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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:Ebolavirus (EBOV) and Marburgvirus (MARV) that compose the filovirus family of negative strand RNA viruses infect a broad range of mammalian cells. Recent studies indicate that cellular entry of this family of viruses requires a series of cellular protein interactions and molecular mechanisms, some of which are unique to filoviruses and others are commonly used by all viral glycoproteins. Details of this entry pathway are highlighted here. Virus entry into cells is initiated by the interaction of the viral glycoprotein(1) subunit (GP(1)) with both adherence factors and one or more receptors on the surface of host cells. On epithelial cells, we recently demonstrated that TIM-1 serves as a receptor for this family of viruses, but the cell surface receptors in other cell types remain unidentified. Upon receptor binding, the virus is internalized into endosomes primarily via macropinocytosis, but perhaps by other mechanisms as well. Within the acidified endosome, the heavily glycosylated GP(1) is cleaved to a smaller form by the low pH-dependent cellular proteases Cathepsin L and B, exposing residues in the receptor binding site (RBS). Details of the molecular events following cathepsin-dependent trimming of GP(1) are currently incomplete; however, the processed GP(1) specifically interacts with endosomal/lysosomal membranes that contain the Niemann Pick C1 (NPC1) protein and expression of NPC1 is required for productive infection, suggesting that GP/NPC1 interactions may be an important late step in the entry process. Additional events such as further GP(1) processing and/or reducing events may also be required to generate a fusion-ready form of the glycoprotein. Once this has been achieved, sequences in the filovirus GP(2) subunit mediate viral/cellular membrane fusion via mechanisms similar to those previously described for other enveloped viruses. This multi-step entry pathway highlights the complex and highly orchestrated path of internalization and fusion that appears unique for filoviruses.

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.

Project description:Peptide T20, which targets the HIV protein gp41, represents the first approved member of the class of HIV drugs known as membrane fusion inhibitors. However, mechanisms which lead to resistance through clinical use of T20 are not well-understood because the structure of the bound complex remains undetermined. In this report, an atomic-level model of a T20-gp41 complex embedded in an explicit DOPC membrane was constructed, and molecular dynamics simulations, followed by binding energy analysis (MM-GBSA method), were performed to delineate structural and energetic features that contribute to drug resistance. Per-residue binding footprints for T20 with wild-type gp41 reveal strong intermolecular van der Waals, Coulombic, and H-bond interactions in striking agreement with clinically observed resistance patterns. In addition, seven deleterious gp41 point mutations (L33Q, L33S, G36V, I37K, V38E, Q40H, and Q40K) were simulated, and all correctly exhibited decreases in the level of binding, including the fact that L33Q and Q40K are most detrimental. Six of the seven simulations yield good quantitative agreement (r(2) = 0.72; N = 6) with available experimental fold resistance data. Results from energy decomposition, heat map analysis, and differential (mutant minus wild-type) footprinting indicate the following. (1) Mutations disrupt intermolecular H-bonding and reduce the level of favorable contact with gp41 at M19. (2) Charged mutations (I37K, Q40K, and V38E) lead to significant Coulombic changes that weaken favorable van der Waals interactions. (3) Q40K is more detrimental than I37K because of interaction differences with a polar/charged patch on T20 in the initial (wild-type) state. (4) Resistance for L33S versus L33Q likely involves side chain packing differences in the final (mutated) state. A valuable finding of the work involves identification of favorable interactions among the C-terminal end of T20 (WNWF motif), residues on gp41 (including the fusion peptide), and headgroups in the adjacent membrane. The results suggest a complete T20 binding site would contribute to a stable complex, which could help to explain why prior studies, which employed truncated gp41 constructs, reported that C-terminal T20 residues may not interact with gp41. A hypothesis resulting from this study is that peptides could be designed to increase the level of favorable contact with both the membrane and gp41 which would lead to enhanced activity.

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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