An intact amber-free HIV-1 system for in-virus protein bioorthogonal click labeling that delineates envelope conformational dynamics

Abstract The HIV-1 envelope (Env) glycoprotein is conformationally dynamic and mediates membrane fusion required for cell entry. Single-molecule fluorescence resonance energy transfer (smFRET) of Env using peptide tags has provided mechanistic insights into the dynamics of Env conformations. Nevertheless, using peptide tags risks potential effects on structural integrity. Here, we aim to establish minimally invasive smFRET systems of Env on the virus by combining genetic code expansion and bioorthogonal click chemistry. Amber stop-codon suppression allows site-specifically incorporating noncanonical/unnatural amino acids (ncAAs) at introduced amber sites into proteins. However, ncAA incorporation into Env (or other HIV-1 proteins) in the virus context has been challenging due to low copies of Env on virions and incomplete amber suppression in mammalian cells. Here, we developed an intact amber-free virus system that overcomes impediments from preexisting ambers in HIV-1. Using this system, we successfully incorporated dual ncAAs at amber-introduced sites into Env on intact virions. Dual-ncAA incorporated Env retained similar neutralization sensitivities to neutralizing antibodies as wildtype. smFRET of click-labeled Env on intact amber-free virions recapitulated conformational profiles of Env. The amber-free HIV-1 infectious system also permits in-virus protein bioorthogonal labeling, compatible with various advanced microscopic studies of virus entry, trafficking, and egress in living cells.<jats:fig position="float" orientation="portrait" fig-type="figure"><jats:graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="530526v1_ufig1" position="float" orientation="portrait" /></jats:fig>Amber-free HIV-1 infectious systems actualized minimal invasive Env tagging for smFRET, versatile for in-virus bioorthogonal click labeling in advanced microscopic studies of virus-host interactions..

Medienart:	Preprint

Erscheinungsjahr:	2023
Erschienen:	2023

Enthalten in:	bioRxiv.org - (2023) vom: 04. März Zur Gesamtaufnahme - year:2023

Sprache:	Englisch

Beteiligte Personen:	Ao, Yuanyun [VerfasserIn] Grover, Jonathan R. [VerfasserIn] Han, Yang [VerfasserIn] Zhong, Guohua [VerfasserIn] Qin, Wenyi [VerfasserIn] Ghimire, Dibya [VerfasserIn] Haque, Anzarul [VerfasserIn] Bhattacharjee, Rajanya [VerfasserIn] Zhang, Baoshan [VerfasserIn] Arthos, James [VerfasserIn] Lemke, Edward A. [VerfasserIn] Kwong, Peter D. [VerfasserIn] Lu, Maolin [VerfasserIn]

Links:	Volltext [kostenfrei]

Themen:	570 Biology

doi:	10.1101/2023.02.28.530526

funding:
Förderinstitution / Projekttitel:

PPN (Katalog-ID):	XBI038848066