An intact amber-free HIV-1 system for in-virus protein bioorthogonal click labeling that delineates envelope conformational dynamics

Abstract

The HIV-1 envelope (Env) glycoprotein is conformationally dynamic and mediates membrane fusion required for cell entry. Single-molecule fluorescence resonance energy transfer (smFRET) of Env using peptide tags has provided mechanistic insights into the dynamics of Env conformations. Nevertheless, using peptide tags risks potential effects on structural integrity. Here, we aim to establish minimally invasive smFRET systems of Env on the virus by combining genetic code expansion and bioorthogonal click chemistry. Amber stop-codon suppression allows site-specifically incorporating noncanonical/unnatural amino acids (ncAAs) at introduced amber sites into proteins. However, ncAA incorporation into Env (or other HIV-1 proteins) in the virus context has been challenging due to low copies of Env on virions and incomplete amber suppression in mammalian cells. Here, we developed an intact amber-free virus system that overcomes impediments from preexisting ambers in HIV-1. Using this system, we successfully incorporated dual ncAAs at amber-introduced sites into Env on intact virions. Dual-ncAA incorporated Env retained similar neutralization sensitivities to neutralizing antibodies as wildtype. smFRET of click-labeled Env on intact amber-free virions recapitulated conformational profiles of Env. The amber-free HIV-1 infectious system also permits in-virus protein bioorthogonal labeling, compatible with various advanced microscopic studies of virus entry, trafficking, and egress in living cells.Amber-free HIV-1 infectious systems actualized minimal invasive Env tagging for smFRET, versatile for in-virus bioorthogonal click labeling in advanced microscopic studies of virus-host interactions.

HIV-1 Capsid Uncoating Is a Multistep Process That Proceeds through Defect Formation Followed by Disassembly of the Capsid Lattice

“…Specificity of CA* labeling is evident from the lack of discernible SiR puncta or background signal, outside of p24 immunostained cores that contain WT CA (Figure S3A). Although recent reports suggest that HIV-1 backbone possesses amber codons in Vif, Vpu, and Rev, the lack of off-target staining of the WT CA vector in our experiments (Figure S3A) likely reflects the absence of these proteins in budding virions.…”

Human Immunodeficiency Virus 1 Capsid Uncoating in the Nucleus Progresses Through Defect Formation in the Capsid Lattice

“…S1A). Although recent reports suggest that HIV-1 backbone is click-labeled, unless all amber codons in Vif, Vpu, and Rev are mutated, 59 the lack of off-target staining of the WT CA vector in our experiments(Fig. 1A) likely reflects the absence of these proteins in budding virions.…”