Genetic code expansion for single noncanonical amino acid incorporation into SARS-CoV-2 Omicron spike on amber-free lentiviral particles.

Fluorescent labeling or tagging of viral proteins is crucial for understanding molecular virology and guiding antiviral interventions. However, traditional methods using large tags are often invasive, impeding visualization of viral proteins in their native states. We detail a methodology combining an amber-free lentiviral packaging system with amber (TAG stop codon) suppression - genetic code expansion and site-specific bioorthogonal click labeling. Amber suppression enables site-specific incorporation of a non-canonical amino acid (ncAA) bearing strained alkynes or alkenes into a target protein. The ncAA-containing viral protein can then be labeled with a fluorophore conjugated with a click-chemistry-reactive functional group via copper-free click chemistry, allowing single-residue fluorescent labeling with minimal impact on protein structure and function. However, amber stop codons that serve as native termination signals in viral genomes can be inadvertently suppressed during genetic code expansion, leading to protein readthrough and unpredictable experimental outcomes. Here, we provide a step-by-step method for constructing a complete amber-free lentivirus, which overcomes this limitation by providing an amber-free viral background. We then describe a stepwise workflow for single ncAA incorporation into the Omicron spike decorated on amber-free lentiviruses, followed by the attachment of organic fluorophores via click chemistry. The methods described here are, in principle, adaptable to other viral surface glycoproteins, non-surface lentiviral proteins, and pseudotyped viral systems, with appropriate optimization and validation as needed.