCN, denoted S′), proline, and alanine, (S′PA). In contrast, the less conserved positions are enriched for serine encoded with AGY codons (denoted S″), glycine and asparagine, (GS″N). We tested this phenomenon in the HIV envelope glycoprotein (gp120), and the V-gene that encodes B-cell receptors/antibodies. These fast evolving proteins both have hypervariable positions, which are under diversifying selection, closely adjacent to highly conserved structural regions. In both instances, we identified an opposite abundance of two groups of serine substitutions, with enrichment of S′PA in the conserved positions, and GS″N in the hypervariable regions. Finally, we analyzed the substitutions across 60,000 individual human exomes to show that, when serine has a specific functional constraint of phosphorylation capability, S′ codons are 32-folds less prone than S″ to substitutions to Threonine or Tyrosine that could potentially retain the phosphorylation site capacity. Combined, our results, that cover evolutionary signals at different temporal scales, demonstrate that through its encoding by two codon sets, serine allows for the existence of alternating substitution patterns within positions of functional maintenance versus sites of rapid diversification.

Tenofovir disoproxil fumarate induces peripheral neuropathy and alters inflammation and mitochondrial biogenesis in the brains of mice

Mounting evidence suggests that antiretroviral therapy (ART) drugs may contribute to the prevalence of HIV-associated neurological dysfunction. The HIV envelope glycoprotein (gp120) is neurotoxic and has been linked to alterations in mitochondrial function and increased inflammatory gene expression, which are common neuropathological findings in HIV+ cases on ART with neurological disorders. Tenofovir disproxil fumarate (TDF) has been shown to affect neurogenesis in brains of mice and mitochondria in neurons. In this study, we hypothesized that TDF contributes to neurotoxicity by modulating mitochondrial biogenesis and inflammatory pathways. TDF administered to wild-type (wt) and GFAP-gp120 transgenic (tg) mice caused peripheral neuropathy, as indicated by nerve conduction slowing and thermal hyperalgesia. Conversely TDF protected gp120-tg mice from cognitive dysfunction. In the brains of wt and gp120-tg mice, TDF decreased expression of mitochondrial transcription factor A (TFAM). However, double immunolabelling revealed that TFAM was reduced in neurons and increased in astroglia in the hippocampi of TDF-treated wt and gp120-tg mice. TDF also increased expression of GFAP and decreased expression of IBA1 in the wt and gp120-tg mice. TDF increased tumor necrosis factor (TNF) α in wt mice. However, TDF reduced interleukin (IL) 1β and TNFα mRNA in gp120-tg mouse brains. Primary human astroglia were exposed to increasing doses of TDF for 24 hours and then analyzed for mitochondrial alterations and inflammatory gene expression. In astroglia, TDF caused a dose-dependent increase in oxygen consumption rate, extracellular acidification rate and spare respiratory capacity, changes consistent with increased metabolism. TDF also reduced IL-1β-mediated increases in IL-1β and TNFα mRNA. These data demonstrate that TDF causes peripheral neuropathy in mice and alterations in inflammatory signaling and mitochondrial activity in the brain.

The Discovery and Development of Oxalamide and Pyrrole Small Molecule Inhibitors of gp120 and HIV Entry - A Review

Human immunodeficiency virus type-1 (HIV-1) is the causative agent responsible for the acquired immunodeficiency syndrome (AIDS) pandemic. More than 60 million infections and 25 million deaths have occurred since AIDS was first identified in the early 1980s. Advances in available therapeutics, in particular combination antiretroviral therapy, have significantly improved the treatment of HIV infection and have facilitated the shift from high mortality and morbidity to that of a manageable chronic disease. Unfortunately, none of the currently available drugs are curative of HIV. To deal with the rapid emergence of drug resistance, off-target effects, and the overall difficulty of eradicating the virus, an urgent need exists to develop new drugs, especially against targets critically important for the HIV-1 life cycle. Viral entry, which involves the interaction of the surface envelope glycoprotein, gp120, with the cellular receptor, CD4, is the first step of HIV-1 infection. Gp120 has been validated as an attractive target for anti-HIV-1 drug design or novel HIV detection tools. Several small molecule gp120 antagonists are currently under investigation as potential entry inhibitors. Pyrrole, piperazine, triazole, pyrazolinone, oxalamide, and piperidine derivatives, among others, have been investigated as gp120 antagonist candidates. Herein, we discuss the current state of research with respect to the design, synthesis and biological evaluation of oxalamide derivatives and five-membered heterocycles, namely, the pyrrole-containing small molecule as inhibitors of gp120 and HIV entry.

Recent Progress in the Development of HIV-1 Entry Inhibitors: From Small Molecules to Potent Anti-HIV Agents

Viral entry, the first process in the reproduction of viruses, primarily involves attachment of the viral envelope proteins to membranes of the host cell. The crucial components that play an important role in viral entry include viral surface glycoprotein gp120, viral transmembrane glycoprotein gp41, host cell glycoprotein (CD4), and host cell chemokine receptors (CCR5 and CXCR4). Inhibition of the multiple molecular interactions of these components can restrain viruses, such as HIV-1, from fusion with the host cell, blocking them from reproducing. This review article specifically focuses on the recent progress in the development of small-molecule HIV-1 entry inhibitors and incorporates important aspects of their structural modification that lead to the discovery of new molecular scaffolds with more potency.