Search

CCR5 is a class A GPCR and serves as one of the coreceptors facilitating HIV-1 entry into host cells. This receptor has vital roles in the immune system and is involved in the pathogenesis of different diseases. Various studies were conducted to understand its activation mechanism, including structural studies in which inactive and active states of the receptor were determined in complex with various binding partners. These determined structures provided opportunities to perform molecular dynamics (MD) simulations and to analyze conformational changes observed in the protein structures. The atomic-level dynamic studies allow us to explore the effects of ionizable residues on the receptor. Here, our aim was to investigate the conformational changes in CCR5 when it forms a complex with either the inhibitor maraviroc (MRV), an approved anti-HIV drug, or HIV-1 envelope protein GP120, and compare these changes to the receptor's apo form. In our simulations, we considered both ionized and protonated states of ionizable binding site residue GLU283 7.39 in CCR5 as the protonation state of this residue was considered ambiguously in previous studies. Our molecular simulations results suggested that in fact, the change in the protonation state of GLU283 7.39 caused interaction profiles to be different between CCR5 and its binding partners, GP120 or MRV. We observed that when the protonated state of GLU283 7.39 was considered in complex with the envelope protein GP120, there were substantial structural changes in CCR5, indicating that it adopts a more active-like conformation. On the other hand, CCR5 in complex with MRV always adopted an inactive conformation regardless of the protonation state. Hence, the CCR5 coreceptor displays conformational heterogeneity not only depending on its binding partner but also influenced by the protonation state of the binding site binding site residue GLU283 7.39 . This outcome is also in accordance with some studies showing that GP120 binding could activate signaling pathways. This outcome could also have significant implications for discovering novel CCR5 inhibitors as anti-HIV drugs using in silico methods such as molecular docking, as it may be necessary to consider the protonated state of GLU283 7.39 .

Targeting the hydrophobic Phe43 pocket of HIV's envelope glycoprotein gp120 is a critical strategy for antiviral interventions due to its role in interacting with the host cell's CD4. Previous inhibitors, including small molecules and CD4 mimetic peptides based on scyllatoxin, have demonstrated significant binding and neutralization capabilities but were often chemically synthesized or contained non-canonical amino acids. Microbial expression using natural amino acids offers advantages such as cost-effectiveness, scalability, and efficient production of fusion proteins. In this study, we enhanced the previous scyllatoxin-based synthetic peptide by substituting natural amino acids and successfully expressed it in E. coli. The peptide was optimized by mutating the C-terminal amidated valine to valine and glutamine, and by reducing the disulfide bonds from three to two. Circular dichroism confirmed proper secondary structure formation, and fluorescence polarization analysis revealed specific, concentration-dependent binding to HIV gp120, supported by molecular dynamics simulations. These findings indicate the potential for scalable microbial production of effective antiviral peptides, with significant applications in pharmaceutical development for HIV treatment., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The HIV-1 envelope glycoprotein (Env) plays crucial role in viral infection by facilitating viral attachment to host cells and inducing fusion of the virus with the host cell membrane. This fusion allows the HIV-1 viral genome to enter the target cell then triggering various stages of the viral life cycle. The native Env directly interacts with the main receptor CD4 and the co-receptor (CCR5 or CXCR4) in human cell membrane then induces membrane fusion. The elucidation of the structure of Env with CD4 and co-receptors in different HIV-1 subtypes is essential for the understanding of the mechanism of virus entry. Here we report the Cryo-EM structure of the CD4-bound HIV-1 heterotrimeric Env from Asia prevalent CRF07_BC CH119 strain. In this structure, the binding of three CD4 molecules with Env induced extensively conformational changes in gp120, resulting in the transformation of the Env from close state to intermediate open state. Additionally, the conformational shift of V1/V2 loops of the heterotrimeric Env allosterically expose the V3 loop and promoting the further interactions with co-receptor CCR5 or CXCR4. These findings not only illustrate the structural complexity and plasticity of HIV-1 Env but also give new insights how the biological trimeric Env initialize the immune recognition and membrane fusion., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Temsavir binds directly to the HIV-1 envelope glycoprotein gp120 and selectively inhibits interactions between HIV-1 and CD4 receptors. Previous studies identified gp120 amino acid positions where substitutions are associated with reduced susceptibility to temsavir. The mechanism by which temsavir susceptibility is altered in these envelope glycoproteins was evaluated. Pseudoviruses encoding gp120 substitutions alone (S375H/I/M/N, M426L, M434I, M475I) or in combination (S375H + M475I) were engineered on a wild-type JRFL background. Temsavir-gp120 and CD4-gp120 binding kinetics and ability of temsavir to block CD4-gp120 binding were evaluated using the purified polymorphic gp120 proteins and a Creoptix® WAVE Delta grating-coupled interferometry system. Fold-change in half-maximal inhibitory concentration (IC 50 ) in JRFL-based pseudoviruses containing the aforementioned polymorphisms relative to that of wild-type ranged from 4-fold to 29,726-fold, while temsavir binding affinity for the polymorphic gp120 proteins varied from 0.7-fold to 73.7-fold relative to wild-type gp120. Strong correlations between temsavir IC 50 and temsavir binding affinity (r = 0.7332; P = 0.0246) as well as temsavir binding on-rate (r = -0.8940; P = 0.0011) were observed. Binding affinity of gp120 proteins for CD4 varied between 0.4-fold and 3.1-fold compared with wild-type gp120; no correlations between temsavir IC 50 and CD4 binding kinetic parameters were observed. For all polymorphic gp120 proteins, temsavir was able to fully block CD4 binding; 3 polymorphs required higher temsavir concentrations. Loss of susceptibility to temsavir observed for gp120 polymorphisms strongly correlated with reductions in temsavir binding on-rate. Nonetheless, temsavir retained the ability to fully block CD4-gp120 engagement given sufficiently high concentrations., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 ViiV Healthcare. Published by Elsevier B.V. All rights reserved.)

Soluble HIV-1 envelope (Env) trimers may serve as effective vaccine immunogens. The widely utilized SOSIP trimers have been paramount for structural studies, but the disulfide bond they feature between gp120 and gp41 constrains intersubunit mobility and may alter antigenicity. Here, we report an alternative strategy to generate stabilized soluble Env trimers free of covalent gp120-gp41 bonds. Stabilization was achieved by introducing an intrasubunit disulfide bond between the inner and outer domains of gp120, defined as interdomain lock (IDL). Correctly folded IDL trimers displaying a native-like antigenic profile were produced for HIV-1 Envs of different clades. Importantly, the IDL design abrogated CD4 binding while not affecting recognition by potent neutralizing antibodies to the CD4-binding site. By cryoelectron microscopy, IDL trimers were shown to adopt a closed prefusion configuration, while single-molecule fluorescence resonance energy transfer documented a high prevalence of native-like conformation. Thus, IDL trimers may be promising candidates as vaccine immunogens., Competing Interests: Declaration of interests P.L. and P.Z. applied for a patent describing HIV-1 envelope trimers stabilized in a native-like, non-CD4-binding conformation (US Provisional Patent Application No. 62/306,006, filed on March 9, 2016, entitled “Recombinant HIV-1 envelope proteins and their use”)., (Published by Elsevier Inc.)

