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.

Viral fusion proteins are indispensable mediators of viral entry that orchestrate the fusion of viral and host membranes, making them primary targets for antiviral interventions. Class I fusion proteins, displayed on the surface of enveloped viruses (such as HIV-1, RSV, SARS-CoV-2, Nipah, influenza, and Ebola viruses), share conserved structural features, including the fusion peptide or loop and heptad repeat regions. These elements are essential for the formation of the post-fusion six-helix bundle during membrane fusion. Peptide inhibitors that mimic heptad repeat motifs have consequently emerged as an effective strategy for blocking the fusion process. This review summarizes design strategies for such inhibitors and highlights how sequence and structural insights have enabled their optimization via α-helical stabilization, hydrocarbon stapling, lactam bridges, lipid conjugation, macrocyclization, and multivalency. Using representative examples across major viral systems, this review illustrates how these strategies have led to the development of potent, stable, and even broad-spectrum antiviral peptides. This review provides insights to guide the rational design of next-generation peptide-based fusion inhibitors targeting viral membrane fusion.