Bioorthogonal click labeling of an amber-free HIV-1 provirus for in-virus single molecule imaging

Yuanyun Ao, Jonathan R. Grover, Levi Gifford, Yang Han, Guohua Zhong, Revansiddha Katte, Wenwei Li, Rajanya Bhattacharjee, Baoshan Zhang, Stephanie Sauve, Wenyi Qin, Dibya Ghimire, Md Anzarul Haque, James Arthos, Mahmoud Moradi, Walther Mothes, Edward A. Lemke, Peter D. Kwong, Gregory B. Melikyan, Maolin Lu

Research output: Contribution to journalArticlepeer-review


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.

Original languageEnglish (US)
Pages (from-to)487-501.e7
JournalCell Chemical Biology
Issue number3
StatePublished - Mar 21 2024
Externally publishedYes


  • amber suppression
  • amber-free provirus
  • bioorthogonal labeling
  • click chemistry
  • envelope glycoprotein
  • genetic code expansion
  • HIV-1
  • single virus tracking
  • single-molecule FRET
  • single-molecule imaging

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Medicine
  • Molecular Biology
  • Pharmacology
  • Drug Discovery
  • Clinical Biochemistry


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