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Discovery of critical residues for viral entry and inhibition through structural Insight of HIV-1 fusion inhibitor CP621-652.

ABSTRACT: The core structure of HIV-1 gp41 is a stable six-helix bundle (6-HB) folded by its trimeric N- and C-terminal heptad repeats (NHR and CHR). We previously identified that the (621)QIWNNMT(627) motif located at the upstream region of gp41 CHR plays critical roles for the stabilization of the 6-HB core and peptide CP621-652 containing this motif is a potent HIV-1 fusion inhibitor, however, the molecular determinants underlying the stability and anti-HIV activity remained elusive. In this study, we determined the high-resolution crystal structure of CP621-652 complexed by T21. We find that the (621)QIWNNMT(627) motif does not maintain the ?-helical conformation. Instead, residues Met(626) and Thr(627) form a unique hook-like structure (denoted as M-T hook), in which Thr(627) redirects the peptide chain to position Met(626) above the left side of the hydrophobic pocket on the NHR trimer. The side chain of Met(626) caps the hydrophobic pocket, stabilizing the interaction between the pocket and the pocket-binding domain. Our mutagenesis studies demonstrate that mutations of the M-T hook residues could completely abolish HIV-1 Env-mediated cell fusion and virus entry, and significantly destabilize the interaction of NHR and CHR peptides and reduce the anti-HIV activity of CP621-652. Our results identify an unusual structural feature that stabilizes the six-helix bundle, providing novel insights into the mechanisms of HIV-1 fusion and inhibition.

SUBMITTER: Chong H

PROVIDER: S-EPMC3370210 | biostudies-literature | 2012 Jun

REPOSITORIES: biostudies-literature

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Discovery of critical residues for viral entry and inhibition through structural Insight of HIV-1 fusion inhibitor CP621-652.

Chong Huihui H Yao Xue X Qiu Zonglin Z Qin Bo B Han Ruiyun R Waltersperger Sandro S Wang Meitian M Cui Sheng S He Yuxian Y

The Journal of biological chemistry 20120416 24

The core structure of HIV-1 gp41 is a stable six-helix bundle (6-HB) folded by its trimeric N- and C-terminal heptad repeats (NHR and CHR). We previously identified that the (621)QIWNNMT(627) motif located at the upstream region of gp41 CHR plays critical roles for the stabilization of the 6-HB core and peptide CP621-652 containing this motif is a potent HIV-1 fusion inhibitor, however, the molecular determinants underlying the stability and anti-HIV activity remained elusive. In this study, we ...[more]

PMID: 22511760

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Project description:The peptide drug enfuvirtide (T20) is the only viral fusion inhibitor used in combination therapy for HIV-1 infection, but it has relatively low antiviral activity and easily induces drug resistance. Emerging studies demonstrate that lipopeptide-based fusion inhibitors, such as LP-11 and LP-19, which mainly target the gp41 pocket site, have greatly improved antiviral potency and in vivo stability. In this study, we focused on developing a T20-based lipopeptide inhibitor that lacks pocket-binding sequence and targets a different site. First, the C-terminal tryptophan-rich motif (TRM) of T20 was verified to be essential for its target binding and inhibition; then, a novel lipopeptide, termed LP-40, was created by replacing the TRM with a fatty acid group. LP-40 showed markedly enhanced binding affinity for the target site and dramatically increased inhibitory activity on HIV-1 membrane fusion, entry, and infection. Unlike LP-11 and LP-19, which required a flexible linker between the peptide sequence and the lipid moiety, addition of a linker to LP-40 sharply reduced its potency, implying different binding modes with the extended N-terminal helices of gp41. Also, interestingly, LP-40 showed more potent activity than LP-11 in inhibiting HIV-1 Env-mediated cell-cell fusion while it was less active than LP-11 in inhibiting pseudovirus entry, and the two inhibitors displayed synergistic antiviral effects. The crystal structure of LP-40 in complex with a target peptide revealed their key binding residues and motifs. Combined, our studies have not only provided a potent HIV-1 fusion inhibitor, but also revealed new insights into the mechanisms of viral inhibition.IMPORTANCE T20 is the only membrane fusion inhibitor available for treatment of viral infection; however, T20 requires high doses and has a low genetic barrier for resistance, and its inhibitory mechanism and structural basis remain unclear. Here, we report the design of LP-40, a T20-based lipopeptide inhibitor that has greatly improved anti-HIV activity and is a more potent inhibitor of cell-cell fusion than of cell-free virus infection. The binding modes of two classes of membrane-anchoring lipopeptides (LP-40 and LP-11) verify the current fusion model in which an extended prehairpin structure bridges the viral and cellular membranes, and their complementary effects suggest a vital strategy for combination therapy of HIV-1 infection. Moreover, our understanding of the mechanism of action of T20 and its derivatives benefits from the crystal structure of LP-40.