Previous data suggest a lack of cross-resistance between the gp120-directed attachment inhibitor temsavir (active moiety of fostemsavir) and the CD4-directed post-attachment inhibitor ibalizumab. Recently, analysis of HIV-1 envelopes with reduced sensitivity to both inhibitors was undertaken to determine whether they shared genotypic correlates of resistance. Sequences from 2 envelopes with reduced susceptibility to both agents were mapped onto a temsavir-bound gp120 structure. Residues within 5.0 Å of the temsavir binding site were evaluated using reverse genetics. Broader applicability and contextual determinants of key substitutions were further assessed using envelopes from participants in the phase 3 BRIGHTE study. Temsavir sensitivity was measured by half-maximal inhibitory concentration (IC 50 ) and ibalizumab sensitivity by IC 50 and maximum percent inhibition (MPI). One envelope required substitutions of E113D and T434M for full restoration of temsavir susceptibility. Neither substitution nor their combination affected ibalizumab sensitivity. However, in the second envelope, an E202 substitution (HXB2, T202) was sufficient for observed loss of susceptibility to both inhibitors. One BRIGHTE participant with no ibalizumab exposure had an emergent K202E substitution at protocol-defined virologic failure, with reduced sensitivity to both inhibitors. Introducing T202E into previously susceptible clinical isolates reduced temsavir potency by ≥ 40-fold and ibalizumab MPI from >99% to ∼80%. Interestingly, introduction of the gp120 V5 region from a highly ibalizumab-susceptible envelope mitigated the E202 effect on ibalizumab but not temsavir. A rare HIV-1 gp120 E202 mutation reduced temsavir susceptibility, and depending on sequence context, could result in reduced susceptibility to ibalizumab., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Objective: This study aims to investigate the functions and mechanistic pathways of Astrocyte Elevated Gene-1 (AEG-1) in the disruption of the blood-retinal barrier (BRB) caused by the HIV-1 envelope glycoprotein gp120., Design: We utilized ARPE-19 cells challenged with gp120 as our model system., Methods: Several analytical techniques were employed to decipher the intricate interactions at play. These included PCR, Western blot, and immunofluorescence assays for the molecular characterization, and transendothelial electrical resistance (TEER) measurements to evaluate barrier integrity., Results: We observed that AEG-1 expression was elevated, whereas the expression levels of tight junction proteins ZO-1, Occludin, and Claudin5 were downregulated in gp120-challenged cells. TEER measurements corroborated these findings, indicating barrier dysfunction. Additional mechanistic studies revealed that the activation of NFκB and MMP2/9 pathways mediated the AEG-1-induced barrier destabilization. Through the use of lentiviral vectors, we engineered cell lines with modulated AEG-1 expression levels. Silencing AEG-1 alleviated gp120-induced downregulation of tight junction proteins and barrier impairment while concurrently inhibiting the NFκB and MMP2/9 pathways. Conversely, overexpression of AEG-1 exacerbated these pathological changes, further compromising the integrity of the BRB., Conclusion: Gp120 upregulates the expression of AEG-1 and activates the NFκB and MMP2/9 pathways. This in turn leads to the downregulation of tight junction proteins, resulting in the disruption of barrier function., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

Apart from mediating viral entry, the function of the free HIV-1 envelope protein (gp120) has yet to be elucidated. Our group previously showed that EP2 derived from one β-strand in gp120 can form amyloid fibrils that increase HIV-1 infectivity. Importantly, gp120 contains ~30 β-strands. We examined whether gp120 might serve as a precursor protein for the proteolytic release of amyloidogenic fragments that form amyloid fibrils, thereby promoting viral infection. Peptide array scanning, enzyme degradation assays, and viral infection experiments in vitro confirmed that many β-stranded peptides derived from gp120 can indeed form amyloid fibrils that increase HIV-1 infectivity. These gp120-derived amyloidogenic peptides, or GAPs, which were confirmed to form amyloid fibrils, were termed gp120-derived enhancers of viral infection (GEVIs). GEVIs specifically capture HIV-1 virions and promote their attachment to target cells, thereby increasing HIV-1 infectivity. Different GAPs can cross-interact to form heterogeneous fibrils that retain the ability to increase HIV-1 infectivity. GEVIs even suppressed the antiviral activity of a panel of antiretroviral agents. Notably, endogenous GAPs and GEVIs were found in the lymphatic fluid, lymph nodes, and cerebrospinal fluid (CSF) of AIDS patients in vivo. Overall, gp120-derived amyloid fibrils might play a crucial role in the process of HIV-1 infectivity and thus represent novel targets for anti-HIV therapeutics., (© 2024. The Author(s), under exclusive licence to CSI and USTC.)

Importance: CD4-mimetic compounds (CD4mcs) are small-molecule inhibitors of human immunodeficiency virus (HIV-1) entry into host cells. CD4mcs target a pocket on the viral envelope glycoprotein (Env) spike that is used for binding to the receptor, CD4, and is highly conserved among HIV-1 strains. Nonetheless, naturally occurring HIV-1 strains exhibit a wide range of sensitivities to CD4mcs. Our study identifies changes distant from the binding pocket that can influence the susceptibility of natural HIV-1 strains to the antiviral effects of multiple CD4mcs. We relate the antiviral potency of the CD4mc against this panel of HIV-1 variants to the ability of the CD4mc to activate entry-related changes in Env conformation prematurely. These findings will guide efforts to improve the potency and breadth of CD4mcs against natural HIV-1 variants., Competing Interests: The authors declare no conflict of interest.

HIV-1 envelope (Env) exhibits distinct conformational changes in response to host receptor (CD4) engagement. Env, a trimer of gp120 and gp41 heterodimers, has been structurally characterized in a closed, prefusion conformation with closely associated gp120s and coreceptor binding sites on gp120 V3 hidden by V1V2 loops 1-4 and in fully saturated CD4-bound open Env conformations with changes including outwardly rotated gp120s and displaced V1V2 loops 3-9 . To investigate changes resulting from substoichiometric CD4 binding, we solved single-particle cryo-electron microscopy (cryo-EM) structures of soluble, native-like heterotrimeric Envs bound to one or two CD4 molecules. Most of the Env trimers bound to one CD4 adopted the closed, prefusion Env state, with a minority exhibiting a heterogeneous partially open Env conformation. When bound to two CD4s, the CD4-bound gp120s exhibited an open Env conformation including a four-stranded gp120 bridging sheet and displaced gp120 V1V2 loops that expose the coreceptor sites on V3. The third gp120 adopted an intermediate, occluded-open state 10 that showed gp120 outward rotation but maintained the prefusion three-stranded gp120 bridging sheet with only partial V1V2 displacement and V3 exposure. We conclude that most of the engagements with one CD4 molecule were insufficient to stimulate CD4-induced conformational changes, whereas binding two CD4 molecules led to Env opening in CD4-bound protomers only. The substoichiometric CD4-bound soluble Env heterotrimer structures resembled counterparts derived from a cryo-electron tomography study of complexes between virion-bound Envs and membrane-anchored CD4 (ref.  11 ), validating their physiological relevance. Together, these results illuminate intermediate conformations of HIV-1 Env and illustrate its structural plasticity., (© 2023. The Author(s).)

Human immunodeficiency virus 1 (HIV-1) infection is initiated by binding of the viral envelope glycoprotein (Env) to the cell-surface receptor CD4 1-4 . Although high-resolution structures of Env in a complex with the soluble domains of CD4 have been determined, the binding process is less understood in native membranes 5-13 . Here we used cryo-electron tomography to monitor Env-CD4 interactions at the membrane-membrane interfaces formed between HIV-1 and CD4-presenting virus-like particles. Env-CD4 complexes organized into clusters and rings, bringing the opposing membranes closer together. Env-CD4 clustering was dependent on capsid maturation. Subtomogram averaging and classification revealed that Env bound to one, two and finally three CD4 molecules, after which Env adopted an open state. Our data indicate that asymmetric HIV-1 Env trimers bound to one and two CD4 molecules are detectable intermediates during virus binding to host cell membranes, which probably has consequences for antibody-mediated immune responses and vaccine immunogen design., (© 2023. The Author(s).)

Cardiovascular complications are seen among human immunodeficiency virus (HIV)-positive individuals, who now survive longer due to successful antiretroviral therapies. Pulmonary arterial hypertension (PAH) is a fatal disease characterized by increased blood pressure in the lung circulation. The prevalence of PAH in the HIV-positive population is dramatically higher than that in the general population. While HIV-1 Group M Subtype B is the most prevalent subtype in western countries, the majority of HIV-1 infections in eastern Africa and former Soviet Union countries are caused by Subtype A. Research on vascular complications in the HIV-positive population in the context of subtype differences, however, has not been rigorous. Much of the research on HIV has focused on Subtype B, and information on the mechanisms of Subtype A is nonexistent. The lack of such knowledge results in health disparities in the development of therapeutic strategies to prevent/treat HIV complications. The present study examined the effects of HIV-1 gp120 of Subtypes A and B on human pulmonary artery endothelial cells by performing protein arrays. We found that the gene expression changes caused by gp120s of Subtypes A and B are different. Subtype A is a more potent downregulator of perostasin, matrix metalloproteinase-2, and ErbB than Subtype B, while Subtype B is more effective in downregulating monocyte chemotactic protein-2 (MCP-2), MCP-3, and thymus- and activation-regulated chemokine proteins. This is the first report of gp120 proteins affecting host cells in an HIV subtype-specific manner, opening up the possibility that complications occur differently in HIV patients throughout the world.

