Bioorthogonal click-chemistry labeling and visualization of dual noncanonical amino acid-incorporated HIV-1 Env on intact virions.

Site-specific fluorescent tagging enables visualization of protein conformational changes and molecular interactions on native biological assemblies. Using the HIV-1 envelope (Env) glycoprotein as an example, this protocol details a site-specific dual-fluorophore tagging strategy suitable for downstream single-molecule Förster resonance energy transfer (smFRET) studies. Genetic code expansion via amber (TAG stop codon) suppression, combined with click chemistry, offers a minimally invasive route to such labeling. However, the sequential incorporation of noncanonical amino acids (ncAAs) at two introduced amber codons often faces low efficiency and off-target readthrough at naturally occurring amber sites in the viral genome. To mitigate these technical constraints, the methods employ an engineered "intact amber-free HIV-1 provirus" in which endogenous TAG codons in essential viral genes are replaced with TAA. This design allows precise incorporation of ncAA trans-cyclooct-2-en-L-lysine (TCO*A) exclusively at two engineered amber sites within Env during virion production in HEK293T cells, followed by labeling via strain-promoted inverse electron-demand Diels-Alder cycloaddition with tetrazine-conjugated fluorophores. The method supports site-specific dual-click labeling for smFRET analysis and single-site labeling for live-cell imaging. The methods presented here provide stepwise procedures for click-chemistry labeling and visualization of Env on intact virions and can be easily adapted for single- or dual-site biorthogonal tagging of other viral and cellular proteins.