Structural and Functional Characterization of the Secondary Mutation N126K Selected by Various HIV-1 Fusion Inhibitors.

Danwei Yu,Yuanmei Zhu,Huihui Chong,Bo Qin,Xiaohui Ding,Yang Su,Yuxian He,Sheng Cui

doi:10.3390/v12030326

Danwei Yu, Yuanmei Zhu + Show 6 more

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https://doi.org/10.3390/v12030326

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Abstract

Peptides derived from the C-terminal heptad repeat (CHR) region of HIV-1 gp41 is potent viral membrane fusion inhibitors, such as the first clinically approved peptide drug T20 and a group of newly-designed peptides. The resistance profiles of various HIV-1 fusion inhibitors were previously characterized, and the secondary mutation N126K in the gp41 CHR was routinely identified during the in vitro and in vivo selections. In this study, the functional and structural relevance of the N126K mutation has been characterized from multiple angles. First, we show that a single N126K mutation across several HIV-1 isolates conferred mild to moderate cross-resistances. Second, the N126K mutation exerted different effects on Env-mediated HIV-1 entry and cell-cell fusion. Third, the N126K mutation did not interfere with the expression and processing of viral Env glycoproteins, but it disrupted the Asn126-based glycosylation site in gp41. Fourth, the N126K mutation was verified to enhance the thermal stability of 6-HB conformation. Fifth, we determined the crystal structure of a 6-HB bearing the N126K mutation, which revealed the interhelical and intrahelical interactions underlying the increased thermostability. Therefore, our data provide new information to understand the mechanism of HIV-1 gp41-mediated cell fusion and its resistance mode to viral fusion inhibitors.

Highlights

Human immunodeficiency virus type 1 (HIV-1) entry into target cells is mediated by its viral envelope (Env) glycoprotein, which is initially synthesized as a fusion-inactive precursor polypeptide and cleaved into the non-covalently surface subunit gp120 and transmembrane subunit gp41 [1,2]
The corresponding Env pseudotyped viruses were generated to determine the effect of the introduced N126K substitution on the sensitivity of diverse HIV-1 isolates to several fusion inhibitors, including T20, C34, SFT, SC22EK, MTSC22EK, and HP23
In the context of the secondary mutation N126K in the C-terminal heptad repeat (CHR) of HIV-1 gp41 was commonly observed in the selection of mutant viruses resistant to various fusion inhibitors, here we characterized its biological relevance and structural properties by multiple approaches

Summary

Introduction

Human immunodeficiency virus type 1 (HIV-1) entry into target cells is mediated by its viral envelope (Env) glycoprotein, which is initially synthesized as a fusion-inactive precursor polypeptide (gp160) and cleaved into the non-covalently surface subunit gp120 and transmembrane subunit gp41 [1,2]. The FP of gp is inserted into the cell membrane, leading an extended prehairpin state that transiently bridges the cell and viral membranes; three CHR helices fold in an antiparallel orientation into the trimeric NHR coiled coil grooves, resulting in a six-helix bundle (6-HB) structure that pulls the two membranes in apposition. Viruses 2020, 12, x FOR PEER REVIEW Viruses. 2020, 12, prehairpin state that transiently bridges the cell and viral membranes; three CHR helices of fold in an antiparallel orientation into the trimeric NHR coiled coil grooves, resulting in a six-helix bundle (6-HB) structure that pulls the two membranes in apposition for fusion [3,4]. Two hydrophobic pockets in the C-terminal portion of the NHR helices play important hydrophobic pockets in the C-terminal portion of the NHR helices play important roles for the roles for the stability of 6-HB, being considered ideal target sites for anti-HIV agents [2,5,6,7,8]

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