Current therapeutic armamentarium for treatment of HIV-1 infection is based on the use of highly active antiretroviral therapy that, unfortunately, does not act as a curative remedy. Moreover, duration of the therapy often results in lack of compliance with the consequent emergence of multidrug resistance. Finally, drug toxicity issues also arise during treatments. In the attempt to achieve a curative effect, in addition to invest substantial resources in finding new anti-HIV-1 agents and in optimizing antiviral lead compounds and drugs currently available, additional efforts should be done to deplete viral reservoir located within host CD4 + T cells. Gp120 binders represent a class of compounds able to affect the interactions between viral envelope proteins and host CD4, thus avoiding virus-to-cell attachment and fusion, and the consequent viral entry into host cells. This review summarizes the efforts done in the last five years to design new gp120 binders, that finally culminated in the approval of fostemsavir as an anti-HIV-1 drug., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Masson SAS. All rights reserved.)

The HIV-1 envelope glycoprotein (Env) trimer is responsible for viral entry into target cells and is the sole target of neutralizing antibodies. The Env protein is therefore the focus of HIV-1 vaccine design. Env consists of two noncovalently linked subunits (gp120 and gp41) that form a trimer of heterodimers and this 6-subunit complex is metastable and conformationally flexible. Several approaches have been pursued to stabilize the Env trimer for vaccine purposes, which include structure-based design, high-throughput screening, and selection by mammalian cell display. Here, we employed directed virus evolution to improve Env trimer stability. Accordingly, we deliberately destabilized the Env gp120-gp41 interface by mutagenesis in the context of replicating HIV-1 LAI virus and virus evolution over time. We identified compensatory changes that pointed at convergent evolution, as they were largely restricted to specific Env regions, namely, the V1V2 domain of gp120 and the HR1 and HR2 domain of gp41. Specifically, S614G in V1V2 and Q567R in HR1 were frequently identified. Interestingly, the majority of the compensatory mutations were at distant locations from the original mutations and most likely strengthen intersubunit interactions. These results show how the virus can overcome Env instability and illuminate the regions that play a dominant role in Env stability. IMPORTANCE A successful HIV-1 vaccine most likely requires an envelope glycoprotein (Env) component, as Env is the only viral protein on the surface of the virus and the target for neutralizing antibodies. However, HIV Env is metastable and flexible because of the weak interactions between the Env subunits, complicating the generation of recombinant mimics of native Env. Here, we used directed viral evolution to study Env stability. We deliberately destabilized the interface between Env subunits and explored the capacity of the virus to repair trimer instability by evolution. We identified compensatory mutations that converged in specific Env locations: the apex and the trimer interface. Selected mutations enhanced the stability of recombinant soluble Env trimer proteins. These results provided clues on understanding the structural mechanisms involved in Env trimer stability, which can guide future immunogen design.

We present the development of alternative scaffolds and validation of their synthetic pathways as a tool for the exploration of new HIV gp120 inhibitors based on the recently discovered inhibitor of this class, NBD-14136. The new synthetic routes were based on isosteric replacements of the amine and acid precursors required for the synthesis of NBD-14136, guided by molecular modeling and chemical feasibility analysis. To ensure that these synthetic tools and new scaffolds had the potential for further exploration, we eventually tested few representative compounds from each newly designed scaffold against the gp120 inhibition assay and cell viability assays., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Masson SAS. All rights reserved.)

The third variable loop region (V3 loop) on gp120 plays an important role in cellular entry of HIV-1. Its interaction with the cellular CD4 and coreceptors is an important hallmark in facilitating the bridging by gp41 and subsequent fusion of membranes for transfer of viral genetic material. Further, the virus phenotype determines the cell tropism via respective co- receptor binding. Thus, coreceptor binding motif of envelope is considered to be a potent anti-viral drug target for viral entry inhibition. However, its high variability in sequence is the major hurdle for developing inhibitors targeting the region. In this study, we have used an in silico Virtual Screening and "Fragment-based" method to design small molecules based on the gp120 V3 loop interactions with a potent broadly neutralizing human monoclonal antibody, 447-52D. From the in silico analysis a potent scaffold, 1,3,5-triazine was identified for further development. Derivatives of 1,3,5-triazine with specific functional groups were designed and synthesized keeping the interaction with co-receptor intact. Finally, preliminary evaluation of molecules for HIV-1 inhibition on two different virus strains (clade C, clade B) yielded IC50 < 5.0 μM. The approach used to design molecules based on broadly neutralizing antibody, was useful for development of target specific potent antiviral agents to prevent HIV entry. The study reported promising inhibitors that could be further developed and studied., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Deciphering the glycosylation of the viral envelope (Env) glycoprotein is critical for evaluating viral escape from the host's immune response and developing vaccines and antiviral drugs. However, it is still challenging to precisely decode the site-specific glycosylation characteristics of the highly glycosylated Env proteins, although glycoproteomics have made significant advances in mass spectrometry techniques and data analysis tools. Here, we present a hybrid dissociation technique, EThcD-sceHCD, by combining electron transfer/higher-energy collisional dissociation (EThcD) and stepped collision energy/higher-energy collisional dissociation (sceHCD) into a sequential glycoproteomic workflow. Following this scheme, we characterized site-specific N/O-glycosylation of the human immunodeficiency virus type 1 (HIV-1) Env protein gp120. The EThcD-sceHCD method increased the number of identified glycopeptides when compared with EThcD, while producing more comprehensive fragment ions than sceHCD for site-specific glycosylation analysis, especially for accurate O-glycosite assignment. Finally, eighteen N-glycosites and five O-glycosites with attached glycans were assigned unambiguously from heavily glycosylated gp120. These results indicate that our workflow can achieve improved performance for analysis of the N/O-glycosylation of a highly glycosylated protein containing numerous potential glycosites in one process. Knowledge of the glycosylation landscape of the Env glycoprotein will be useful for understanding of HIV-1 infection and development of vaccines and drugs., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Zhang, Zheng, Zhao, Mao, Cao, Zeng, Liu, Hu, Gong, Cheng, Chen and Yang.)

Background: We previously developed drug-like peptide triazoles (PTs) that target HIV-1 Envelope (Env) gp120, potently inhibit viral entry, and irreversibly inactivate virions. Here, we investigated potential mechanisms of viral escape from this promising class of HIV-1 entry inhibitors., Results: HIV-1 resistance to cyclic (AAR029b) and linear (KR13) PTs was obtained by dose escalation in viral passaging experiments. High-level resistance for both inhibitors developed slowly (relative to escape from gp41-targeted C-peptide inhibitor C37) by acquiring mutations in gp120 both within (Val255) and distant to (Ser143) the putative PT binding site. The similarity in the resistance profiles for AAR029b and KR13 suggests that the shared IXW pharmacophore provided the primary pressure for HIV-1 escape. In single-round infectivity studies employing recombinant virus, V255I/S143N double escape mutants reduced PT antiviral potency by 150- to 3900-fold. Curiously, the combined mutations had a much smaller impact on PT binding affinity for monomeric gp120 (four to ninefold). This binding disruption was entirely due to the V255I mutation, which generated few steric clashes with PT in molecular docking. However, this minor effect on PT affinity belied large, offsetting changes to association enthalpy and entropy. The escape mutations had negligible effect on CD4 binding and utilization during entry, but significantly altered both binding thermodynamics and inhibitory potency of the conformationally-specific, anti-CD4i antibody 17b. Moreover, the escape mutations substantially decreased gp120 shedding induced by either soluble CD4 or AAR029b., Conclusions: Together, the data suggest that the escape mutations significantly modified the energetic landscape of Env's prefusogenic state, altering conformational dynamics to hinder PT-induced irreversible inactivation of Env. This work therein reveals a unique mode of virus escape for HIV-1, namely, resistance by altering the intrinsic conformational dynamics of the Env trimer., (© 2021. The Author(s).)

