Enfuvirtide (T20)-Based Lipopeptide Is a Potent HIV-1 Cell Fusion Inhibitor: Implications for Viral Entry and Inhibition

ABSTRACT 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.

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Ability of antibodies specific to the HIV-1 envelope glycoprotein to block the fusion inhibitor T20 in a cell–cell fusion assay

Immunobiology ◽

10.1016/j.imbio.2012.01.007 ◽

2012 ◽

Vol 217 (10) ◽

pp. 943-950 ◽

Cited By ~ 5

Author(s):

Nadine Vincent ◽

Etienne Malvoisin

Keyword(s):

Cell Fusion ◽

Envelope Glycoprotein ◽

Fusion Inhibitor ◽

A Cell ◽

Hiv 1 ◽

Cell Cell

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Faculty Opinions recommendation of Structural basis for coronavirus-mediated membrane fusion. Crystal structure of mouse hepatitis virus spike protein fusion core.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1018917.215498 ◽

2004 ◽

Author(s):

Peter Colman

Keyword(s):

Crystal Structure ◽

Membrane Fusion ◽

Hepatitis Virus ◽

Mouse Hepatitis Virus ◽

Spike Protein ◽

Structural Basis ◽

Protein Fusion

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Faculty Opinions recommendation of Structural basis of eukaryotic cell-cell fusion.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.718348654.793494093 ◽

2014 ◽

Author(s):

David Tareste

Keyword(s):

Cell Fusion ◽

Eukaryotic Cell ◽

Structural Basis ◽

Cell Cell

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Prefusion structure of human cytomegalovirus glycoprotein B and structural basis for membrane fusion

Science Advances ◽

10.1126/sciadv.abf3178 ◽

2021 ◽

Vol 7 (10) ◽

pp. eabf3178

Author(s):

Yuhang Liu ◽

Kyle P. Heim ◽

Ye Che ◽

Xiaoyuan Chi ◽

Xiayang Qiu ◽

...

Keyword(s):

Membrane Fusion ◽

Human Cytomegalovirus ◽

Transmembrane Domain ◽

Proximal Region ◽

Viral Envelope ◽

Vaccine Antigen ◽

Glycoprotein B ◽

Structural Basis ◽

Fusion Inhibitor ◽

Host Cell Membrane

Human cytomegalovirus (HCMV) causes congenital disease with long-term morbidity. HCMV glycoprotein B (gB) transitions irreversibly from a metastable prefusion to a stable postfusion conformation to fuse the viral envelope with a host cell membrane during entry. We stabilized prefusion gB on the virion with a fusion inhibitor and a chemical cross-linker, extracted and purified it, and then determined its structure to 3.6-Å resolution by electron cryomicroscopy. Our results revealed the structural rearrangements that mediate membrane fusion and details of the interactions among the fusion loops, the membrane-proximal region, transmembrane domain, and bound fusion inhibitor that stabilized gB in the prefusion state. The structure rationalizes known gB antigenic sites. By analogy to successful vaccine antigen engineering approaches for other viral pathogens, the high-resolution prefusion gB structure provides a basis to develop stabilized prefusion gB HCMV vaccine antigens.

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Cell–cell fusion and internalization of the CNS-based, HIV-1 co-receptor, APJ

Virology ◽

10.1016/s0042-6822(02)00021-1 ◽

2003 ◽

Vol 307 (1) ◽

pp. 22-36 ◽

Cited By ~ 41

Author(s):

Naiming Zhou ◽

Xuejun Fan ◽

Muhammad Mukhtar ◽

Jianhua Fang ◽

Charvi A Patel ◽

...

Keyword(s):

Cell Fusion ◽

Hiv 1 ◽

Cell Cell

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Molecular mechanism of HIV-1 resistance to sifuvirtide, a clinical trial–approved membrane fusion inhibitor

Journal of Biological Chemistry ◽

10.1074/jbc.ra118.003538 ◽

2018 ◽

Vol 293 (33) ◽

pp. 12703-12718 ◽

Cited By ~ 11

Author(s):

Danwei Yu ◽

Xiaohui Ding ◽

Zixuan Liu ◽

Xiyuan Wu ◽

Yuanmei Zhu ◽

...

Keyword(s):

Clinical Trial ◽

Membrane Fusion ◽

Molecular Mechanism ◽

Fusion Inhibitor ◽

Hiv 1

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Therapeutic Efficacy and Resistance Selection of a Lipopeptide Fusion Inhibitor in Simian Immunodeficiency Virus-Infected Rhesus Macaques

Journal of Virology ◽

10.1128/jvi.00384-20 ◽

2020 ◽

Vol 94 (15) ◽

Cited By ~ 1

Author(s):

Danwei Yu ◽

Jing Xue ◽

Huamian Wei ◽

Zhe Cong ◽

Ting Chen ◽

...

