%0 Journal Article
%T Bioorthogonal click labeling of an amber-free HIV-1 provirus for in-virus single molecule imaging.
%A Ao Y
%A Grover JR
%A Gifford L
%A Han Y
%A Zhong G
%A Katte R
%A Li W
%A Bhattacharjee R
%A Zhang B
%A Sauve S
%A Qin W
%A Ghimire D
%A Haque MA
%A Arthos J
%A Moradi M
%A Mothes W
%A Lemke EA
%A Kwong PD
%A Melikyan GB
%A Lu M
%J Cell Chem Biol
%V 31
%N 3
%D 2024 Mar 21
%M 38232732
%F 9.039
%R 10.1016/j.chembiol.2023.12.017
%X Structural dynamics of human immunodeficiency virus 1 (HIV-1) envelope (Env) glycoprotein mediate cell entry and facilitate immune evasion. Single-molecule FRET using peptides for Env labeling revealed structural dynamics of Env, but peptide use risks potential effects on structural integrity/dynamics. While incorporating noncanonical amino acids (ncAAs) into Env by amber stop-codon suppression, followed by click chemistry, offers a minimally invasive approach, this has proved to be technically challenging for HIV-1. Here, we develope an intact amber-free HIV-1 system that overcomes hurdles of preexisting viral amber codons. We achieved dual-ncAA incorporation into Env on amber-free virions, enabling single-molecule Förster resonance energy transfer (smFRET) studies of click-labeled Env that validated the previous peptide-based labeling approaches by confirming the intrinsic propensity of Env to dynamically sample multiple conformational states. Amber-free click-labeled Env also enabled real-time tracking of single virion internalization and trafficking in cells. Our system thus permits in-virus bioorthogonal labeling of proteins, compatible with studies of virus entry, trafficking, and egress from cells.