Removal of the membrane-tethering signal peptides that target secretory proteins to the endoplasmic reticulum is a prerequisite for proper folding. While generally thought to be removed co-translationally, we report two additional post-targeting functions for the HIV-1 gp120 signal peptide, which remains attached until gp120 folding triggers its removal. First, the signal peptide improves folding fidelity by enhancing conformational plasticity of gp120 by driving disulfide isomerization through a redox-active cysteine. Simultaneously, the signal peptide delays folding by tethering the N terminus to the membrane, until assembly with the C terminus. Second, its carefully timed cleavage represents intramolecular quality control and ensures release of (only) natively folded gp120. Postponed cleavage and the redox-active cysteine are both highly conserved and important for viral fitness. Considering the ∼15% proteins with signal peptides and the frequency of N-to-C contacts in protein structures, these regulatory roles of signal peptides are bound to be more common in secretory-protein biogenesis., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

The first step in HIV-1 entry is the attachment of the envelope (Env) trimer to target cell CD4. As such, the CD4-binding site (CD4bs) remains one of the few universally accessible sites for antibodies (Abs). We recently described a method of isolating Abs directly from the circulating plasma and described a panel of broadly neutralizing Abs (bnAbs) from an HIV-1 "elite neutralizer" referred to as patient N49 (N49 Ab lineage [M. M. Sajadi, A. Dashti, Z. R. Tehrani, W. D. Tolbert, et al., Cell 173:1783-1795.e14, 2018, https://doi.org/10.1016/j.cell.2018.03.061]). Here, we describe the molecular details of antigen recognition by N49P6, an Ab of the N49 lineage that recapitulates most of the neutralization breadth and potency of the donor's plasma IgG. Our studies done in the context of monomeric and trimeric antigens indicate that N49P6 combines many characteristics of known CD4bs-specific bnAbs with features that are unique to the N49 Ab lineage to achieve its remarkable neutralization breadth. These include the omission of the CD4 Phe 43 cavity and dependence instead on interactions with highly conserved gp120 inner domain layer 3. Interestingly, when bound to BG505 SOSIP, N49P6 closely mimics the initial contact of host receptor CD4 to the adjacent promoter of the HIV-1 Env trimer to lock the trimer in the closed conformation. Altogether, N49P6 defines a new class of near-pan-neutralizing, plasma deconvoluted CD4bs Abs that we refer to as the N49P series. The details of the mechanisms of action of this new Ab class pave the way for the next generation of HIV-1 bnAbs that can be used as vaccine components of therapeutics. IMPORTANCE Binding to target cell CD4 is the first crucial step required for HIV-1 infection. Thus, the CD4-binding site (CD4bs) is one of the most accessible sites for antibodies (Abs). However, due to steric constraints, only a few Abs are capable of targeting this site. Here, we show that the exceptional neutralization breadth and potency of N49P6, a near-pan-neutralizing Ab targeting the CD4bs isolated from the plasma of an HIV-1 "elite neutralizer," patient N49, are due to its signature combination of more typical CD4bs Ab-binding characteristics with unique interactions with the highly conserved gp120 inner domain. In addition, we also present a structural analysis of N49P6 in complex with the BG505 SOSIP trimer to show that N49P6 exhibits remarkable breadth in part by mimicking CD4's quaternary interaction with the neighboring gp120 protomer. In its mode of antigen interaction, N49P6 is unique and represents a new class of CD4bs-specific bnAbs.

Proinflammatory microenvironmental is crucial for the Human Immunodeficiency Virus Type 1 (HIV-1) pathogenesis. The viral glycoprotein 120 (gp120) must interact with the CD4+ T cell chemokine receptor (CCR5) and a co-receptor C-X-C chemokine receptor type 4 (CXCR4) to let the virus entry into the host cells. However, the interaction of the viral particle with other cell surface receptors is mandatory for its attachment and subsequently entry. Tumor Necrosis Factor receptor type I (TNFR1), type II (TNFR2) and Fas are a superfamily of transmembrane proteins involved in canonical inflammatory pathway and cell death by apoptosis as responses against viral pathogens. In our study, we performed an in silico evaluation of the molecular interactions between viral protein gp120 and TNF receptors (TNFR1, TNFR2 and Fas). Protein structures were retrieved from Protein Databank (PDB), and Molecular Docking and dynamics were performed using ClusPro 2.0 server and GROMACS software, respectively. We observed that gp120 is able to bind TNFR1, TNFR2 and Fas receptors, although only the TNFR2-gp120 complex demonstrated to produce a stable and durable binding. Our findings suggest that gp120 may act as an agonist to TNF-α and also function as an attachment factor in HIV-1 entry process. These molecular interaction by gp120 may be the key to HIV-1 immunopathogenesis. In conclusion, gp120 may stimulate pro-inflammatory and apoptotic signaling transduction pathways mediated by TNFR2 and may act as an attachment factor retaining HIV-1 viral particles on the host cell surface., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The entry of HIV-1 into host cells is initiated by the interaction of the viral envelope (Env) spike with the CD4 receptor. During this process, the spike undergoes a series of conformational changes that eventually lead to the exposure of the fusion peptide located at the N-terminus of the transmembrane glycoprotein, gp41. Recent structural and functional studies have provided important insights into the interaction of Env with CD4 at various stages. However, a fine elucidation of the earliest events of CD4 contact and its immediate effect on the Env conformation remains a challenge for investigation. Here, we summarize the discovery of the quaternary nature of the CD4-binding site in the HIV-1 Env and the role of quaternary contact in the functional interaction with the CD4 receptor. We propose two models for this initial contact based on the current knowledge and discuss how a better understanding of the quaternary interaction may lead to improved immunogens and antibodies targeting the CD4-binding site.

The chemokine receptor CCR5 is a key player in HIV-1 infection. The cryo-EM 3D structure of HIV-1 envelope glycoprotein (Env) subunit gp120 in complex with CD4 and CCR5 has provided important structural insights into HIV-1/host cell interaction, yet it has not explained the signaling properties of Env nor the fact that CCR5 exists in distinct forms that show distinct Env binding properties. We used classical molecular dynamics and site-directed mutagenesis to characterize the CCR5 conformations stabilized by four gp120s, from laboratory-adapted and primary HIV-1 strains, and which were previously shown to bind differentially to distinct CCR5 forms and to exhibit distinct cellular tropisms. The comparative analysis of the simulated structures reveals that the different gp120s do indeed stabilize CCR5 in different conformational ensembles. They differentially reorient extracellular loops 2 and 3 of CCR5 and thus accessibility to the transmembrane binding cavity. They also reshape this cavity differently and give rise to different positions of intracellular ends of transmembrane helices 5, 6 and 7 of the receptor and of its third intracellular loop, which may in turn influence the G protein binding region differently. These results suggest that the binding of gp120s to CCR5 may have different functional outcomes, which could result in different properties for viruses.

A major challenge in developing an effective vaccine against HIV-1 is the genetic diversity of its viral envelope. Because of the broad range of sequences exhibited by HIV-1 strains, protective antibodies must be able to bind and neutralize a widely mutated viral envelope protein. No vaccine has yet been designed which induces broadly neutralizing or protective immune responses against HIV in humans. Nanomaterial-based vaccines have shown the ability to generate antibody and cellular immune responses of increased breadth and neutralization potency. Thus, we have developed supramolecular nanofiber-based immunogens bearing the HIV gp120 envelope glycoprotein. These immunogens generated antibody responses that had increased magnitude and binding breadth compared to soluble gp120. By varying gp120 density on nanofibers, we determined that increased antigen valency was associated with increased antibody magnitude and germinal center responses. This study presents a proof-of-concept for a nanofiber vaccine platform generating broad, high binding antibody responses against the HIV-1 envelope glycoprotein., (© 2021. The Author(s).)

Human immunodeficiency virus 1 (HIV-1) infection can cause several HIV-associated neurocognitive disorders a variety of neurological impairments characterized by the loss of cortical and subcortical neurons and decreased cognitive and motor function. HIV-1 gp120, the major envelope glycoprotein on viral particles, acts as a binding protein for viral entry and is known to be an agent of neuronal cell death. To determine the mechanism of HIV-1 gp120-induced memory dysfunction, we performed mouse intracerebroventricular (i.c.v.) infusion with HIV-1 gp120 protein (300 ng per mouse) and investigated memory impairment and amyloidogenesis. Infusion of the HIV-1 gp120 protein induced memory dysfunction, which was evaluated using passive avoidance and water maze tests. Infusion of HIV-1 gp120 induced neuroinflammation, such as the release of iNOS and COX-2 and the activation of astrocytes and microglia and increased the mRNA and protein levels of IL-6, ICAM-1, M-CSF, TIM, and IL-2. In particular, we found that the infusion of HIV-1 gp120 induced the accumulation of amyloid plaques and signs of elevated amyloidogenesis, such as increased expression of amyloid precursor protein and BACE1 and increased β-secretase activity. Therefore, these studies suggest that HIV-1 gp120 may induce memory impairment through Aβ accumulation and neuroinflammation., (© 2021. The Author(s).)

