Protocol for click labeling of HIV-1 envelope on amber-free virions prepared using genetic code expansion

Site-specific fluorescent labeling of HIV-1 proteins enables direct visualization of their dynamics and trafficking. Here, we present a protocol to label HIV-1 envelope (Env) glycoproteins on virions using genetic code expansion and click chemistry. We describe steps for using HEK293T cells to produce amber-free HIV-1 carrying a noncanonical amino acid (ncAA) and for fluorophore conjugation to the ncAA. This protocol offers a practical way to apply amber suppression and click chemistry to track viral proteins during the infection lifecycle. For complete details on the use and execution of this protocol, please refer to Ao et al.¹.