Project description:Development of highly effective antivirals that are robust to viral evolution is a practical strategy for combating the continuously evolved severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Inspired by viral multistep entry process, we here focus on developing a bispecific SARS-CoV-2 entry inhibitor, which acts on the cell receptor angiotensin converting enzyme 2 (ACE2) and viral S2 fusion protein. First, we identified a panel of diverse spike (S) receptor-binding domains (RBDs) and found that the RBD derived from Guangdong pangolin coronavirus (PCoV-GD) possessed the most potent antiviral potency. Next, we created a bispecific inhibitor termed RBD-IPB01 by genetically linking a peptide fusion inhibitor IPB01 to the C-terminal of PCoV-GD RBD, which exhibited greatly increased antiviral potency via cell membrane ACE2 anchoring. Promisingly, RBD-IPB01 had a uniformly bifunctional inhibition on divergent pseudo- and authentic SARS-CoV-2 variants, including multiple Omicron subvariants. RBD-IPB01 also showed consistently cross-inhibition of other sarbecoviruses, including SARS-CoV, PCoV-GD, and Guangxi pangolin coronavirus (PCoV-GX). RBD-IPB01 displayed low cytotoxicity, high trypsin resistance, and favorable metabolic stability. Combined, our studies have provided a tantalizing insight into the design of broad-spectrum and potent antiviral agent. IMPORTANCE Ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) evolution and spillover potential of a wide variety of sarbecovirus lineages indicate the importance of developing highly effective antivirals with broad capability. By directing host angiotensin converting enzyme 2 receptor and viral S2 fusion protein, we have created a dual-targeted virus entry inhibitor with high antiviral potency and breadth. The inhibitor receptor-binding domain (RBD)-IPB01 with the Guangdong pangolin coronavirus (PCoV-GD) spike RBD and a fusion inhibitor IPB01 displays bifunctional cross-inhibitions on pseudo- and authentic SARS-CoV-2 variants including Omicron, as well as on the sarbecoviruses SARS-CoV, PCoV-GD, and Guangxi pangolin coronavirus. RBD-IPB01 also efficiently inhibits diverse SARS-CoV-2 infection of human Calu-3 cells and blocks viral S-mediated cell-cell fusion with a dual function. Thus, the creation of such a bifunctional inhibitor with pan-sarbecovirus neutralizing capability has not only provided a potential weapon to combat future SARS-CoV-2 variants or yet-to-emerge zoonotic sarbecovirus, but also verified a viable strategy for the designing of antivirals against infection of other enveloped viruses.

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Project description:Herpesvirus entry and spread requires fusion of viral and host cell membranes, which is mediated by the conserved surface glycoprotein B (gB). Upon activation, gB undergoes a major conformational change and transits from a metastable prefusion to a stable postfusion conformation. Although gB is a structural homolog of low-pH-triggered class III fusogens, its fusion activity depends strictly on the presence of the conserved regulatory gH/gL complex and nonconserved receptor binding proteins, which ensure that fusion occurs at the right time and space. How gB maintains its prefusion conformation and how gB fusogenicity is controlled remain poorly understood. Here, we report the isolation and characterization of a naturally selected pseudorabies virus (PrV) gB able to mediate efficient gH/gL-independent virus-cell and cell-cell fusion. We found that the control exerted on gB by the accompanying viral proteins is mediated via its cytosolic domain (CTD). Whereas gB variants lacking the CTD are inactive, a single mutation of a conserved asparagine residue in an alpha-helical motif of the ectodomain recently shown to be at the core of the gB prefusion trimer compensated for CTD absence and uncoupled gB from regulatory viral proteins, resulting in a hyperfusion phenotype. This phenotype was transferred to gB homologs from different alphaherpesvirus genera. Overall, our data propose a model in which the central helix acts as a molecular switch for the gB pre-to-postfusion transition by conveying the structural status of the endo- to the ectodomain, thereby governing their cross talk for fusion activation, providing a new paradigm for herpesvirus fusion regulation.IMPORTANCE The class III fusion protein glycoprotein B (gB) drives membrane fusion during entry and spread of herpesviruses. To mediate fusion, gB requires activation by the conserved gH/gL complex by a poorly defined mechanism. A detailed molecular-level understanding of herpesvirus membrane fusion is of fundamental virological interest and has considerable potential for the development of new therapeutics blocking herpesvirus cell invasion and spread. Using in vitro evolution and targeted mutagenesis of three different animal alphaherpesviruses, we identified a single conserved amino acid in a regulatory helix in the center of the gB ectodomain that enables efficient gH/gL-independent entry and plays a crucial role in the pre-to-postfusion transition of gB. Our results propose that the central helix is a key regulatory element involved in the intrastructural signal transduction between the endo- and ectodomain for fusion activation. This study expands our understanding of herpesvirus membrane fusion and uncovers potential targets for therapeutic interventions.

Dataset Information

Discovery of critical residues for viral entry and inhibition through structural Insight of HIV-1 fusion inhibitor CP621-652.

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Discovery of critical residues for viral entry and inhibition through structural Insight of HIV-1 fusion inhibitor CP621-652.

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