Molecular interactions of the variable envelope gp120 subunit of HIV-1 with two cellular receptors are the first step of viral infection, thereby playing pivotal roles in determining viral infectivity and cell tropism. However, the underlying regulatory mechanisms for interactions under gp120 spontaneous variations largely remain unknown. Here, we show an allosteric mechanism in which a single gp120 mutation remotely controls the ternary interactions between gp120 and its receptors for the switch of viral cell tropism. Virological analyses showed that a G310R substitution at the tip of the gp120 V3 loop selectively abolished the viral replication ability in human cells, despite evoking enhancement of viral replication in macaque cells. Molecular dynamics (MD) simulations predicted that the G310R substitution at a site away from the CD4 interaction site selectively impeded the binding ability of gp120 to human CD4. Consistently, virions with the G310R substitution exhibited a reduced binding ability to human lymphocyte cells. Furthermore, the G310R substitution influenced the gp120-CCR5 interaction in a CCR5-type dependent manner as assessed by MD simulations and an infectivity assay using exogenously expressed CCR5s. Interestingly, an I198M mutation in human CCR5 restored the infectivity of the G310R virus in human cells. Finally, MD simulation predicted amino acid interplays that physically connect the V3 loop and gp120 elements for the CD4 and CCR5 interactions. Collectively, these results suggest that the V3 loop tip is a cis -allosteric regulator that remotely controls intra- and intermolecular interactions of HIV-1 gp120 for balancing ternary interactions with CD4 and CCR5. IMPORTANCE Understanding the molecular bases for viral entry into cells will lead to the elucidation of one of the major viral survival strategies, and thus to the development of new effective antiviral measures. As shown recently, HIV-1 is highly mutable and adaptable in growth-restrictive cells, such as those of macaque origin. HIV-1 initiates its infection by sequential interactions of Env-gp120 with two cell surface receptors, CD4 and CCR5. A recent epoch-making structural study has disclosed that CD4-induced conformation of gp120 is stabilized upon binding of CCR5 to the CD4-gp120 complex, whereas the biological significance of this remains totally unknown. Here, from a series of mutations found in our extensive studies, we identified a single-amino acid adaptive mutation at the V3 loop tip of Env-gp120 critical for its interaction with both CD4 and CCR5 in a host cell species-specific way. This remarkable finding could certainly provoke and accelerate studies to precisely clarify the HIV-1 entry mechanism.

Broadly neutralizing antibodies (bNAbs) are the focus of increasing interest for human immunodeficiency virus type 1 (HIV-1) prevention and treatment. Although several bNAbs are already under clinical evaluation, the development of antibodies with even greater potency and breadth remains a priority. Recently, we reported a novel strategy for improving bNAbs against the CD4-binding site (CD4bs) of gp120 by engraftment of the elongated framework region 3 (FR3) from VRC03, which confers the ability to establish quaternary interactions with a second gp120 protomer. Here, we applied this strategy to a new series of anti-CD4bs bNAbs (N49 lineage) that already possess high potency and breadth. The resultant chimeric antibodies bound the HIV-1 envelope (Env) trimer with a higher affinity than their parental forms. Likewise, their neutralizing capacity against a global panel of HIV-1 Envs was also increased. The introduction of additional modifications further enhanced the neutralization potency. We also tried engrafting the elongated CDR1 of the heavy chain from bNAb 1-18, another highly potent quaternary-binding antibody, onto several VRC01-class bNAbs, but none of them was improved. These findings point to the highly selective requirements for the establishment of quaternary contact with the HIV-1 Env trimer. The improved anti-CD4bs antibodies reported here may provide a helpful complement to current antibody-based protocols for the therapy and prevention of HIV-1 infection. IMPORTANCE Monoclonal antibodies represent one of the most important recent innovations in the fight against infectious diseases. Although potent antibodies can be cloned from infected individuals, various strategies can be employed to improve their activity or pharmacological features. Here, we improved a lineage of very potent antibodies that target the receptor-binding site of HIV-1 by engineering chimeric molecules containing a fragment from a different monoclonal antibody. These engineered antibodies are promising candidates for development of therapeutic or preventive approaches against HIV/AIDS., (Copyright © 2021 American Society for Microbiology.)

Due to the important pathological roles of the HIV-1 gp120, the protein has been intensively used in the research of HIV. However, recombinant gp120 preparation has proven to be difficult because of extremely low expression levels. In order to facilitate gp120 expression, previous methods predominantly involved the replacement of native signal peptide with a heterologous one, resulting in very limited improvement. Currently, preparation of recombinant gp120 with native glycans relies solely on transient expression systems, which are not amendable for large scale production. In this work, we employed a different approach for gp120 expression. Besides replacing the native gp120 signal peptide with that of rat serum albumin and optimizing its codon usage, we generated a stable gp120-expressing cell line in a glutamine synthetase knockout HEK293T cell line that we established for the purpose of amplification of recombinant gene expressions. The combined usage of these techniques dramatically increased gp120 expression levels and yielded a functional product with human cell derived glycan. This method may be applicable to large scale preparation of other viral envelope proteins, such as that of the emerging SARS-CoV-2, or other glycoproteins which require the presence of authentic human glycans., (Published by Elsevier Inc.)

Co-infection with the human pegivirus 1 (HPgV-1) often has a beneficial effect on disease progression in HIV-1-infected individuals. Several HPgV-1 proteins and peptides, including a 20-mer peptide (P6-2) derived from the N-terminal region of the HPgV-1 surface protein E2, have been associated with this phenomenon, which is referred to as viral interference. We identified the cysteine residues, the hydrophobic core tetrapeptide, as well as the C-terminal negative charge as key factors for the HIV-1 inhibitory activity of P6-2. Analysis of mutations in P6-2-resistant HIV-1 indicated a binding site for the peptide in the HIV-1 envelope glycoprotein gp120. In fact, P6-2 was shown to bind to soluble gp120, as well as to a peptide presenting the gp120 V3 loop. Furthermore, the HIV-1 inhibitory activity of P6-2 could be revoked by the V3 loop peptide, thus indicating a molecular mechanism that involves interaction of P6-2 with the gp120 V3 loop., (© 2020 The Authors. ChemMedChem published by Wiley-VCH GmbH.)

Human immunodeficiency virus-1 (HIV-1), the causative agent of AIDS, impacts millions of people. Entry into target cells is mediated by the HIV-1 envelope (Env) glycoprotein interacting with host receptor CD4, which triggers conformational changes allowing binding to a coreceptor and subsequent membrane fusion. Small molecule or peptide CD4-mimetic drugs mimic CD4's Phe43 interaction with Env by inserting into the conserved Phe43 pocket on Env subunit gp120. Here, we present single-particle cryo-EM structures of CD4-mimetics BNM-III-170 and M48U1 bound to a BG505 native-like Env trimer plus the CD4-induced antibody 17b at 3.7 Å and 3.9 Å resolution, respectively. CD4-mimetic-bound BG505 exhibits canonical CD4-induced conformational changes including trimer opening, formation of the 4-stranded gp120 bridging sheet, displacement of the V1V2 loop, and formation of a compact and elongated gp41 HR1C helical bundle. We conclude that CD4-induced structural changes on both gp120 and gp41 Env subunits are induced by binding to the gp120 Phe43 pocket.

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., Competing Interests: The authors have declared that no competing interests exists.

