{Reference Type}: Journal Article
{Title}: Bioorthogonal click labeling of an amber-free HIV-1 provirus for in-virus single molecule imaging.
{Author}: Ao Y;Grover JR;Gifford L;Han Y;Zhong G;Katte R;Li W;Bhattacharjee R;Zhang B;Sauve S;Qin W;Ghimire D;Haque MA;Arthos J;Moradi M;Mothes W;Lemke EA;Kwong PD;Melikyan GB;Lu M;
{Journal}: Cell Chem Biol
{Volume}: 31
{Issue}: 3
{Year}: 2024 Mar 21
{Factor}: 9.039
{DOI}: 10.1016/j.chembiol.2023.12.017
{Abstract}: 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.