Keyword(s):

Membrane Fusion ◽

Therapeutic Efficacy ◽

Rhesus Macaques ◽

Heptad Repeat ◽

Fusion Inhibitor ◽

Resistance Mutations ◽

Fusion Inhibitors ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT We recently reported a group of lipopeptide-based membrane fusion inhibitors with potent antiviral activities against human immunodeficiency virus type 1 (HIV-1), HIV-2, and simian immunodeficiency virus (SIV). In this study, the in vivo therapeutic efficacy of such a lipopeptide, LP-52, was evaluated in rhesus macaques chronically infected with pathogenic SIVmac239. In a pilot study with one monkey, monotherapy with low-dose LP-52 rapidly reduced the plasma viral loads to below the limit of detection and maintained viral suppression during three rounds of structurally interrupted treatment. The therapeutic efficacy of LP-52 was further verified in four infected monkeys; however, three out of the monkeys had viral rebounds under the LP-52 therapy. We next focused on characterizing SIV mutants responsible for the in vivo resistance. Sequence analyses revealed that a V562A or V562M mutation in the N-terminal heptad repeat (NHR) and a E657G mutation in the C-terminal heptad repeat (CHR) of SIV gp41 conferred high resistance to LP-52 and cross-resistance to the peptide drug T20 and two newly designed lipopeptides (LP-80 and LP-83). Moreover, we showed that the resistance mutations greatly reduced the stability of diverse fusion inhibitors with the NHR site, and V562A or V562M in combination with E657G could significantly impair the functionality of viral envelopes (Envs) to mediate SIVmac239 infection and decrease the thermostability of viral six-helical bundle (6-HB) core structure. In conclusion, the present data have not only facilitated the development of novel anti-HIV drugs that target the membrane fusion step, but also help our understanding of the mechanism of viral evolution to develop drug resistance. IMPORTANCE The anti-HIV peptide drug T20 (enfuvirtide) is the only membrane fusion inhibitor available for treatment of viral infection; however, it exhibits relatively weak antiviral activity, short half-life, and a low genetic barrier to inducing drug resistance. Design of lipopeptide-based fusion inhibitors with extremely potent and broad antiviral activities against divergent HIV-1, HIV-2, and SIV isolates have provided drug candidates for clinical development. Here, we have verified a high therapeutic efficacy for the lipopeptide LP-52 in SIVmac239-infected rhesus monkeys. The resistance mutations selected in vivo have also been characterized, providing insights into the mechanism of action of newly designed fusion inhibitors with a membrane-anchoring property. For the first time, the data show that HIV-1 and SIV can share a similar genetic pathway to develop resistance, and that a lipopeptide fusion inhibitor could have a same resistance profile as its template peptide.

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Cell–cell and virus–cell fusion assay–based analyses of alanine insertion mutants in the distal α9 portion of the JRFL gp41 subunit from HIV-1

Journal of Biological Chemistry ◽

10.1074/jbc.ra118.004579 ◽

2019 ◽

Vol 294 (14) ◽

pp. 5677-5687 ◽

Cited By ~ 9

Author(s):

Mizuki Yamamoto ◽

Qingling Du ◽

Jiping Song ◽

Hongyun Wang ◽

Aya Watanabe ◽

...

Keyword(s):

Cell Fusion ◽

Hiv 1 ◽

Insertion Mutants ◽

Cell Cell

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Mechanism of HIV-1 Resistance to an Electronically Constrained α-Helical Peptide Membrane Fusion Inhibitor

Journal of Virology ◽

10.1128/jvi.02044-17 ◽

2018 ◽

Vol 92 (7) ◽

Cited By ~ 9

Author(s):

Xiyuan Wu ◽

Zixuan Liu ◽

Xiaohui Ding ◽

Danwei Yu ◽

Huamian Wei ◽

...

Keyword(s):

Crystal Structure ◽

Cross Resistance ◽

Fusion Inhibitor ◽

Viral Fusion ◽

Wild Type ◽

Genetic Pathway ◽

Fusion Inhibitors ◽

Helical Peptide ◽

Mimic Peptide ◽

Hiv 1

ABSTRACTSC29EK is an electronically constrained α-helical peptide HIV-1 fusion inhibitor that is highly effective against both wild-type and enfuvirtide (T20)-resistant viruses. In this study, we focused on investigating the mechanism of HIV-1 resistance to SC29EK by two approaches. First, SC29EK-escaping HIV-1 variants were selected and characterized. Three mutant viruses, which possessed two (N43K/E49A) or three (Q39R/N43K/N126K and N43K/E49A/N126K) amino acid substitutions in the N- and C-terminal repeat regions of gp41 were identified as conferring high resistance to SC29EK and cross-resistance to the first-generation (T20 and C34) and newly designed (sifuvirtide, MT-SC29EK, and 2P23) fusion inhibitors. The resistance mutations could reduce the binding stability of SC29EK, impair viral Env-mediated cell fusion and entry, and change the conformation of the gp41 core structure. Further, we determined the crystal structure of SC29EK in complex with a target mimic peptide, which revealed the critical intra- and interhelical interactions underlying the mode of action of SC29EK and the genetic pathway to HIV-1 resistance. Taken together, the present data provide new insights into the structure and function of gp41 and the structure-activity relationship (SAR) of viral fusion inhibitors.IMPORTANCET20 is the only membrane fusion inhibitor available for treatment of viral infection, but it has relatively low anti-HIV activity and genetic barriers for resistance, thus calling for new drugs blocking the viral fusion process. As an electronically constrained α-helical peptide, SC29EK is highly potent against both wild-type and T20-resistant HIV-1 strains. Here, we report the characterization of HIV-1 variants resistant to SC29EK and the crystal structure of SC29EK. The key mutations mediating high resistance to SC29EK and cross-resistance to the first and new generations of fusion inhibitors as well as the underlying mechanisms were identified. The crystal structure of SC29EK bound to a target mimic peptide further revealed its action mode and genetic pathway to inducing resistance. Hence, our data have shed new lights on the mechanisms of HIV-1 fusion and its inhibition.

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