A strategy has been established for the synthesis of a family of bifunctional HIV-1 inhibitor covalent conjugates with the potential to bind simultaneously to both the gp120 and gp41 subunits of the HIV-1 envelope glycoprotein trimeric complex (Env). One component of the conjugates is derived from BNM-III-170, a small-molecule CD4 mimic that binds to gp120. The second component, comprised of the peptide DKWASLWNW ("Trp3"), was derived from the N-terminus of the HIV-1 gp41 Membrane Proximal External Region (MPER) and found previously to bind to the gp41 subunit of Env. The resulting bifunctional conjugates were shown to inhibit virus cell infection with low micromolar potency and to induce lysis of the HIV-1 virion. Crucially, virolysis was found to be dependent on the covalent linkage of the BNM-III-170 and Trp3 domains, as coadministration of a mixture of the un-cross-linked components proved to be nonlytic. However, a significant magnitude of lytic activity was observed in Env-negative and other control pseudoviruses, suggesting parallel mechanisms of action of the conjugates involving Env interaction and direct membrane disruption. Computational modeling suggested strong membrane-binding activity of BNM-III-170, which may underly the nonspecific virolytic effects of the conjugates. To investigate the scope of the membrane effect, cell-based cytotoxicity and membrane permeability assays were performed employing flow cytometry. Here, we observed a dose-dependent and specific cytotoxic effect on HIV-1 Env-expressing cells by the small-molecule bifunctional inhibitor. Most importantly, Env-negative cells were not susceptible to the cytotoxic effect upon exposure to this construct at concentrations where cell-killing effects were observed for Env-positive cells. Computational structural modeling supports a mechanism in which the bifunctional inhibitors bind to the gp120 and gp41 subunits in tandem in open-state Env trimers and induce relative motion of the gp120 subunits consistent with models of Env inactivation. This observation supports the idea that the cell-killing effect of the small-molecule bifunctional inhibitor is due to specific Env conformational triggering. This work lays important groundwork to advance a small-molecule bifunctional inhibitor approach for eliminating Env-expressing infected cells and the eradication of HIV-1.

CD4-mimetic HIV-1 entry inhibitors are small sized molecules which imitate similar conformational flexibility, in gp120, to the CD4 receptor. However, the mechanism of the conformational flexibility instigated by these small sized inhibitors is little known. Likewise, the effect of the antibody on the function of these inhibitors is also less studied. In this study, we present a thorough inspection of the mechanism of the conformational flexibility induced by a CD4-mimetic inhibitor, NBD-557, using Molecular Dynamics Simulations and free energy calculations. Our result shows the functional importance of Asn425 in substrate induced conformational dynamics in gp120. The MD simulations of Asn425Gly mutant provide a less dynamic gp120 in the presence of NBD-557 without incapacitating the binding enthalpy of NBD-557. The MD simulations of complexes with the antibody clearly show the enhanced affinity of NBD-557 due to the presence of the antibody, which is in good agreement with experimental Isothermal Titration Calorimetry results ( Biochemistry 2006 , 45 , 10973-10980).

Despite current pharmacological intervention strategies, patients with HIV still suffer from chronic inflammation. The nicotinic acetylcholine receptors (nAChRs) are widely distributed throughout the nervous and immune systems. In macrophages, activation of alpha7-nAChR (α7-nAChR) controls inflammatory processes through the cholinergic anti-inflammatory response (CAR). Given that this innate immune response controls inflammation and α7-nAChR plays a critical role in the regulation of systemic inflammation, we investigated the effects of an R5-tropic HIV soluble component, gp120 JRFL , on the CAR functioning. We previously demonstrated that X4-tropic HIV-1 gp120 IIIB disrupts the CAR as well as inducing upregulation of the α7-nAChR in vitro in monocyte-derived macrophages (MDMs), which correlates with the upregulation observed in monocytes, T-lymphocytes, and MDMs recovered from HIV-infected people. We demonstrate here using imaging and molecular assays that the R5-tropic HIV-1 glycoprotein gp120 JRFL upregulates the α7-nAChR in MDMs dependent on CD4 and/or CCR5 activation. This upregulation was also dependent on MEK1 since its inhibition attenuates the upregulation of α7-nAChR induced by gp120 JRFL and was concomitant with an increase in basal calcium levels, which did not result in apoptosis. Moreover, the CAR was determined to be disrupted, since α7-nAChR activation in MDMs did not reduce the production of the proinflammatory cytokines IL-6, GRO-α, or I-309. Furthermore, a partial antagonist of α7-nAChR, bupropion, rescued IL-6 but not GRO-α or I-309 production. Together, these results demonstrate that gp120 JRFL disrupts the CAR in MDMs. Other medications targeting the α7-nAChR need to be tested to reactivate the CAR to ameliorate inflammation in HIV-infected subjects., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Acute HIV infection is characterized by rapid viral seeding of immunologic inductive sites in the gut followed by the severe depletion of gut CD4 + T cells. Trafficking of α 4 β 7 -expressing lymphocytes to the gut is mediated by MAdCAM, the natural ligand of α 4 β 7 that is expressed on gut endothelial cells. MAdCAM signaling through α 4 β 7 costimulates CD4 + T cells and promotes HIV replication. Similar to MAdCAM, the V2 domain of the gp120 HIV envelope protein binds to α 4 β 7 In this study, we report that gp120 V2 shares with MAdCAM the capacity to signal through α 4 β 7 resulting in CD4 + T cell activation and proliferation. As with MAdCAM-mediated costimulation, cellular activation induced by gp120 V2 is inhibited by anti-α 4 β 7 monoclonal antibodies (mAbs). It is also inhibited by anti-V2 domain antibodies including nonneutralizing mAbs that recognize an epitope in V2 that has been linked to reduced risk of acquisition in the RV144 vaccine trial. The capacity of the V2 domain of gp120 to mediate signaling through α 4 β 7 likely impacts early events in HIV infection. The capacity of nonneutralizing V2 antibodies to block this activity reveals a previously unrecognized mechanism whereby such antibodies might impact HIV transmission and pathogenesis., Competing Interests: The authors declare no competing interest.

HIV-1 V2 domain binds α4β7, which assists lymphocyte homing to gut-associated lymphoid tissue. This triggers bacterial translocation, thus contributing to immune activation. We investigated whether variability of V2 179-181 binding site could influence plasma levels of lipopolysaccharide (LPS) and soluble cluster of differentiation 14 (sCD14), markers of microbial translocation/immune activation. HIV gp120 sequences from antiretroviral naïve patients were analyzed for V2 tripeptide composition, length, net charge, and potential N-linked-glycosylation sites. LPS and sCD14 plasma levels were quantified. Clinical/immuno-virologic data were retrieved. Overall, 174 subjects were enrolled, 8% with acute infection, 71% harboring a subtype B. LDV 179-181 was detected in 41% and LDI in 27%. No difference was observed between levels of LPS or sCD14 according to different mimotopes or according to other sequence characteristics. By multivariable analysis, only acute infection was significantly associated with higher sCD14 levels. In conclusion, no association was observed between V2 tripeptide composition and extent of bacterial translocation/immune activation., (© 2020 Wiley Periodicals LLC.)

We previously discovered a class of recombinant lectin conjugates, denoted lectin DLIs ('dual-acting lytic inhibitors') that bind to the HIV-1 envelope (Env) protein trimer and cause both lytic inactivation of HIV-1 virions and cytotoxicity of Env-expressing cells. To facilitate mechanistic investigation of DLI function, we derived the simplified prototype microvirin (MVN)-DLI, containing an MVN domain that binds high-mannose glycans in Env, connected to a DKWASLWNW sequence (denoted 'Trp3') derived from the membrane-associated region of gp41. The relatively much stronger affinity of the lectin component than Trp3 argues that the lectin functions to capture Env to enable Trp3 engagement and consequent Env membrane disruption and virolysis. The relatively simplified engagement pattern of MVN with Env opened up the opportunity, pursued here, to use recombinant glycan knockout gp120 variants to identify the precise Env binding site for MVN that drives DLI engagement and lysis. Using mutagenesis combined with a series of biophysical and virological experiments, we identified a restricted set of residues, N262, N332 and N448, all localized in a cluster on the outer domain of gp120, as the essential epitope for MVN binding. By generating these mutations in the corresponding HIV-1 virus, we established that the engagement of this glycan cluster with the lectin domain of MVN*-DLI is the trigger for DLI-derived virus and cell inactivation. Beyond defining the initial encounter step for lytic inactivation, this study provides a guide to further elucidate DLI mechanism, including the stoichiometry of Env trimer required for function, and downstream DLI optimization., (© 2020 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

