biostudies-literatureAo YNational Institute of Allergy and Infectious DiseasesGilead Research Scholars ProgramamfARVaccine Research CenterAmerican Foundation for Aging ResearchNIAID NIH HHSNational Institutes of HealthEngineering Research CentersNational Institute of General Medical SciencesGilead SciencesNIGMS NIH HHSDivision of Intramural Research487-501.e7https://www.ebi.ac.uk/biostudies/studies/S-EPMC10960674biostudies-literatureUnknown31(3)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 thCell chemical biologyBioorthogonal click labeling of an amber-free HIV-1 provirus for in-virus single molecule imaging.PMC10960674R01 AI129862R01 AI181600U54 AI170752R35 GM147423R56 AI170101R37 AI150560R35GM147423R01 AI150560Arthos JHan YQin WHaque MALemke EALi WMothes WLu MKatte RMelikyan GBAo YZhong GGrover JRGifford LSauve SGhimire DZhang BKwong PDBhattacharjee RMoradi MfalseBioorthogonal click labeling of an amber-free HIV-1 provirus for in-virus single molecule imaging.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 th2024-01-01T00:00:00Z2024 Mar2026-06-01T19:04:27.466Z2025-04-04T03:03:45.199ZS-EPMC109606743823273210.1016/j.chembiol.2023.12.017