Dataset Information

Conserved Residue Asn-145 in the C-Terminal Heptad Repeat Region of HIV-1 gp41 is Critical for Viral Fusion and Regulates the Antiviral Activity of Fusion Inhibitors.

ABSTRACT: Entry of HIV-1 into target cells is mediated by its envelope (Env) glycoprotein composed of the receptor binding subunit gp120 and the fusion protein gp41. Refolding of the gp41 N- and C-terminal heptad repeats (NHR and CHR) into a six-helix bundle (6-HB) conformation drives the viral and cellular membranes in close apposition and generates huge amounts of energy to overcome the kinetic barrier leading to membrane fusion. In this study, we focused on characterizing the structural and functional properties of a single Asn-145 residue, which locates at the middle CHR site of gp41 and is extremely conserved among all the HIV-1, HIV-2, and simian immunodeficiency virus (SIV) isolates. By mutational analysis, we found that Asn-145 plays critical roles for Env-mediated cell-cell fusion and HIV-1 entry. As determined by circular dichroism (CD) spectroscopy and isothermal titration calorimetry (ITC), the substitution of Asn-145 with alanine (N145A) severely impaired the interactions between the NHR and CHR helices. Asn-145 was also verified to be important for the antiviral activity of CHR-derived peptide fusion inhibitors and served as a turn-point for the inhibitory potency. Intriguingly, Asn-145 could regulate the functionality of the M-T hook structure at the N-terminus of the inhibitors and displayed comparable activities with the C-terminal IDL anchor. Crystallographic studies further demonstrated the importance of Asn-145-mediated interhelical and intrahelical interactions in the 6-HB structure. Combined, the present results have provided valuable information for the structure-function relationship of HIV-1 gp41 and the structure-activity relationship of gp41-dependent fusion inhibitors.

SUBMITTER: Geng X

PROVIDER: S-EPMC6669600 | biostudies-literature | 2019 Jul

REPOSITORIES: biostudies-literature

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Conserved Residue Asn-145 in the C-Terminal Heptad Repeat Region of HIV-1 gp41 is Critical for Viral Fusion and Regulates the Antiviral Activity of Fusion Inhibitors.

Geng Xiuzhu X Liu Zixuan Z Yu Danwei D Qin Bo B Zhu Yuanmei Y Cui Sheng S Chong Huihui H He Yuxian Y

Viruses 20190703 7

Entry of HIV-1 into target cells is mediated by its envelope (Env) glycoprotein composed of the receptor binding subunit gp120 and the fusion protein gp41. Refolding of the gp41 N- and C-terminal heptad repeats (NHR and CHR) into a six-helix bundle (6-HB) conformation drives the viral and cellular membranes in close apposition and generates huge amounts of energy to overcome the kinetic barrier leading to membrane fusion. In this study, we focused on characterizing the structural and functional ...[more]

PMID: 31277353

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Project description:BACKGROUND:The native pre-fusion structure of gp120/gp41 complex of human immunodeficiency virus type 1 was recently revealed. In the model, the helices of gp41 (?6, ?7, ?8, and ?9) form a four-helix collar underneath trimeric gp120. Gp41 is a class I fusion protein and mediates membrane fusion by forming a post-fusion structure called the six-helix bundle (6HB). The comparison of the pre- and post-fusion structures revealed the large conformational changes in gp41 during the antiparallel packing of the N- and C-terminal heptad repeats (NHRs and CHRs) in membrane fusion. Several mutagenesis studies of gp41 performed in the past were interpreted based on 6HB, the only available structure at that time. To obtain an insight about the current pre-fusion structural model and conformational changes during membrane fusion, alanine insertion mutagenesis of the NHR, CHR and connecting loop regions of HXB2 gp41 was performed. The effects of mutations on biosynthesis and membrane fusion were analyzed by immunoblotting and fusion assays, respectively. The extent of membrane fusion was evaluated by split luciferase-based pore formation and syncytia formation assays, respectively. RESULTS:Consistent with the current structural model, drastic negative effects of mutations on biosynthesis and membrane fusion were observed for NHR, loop, and proximal regions of CHR (up to amino acid position 643). The insertions in ?9 after it leaves the four-helix collar were tolerable for biosynthesis. These CHR mutants showed varying effects on membrane fusion. Insertion at position 644 or 645 resulted in poor pore and syncytia formation. Efficient pore and syncytia formation almost similar to that of the wild type was observed for insertion at position 647, 648 or 649. However, recovery of virus infectivity was only observed for the insertions beyond position 648. CONCLUSIONS:The mutagenesis data for HXB2 gp41 is in agreement with the recent pre-fusion structure model. The virus infection data suggested that fusion pores sufficiently large enough for the release of the virus genome complex are formed after the completion of 6HB beyond position 648.

Project description:The objective of this study was to track the evolution of sequence changes in both the heptad region 1 (HR1) and HR2 domains of gp41 associated with resistance to enfuvirtide (ENF) in a patient cohort receiving long-term ENF treatment. We studied 17 highly antiretroviral agent-experienced patients receiving long-term ENF treatment with virological rebound or a lack of suppression. Sixty-two samples obtained after between 5 and 107 weeks of ENF therapy were analyzed. Baseline samples from 15 of these 17 patients were available for analysis. Viruses from five samples from four patients were also sequenced after the cessation of ENF therapy. Drug susceptibilities were assessed by a pseudotype virus reporter assay. We identified HR1 and HR2 sequence changes over time in relation to the baseline sequences. Mutations in HR1 (amino acids 36 to 45) were noted in all cases, including previously unreported changes N42Q/H and N43Q. In addition to a range of HR2 sequence changes at polymorphic sites, isolates from 6 of 17 (35%) patients developed an S138A substitution in the HR2 domain at least 8 weeks after the start of ENF treatment and also subsequent to the first emergence of HR1 mutations. In most, but not all, cases the S138A mutation accompanied HR1 mutations at position 43. Molecular modeling demonstrates the close proximity of S138A with amino acids 40 and 45 in HR1. Of note, isolates in samples available from four patients demonstrated the loss of both the HR1 and the S138A HR2 mutations following the cessation of therapy. We show that the S138A HR2 mutation increased the level of resistance by approximately threefold over that conferred by the HR1 mutation N43D. Continual evolution of HR1 in the domain from amino acids 36 to 45 was observed during long-term ENF therapy. We have identified, for the first time, an ENF resistance-associated HR2 mutation, S138A, which appeared in isolates from 6 of 17 patients with virological failure and demonstrated its potential to contribute to drug resistance. We propose that this represents a possible secondary and/or compensatory mutation, particularly when it coexists with mutations at position 43 in HR-1.

Dataset Information

Conserved Residue Asn-145 in the C-Terminal Heptad Repeat Region of HIV-1 gp41 is Critical for Viral Fusion and Regulates the Antiviral Activity of Fusion Inhibitors.

Publications

Conserved Residue Asn-145 in the C-Terminal Heptad Repeat Region of HIV-1 gp41 is Critical for Viral Fusion and Regulates the Antiviral Activity of Fusion Inhibitors.

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