HIV-1 envelope (Env) glycoprotein gp160 exists as a trimer of heterodimers on the viral surface. In most structures of the soluble ectodomain of trimeric HIV-1 envelope glycoprotein, the regions from 512 to 517 of the fusion peptide and from 547 to 568 of the N-heptad repeat are disordered. We used aspartate scanning mutagenesis of subtype B strain JRFL Env as an alternate method to probe residue burial in the context of cleaved, cell surface-expressed Env, as buried residues should be intolerant to substitution with Asp. The data are inconsistent with a fully disordered 547 to 568 stretch, as residues 548, 549, 550, 555, 556, 559, 562, and 566 to 569 are all sensitive to Asp substitution. In the fusion peptide region, residues 513 and 515 were also sensitive to Asp substitution, suggesting that the fusion peptide may not be fully exposed in native Env. gp41 is metastable in the context of native trimer. Introduction of Asp at residues that are exposed in the prefusion state but buried in the postfusion state is expected to destabilize the postfusion state and any intermediate states where the residue is buried. We therefore performed soluble CD4 (sCD4)-induced gp120 shedding experiments to identify Asp mutants at residues 551, 554 to 559, 561 to 567, and 569 that could prevent gp120 shedding. We also observed similar mutational effects on shedding for equivalent mutants in the context of clade C Env from isolate 4-2J.41. These substitutions can potentially be used to stabilize native-like trimer derivatives that are used as HIV-1 vaccine immunogens. IMPORTANCE In most crystal structures of the soluble ectodomain of the HIV-1 Env trimer, some residues in the fusion and N-heptad repeat regions are disordered. Whether this is true in the context of native, functional Env on the virion surface is not known. This knowledge may be useful for stabilizing Env in its prefusion conformation and will also help to improve understanding of the viral entry process. Burial of the charged residue Asp in a protein structure is highly destabilizing. We therefore used Asp scanning mutagenesis to probe the burial of apparently disordered residues in native Env and to examine the effect of mutations in these regions on Env stability and conformation as probed by antibody binding to cell surface-expressed Env, CD4-induced shedding of HIV-1 gp120, and viral infectivity studies. Mutations that prevent shedding can potentially be used to stabilize native-like Env constructs for use as vaccine immunogens., (Copyright © 2020 American Society for Microbiology.)

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., (Copyright © 2020 American Society for Microbiology.)

As the only exposed viral protein at the membrane surface of HIV, envelope glycoprotein gp120 is responsible for recognizing host cells and mediating virus-cell membrane fusion. Available structures of gp120 indicate that it exhibits two distinct conformational states, called closed and open states. Although experimental data demonstrates that CD4 binding stabilizes open state of gp120, detailed structural dynamics and kinetics of gp120 during this process remain elusive. Here, two open-state gp120 simulation systems, one without any ligands (ligand-free) and the other complexed with CD4 (CD4-bound), were subjected to microsecond-scale molecular dynamics simulations following the conformational transitions and allosteric pathways of gp120 evaluated by using the Markov state model and a network-based method, respectively. Our results provide an atomic-resolution description of gp120 conformational transitions, suggesting that gp120 is intrinsically dynamic from the open state to closed state, whereas CD4 binding blocks these transitions. Consistent with experimental structures, five metastable conformations with different orientations of the V1/V2 region and V3 loop have been extracted. The binding of CD4 significantly enhances allosteric communications from the CD4-binding site to V3 loop and β20-21 hairpin, resulting in high-affinity interactions with coreceptors and activation of the conformational transitions switcher, respectively. This study will facilitate the structural understanding of the CD4-binding effects on conformational transitions and allosteric pathways of gp120., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

During human immunodeficiency virus type 1 (HIV-1) entry into cells, the viral envelope glycoprotein (Env) trimer [(gp120/gp41) 3 ] binds the receptors CD4 and CCR5 and fuses the viral and cell membranes. CD4 binding changes Env from a pretriggered (state-1) conformation to more open downstream conformations. BMS-378806 (here called BMS-806) blocks CD4-induced conformational changes in Env important for entry and is hypothesized to stabilize a state-1-like Env conformation, a key vaccine target. Here, we evaluated the effects of BMS-806 on the conformation of Env on the surface of cells and virus-like particles. BMS-806 strengthened the labile, noncovalent interaction of gp120 with the Env trimer, enhanced or maintained the binding of most broadly neutralizing antibodies, and decreased the binding of poorly neutralizing antibodies. Thus, in the presence of BMS-806, the cleaved Env on the surface of cells and virus-like particles exhibits an antigenic profile consistent with a state-1 conformation. We designed novel BMS-806 analogues that stabilized the Env conformation for several weeks after a single application. These long-acting BMS-806 analogues may facilitate enrichment of the metastable state-1 Env conformation for structural characterization and presentation to the immune system. IMPORTANCE The envelope glycoprotein (Env) spike on the surface of human immunodeficiency virus type 1 (HIV-1) mediates the entry of the virus into host cells and is also the target for antibodies. During virus entry, Env needs to change shape. Env flexibility also contributes to the ability of HIV-1 to evade the host immune response; many shapes of Env raise antibodies that cannot recognize the functional Env and therefore do not block virus infection. We found that an HIV-1 entry inhibitor, BMS-806, stabilizes the functional shape of Env. We developed new variants of BMS-806 that stabilize Env in its natural state for long periods of time. The availability of such long-acting stabilizers of Env shape will allow the natural Env conformation to be characterized and tested for efficacy as a vaccine., (Copyright © 2020 American Society for Microbiology.)

At least half of human immunodeficiency virus (HIV)-infected individuals suffer from a wide range of cognitive, behavioral and motor deficits, collectively known as HIV-associated neurocognitive disorders (HAND). The molecular mechanisms that amplify damage within the brain of HIV-infected individuals are unknown. Recently, we described that HIV augments the opening of connexin-43 (Cx43) hemichannels in cultured human astrocytes, which result in the collapse of neuronal processes. Whether HIV soluble viral proteins such as gp120, can regulate hemichannel opening in astrocytes is still ignored. These channels communicate the cytosol with the extracellular space during pathological conditions. We found that gp120 enhances the function of both Cx43 hemichannels and pannexin-1 channels in mouse cortical astrocytes. These effects depended on the activation of IL-1β/TNF-α, p38 MAP kinase, iNOS, cytoplasmic Ca 2+ and purinergic signaling. The gp120-induced channel opening resulted in alterations in Ca 2+ dynamics, nitric oxide production and ATP release. Although the channel opening evoked by gp120 in astrocytes was reproduced in ex vivo brain preparations, these responses were heterogeneous depending on the CA1 region analyzed. We speculate that soluble gp120-induced activation of astroglial Cx43 hemichannels and pannexin-1 channels could be crucial for the pathogenesis of HAND.

The high mannose patch (HMP) of the HIV envelope protein (Env) is the structure most frequently targeted by broadly neutralizing antibodies; therefore, many researchers have attempted to use mimics of this region as a vaccine immunogen. In our previous efforts, vaccinating rabbits with evolved HMP mimic glycopeptides containing Man 9 resulted in an overall antibody response targeting the glycan core and linker rather than the full glycan or Manα1→2Man tips of Man 9 glycans. A possible reason could be processing of our immunogen by host serum mannosidases. We sought to test whether more prolonged dosing could increase the antibody response to intact glycans, possibly by increasing the availability of intact Man 9 to germinal centers. Here, we describe a study investigating the impact of immunization regimen on antibody response by testing immunogen delivery through bolus, an exponential series of mini doses, or a continuously infusing mini-osmotic pump. Our results indicate that, with our glycopeptide immunogens, standard bolus immunization elicited the strongest HIV Env-binding antibody response, even though higher overall titers to the glycopeptide were elicited by the exponential and pump regimens. Antibody selectivity for intact glycan was, if anything, slightly better in the bolus-immunized animals.

The entry of human immunodeficiency virus type I (HIV-1) into host cells is initiated by binding to the cell-surface receptor CD4, which induces a conformational transition of the envelope (Env) glycoprotein gp120 from the closed, unliganded state to the open, CD4-bound state. Despite many available structures in these two states, detailed aspects on the dynamics and thermodynamics of gp120 remain elusive. Here, we performed microsecond-scale (μs-scale) multiple-replica molecular dynamics (MD) simulations to explore the differences in the conformational dynamics, protein motions, and thermodynamics between the unliganded and CD4-bound/complexed forms of gp120. Comparative analyses of MD trajectories reveal that CD4 binding promotes the structural deviations/changes and conformational flexibility, loosens the structural packing, and complicates the molecular motions of gp120. Comparison of the constructed free energy landscapes (FELs) reveals that the CD4-complexed gp120 has more conformational substates, larger conformational entropy, and lower thermostability than the unliganded form. Therefore, the unliganded conformation represents a structurally and energetically stable "ground state" for the full-length gp120. The observed great increase in the mobility of V1/V2 and V3 along with their more versatile movement directions in the CD4-bound gp120 compared to the unliganded form suggests that their orientations with respect to each other and to the structural core determine the differences in the conformational dynamics and thermodynamics between the two gp120 forms. The results presented here provide a basis by which to better understand the functional and immunological properties of gp120 and, furthermore, to deploy appropriate strategies for the development of anti-HIV-1 drugs or vaccines.

Generating a soluble and native-like trimeric envelope glycoprotein (Env) with high efficacy as an immunogen has been a major focus for developing an effective vaccine against HIV-1. The Env immunogen is a heavily glycosylated protein composed of 3 identical surface gp120 and gp41 subunits that form into a trimer of heterodimers (3 × 28 N-glycan sites). During Env immunogen production, endogenous furin works to cleave a hexa-arginine motif connecting the gp120 and gp41 subunits, which is needed to ensure proper protein folding and a native-like conformation of Env. Verification of the overall identity and proteolytic cleavage of Env is therefore important for HIV-1 vaccine development and product quality. Herein, we report the first work using LC-MS to (1) achieve fast and accurate intact mass measurement of Env after deglycosylation and (2) confidently identify the furin cleavage sites.

Purinergic receptors are well-established modulators of inflammatory processes, primarily through detection of extracellular nucleotides that are released by dying or infected cells. Emerging literature has demonstrated that inhibition of these inflammatory receptors can block HIV-1 productive infection and HIV-1-associated inflammation. The specificity of receptor type and mechanism of interaction has not yet been determined. Here, we characterize the inhibitory activity of P2X1 receptor antagonists, NF279 and NF449, in cell lines, primary cells, and a variety of HIV-1 envelope (Env) clades. NF279 and NF449 blocked productive infection at the level of viral membrane fusion, with a range of inhibitory activities against different HIV-1 Env isolates. A mutant virus carrying a truncation deletion of the C-terminal tail of HIV-1 Env glycoprotein 41 (gp41) showed reduced sensitivity to P2X1 antagonists, indicating that the sensitivity of inhibition by these molecules may be modulated by Env conformation. In contrast, a P2X7 antagonist, A438079, had a limited effect on productive infection and fusion. NF279 and NF449 interfered with the ability of the gp120 variable regions 1 and 2 (V1V2)-targeted broadly neutralizing antibody PG9 to block productive infection, suggesting that these drugs may antagonize HIV-1 Env at gp120 V1V2 to block viral membrane fusion. Our observations indicate that P2X1 antagonism can inhibit HIV-1 replication at the level of viral membrane fusion through interaction with Env. Future studies will probe the nature of these compounds in inhibiting HIV-1 fusion and the development of small molecules to block HIV-1 entry via this mechanism. IMPORTANCE While effective treatment can lower the severe morbidity and mortality associated with HIV-1 infection, patients infected with HIV-1 suffer from significantly higher rates of noncommunicable comorbidities associated with chronic inflammation. Emerging literature suggests a key role for P2X1 receptors in mediating this chronic inflammation, but the mechanism is still unknown. Here, we demonstrate that HIV-1 infection is reduced by P2X1 receptor antagonism. This inhibition is mediated by interference with HIV-1 Env and can impact a variety of viral clades. These observations highlight the importance of P2X1 antagonists as potential novel therapeutics that could serve to block a variety of different viral clades with additional benefits for their anti-inflammatory properties., (Copyright © 2020 American Society for Microbiology.)

We previously reported a milestone in the optimization of NBD-11021, an HIV-1 gp120 antagonist, by developing a new and novel analogue, NBD-14189 ( Ref1 ), which showed antiviral activity against HIV-1 HXB2 , with a half maximal inhibitory concentration of 89 nM. However, cytotoxicity remained high, and the absorption, distribution, metabolism, and excretion (ADME) data showed relatively poor aqueous solubility. To optimize these properties, we replaced the phenyl ring in the compound with a pyridine ring and synthesized a set of 48 novel compounds. One of the new analogues, NBD-14270 ( 8 ), showed a marked improvement in cytotoxicity, with 3-fold and 58-fold improvements in selectivity index value compared with that of Ref1 and NBD-11021, respectively. Furthermore, the in vitro ADME data clearly showed improvements in aqueous solubility and other properties compared with those for Ref1 . The data for 8 indicated that the pyridine scaffold is a good bioisostere for phenyl, allowing the further optimization of this molecule.

Attachment of envelope glycoprotein gp120 to the host cell receptor CD4 is the first step during the human immunodeficiency virus-1 (HIV-1) entry into the host cells that makes it a promising target for drug design. To elucidate the crucial three dimensional (3D) structural features of reported HIV-1 gp120 CD4 binding inhibitors, 3D pharmacophores were generated and receptor based approach was employed to quantify these structural features. A four-partial least square factor model with good statistics and predictive ability was generated for the dataset of 100 molecules. To further ascertain the structural requirement for gp120-CD4 binding inhibition, molecular interaction studies of inhibitors with gp120 was carried out by performing molecular docking using Glide 5.6. Based on these studies, structural requirements were drawn and new molecules were designed accordingly to yield new sulphonamides derivatives. A water based green synthetic approach was adopted to obtain these compounds which were evaluated for their HIV-1 gp120 CD4 binding inhibition. The newly synthesized compounds exhibited remarkable activity (10-fold increase) when compared with the standard BMS 806. Further the stability of newly synthesized derivatives with HIV-1 gp120 was also investigated through molecular dynamics simulation studies. This provides a proof of concept for molecular modeling based design of new inhibitors for inhibition of HIV-1 gp120 CD4 interaction.

Inducing immune responses protecting from HIV infection or at least controlling replication poses a huge challenge to modern vaccinology. An increasingly discussed strategy to elicit a potent and broad neutralizing antibody response is the immobilization of HIV's trimeric envelope (Env) surface receptor on a nanoparticulate carrier. As a conceptual proof, we attached an Env variant (BG505 SOSIP.664) to highly stable and biocompatible silica nanoparticles (SiNPs) via site-specific covalent conjugation or nonspecific adsorption to SiNPs. First, we demonstrated the feasibility of SiNPs as platform for Env presentation by a thorough characterization process during which Env density, attachment stability, and antigenicity were evaluated for both formulations. Binding affinities to selected antibodies were in the low nanomolar range for both formulations confirming that the structural integrity of Env is retained after attachment. Second, we explored the recognition of SiNP conjugates by antigen presenting cells. Here, the uptake of Env attached to SiNPs via a site-specific covalent conjugation was 4.5-fold enhanced, whereas adsorbed Env resulted only in a moderate 1.4-fold increase compared with Env in its soluble form. Thus, we propose SiNPs with site-specifically and covalently conjugated Env preferably in a high density as a promising candidate for further investigations as vaccine platform., (Copyright © 2020 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

The oral, cervical, and genital mucosa, covered by stratified squamous epithelia with polarized organization and strong tight and adherens junctions, play a critical role in preventing transmission of viral pathogens, including human immunodeficiency virus (HIV). HIV-1 interaction with mucosal epithelial cells may depolarize epithelia and disrupt their tight and adherens junctions; however, the molecular mechanism of HIV-induced epithelial disruption has not been completely understood. We showed that prolonged interaction of cell-free HIV-1 virions, and viral envelope and transactivator proteins gp120 and tat, respectively, with tonsil, cervical, and foreskin epithelial cells induces an epithelial-mesenchymal transition (EMT). EMT is an epigenetic process leading to the disruption of mucosal epithelia and allowing the paracellular spread of viral and other pathogens. Interaction of cell-free virions and gp120 and tat proteins with epithelial cells substantially reduced E-cadherin expression and activated vimentin and N-cadherin expression, which are well-known mesenchymal markers. HIV gp120- and tat-induced EMT was mediated by SMAD2 phosphorylation and activation of transcription factors Slug, Snail, Twist1 and ZEB1. Activation of TGF-β and MAPK signaling by gp120, tat, and cell-free HIV virions revealed the critical roles of these signaling pathways in EMT induction. gp120- and tat-induced EMT cells were highly migratory via collagen-coated membranes, which is one of the main features of mesenchymal cells. Inhibitors of TGF-β1 and MAPK signaling reduced HIV-induced EMT, suggesting that inactivation of these signaling pathways may restore the normal barrier function of mucosal epithelia., Competing Interests: The authors have declared that no competing interests exist.