PDF(Pubmed)
Abstract：
Structural dynamics of human immunodeficiency virus 1 (HIV-1) envelope (Env) glycoprotein mediate cell entry and facilitate immune evasion. Single-molecule FRET using peptides for Env labeling revealed structural dynamics of Env, but peptide use risks potential effects on structural integrity/dynamics. While incorporating noncanonical amino acids (ncAAs) into Env by amber stop-codon suppression, followed by click chemistry, offers a minimally invasive approach, this has proved to be technically challenging for HIV-1. Here, we develope an intact amber-free HIV-1 system that overcomes hurdles of preexisting viral amber codons. We achieved dual-ncAA incorporation into Env on amber-free virions, enabling single-molecule Förster resonance energy transfer (smFRET) studies of click-labeled Env that validated the previous peptide-based labeling approaches by confirming the intrinsic propensity of Env to dynamically sample multiple conformational states. Amber-free click-labeled Env also enabled real-time tracking of single virion internalization and trafficking in cells. Our system thus permits in-virus bioorthogonal labeling of proteins, compatible with studies of virus entry, trafficking, and egress from cells.
摘要：
人类免疫缺陷病毒1（HIV-1）包膜（Env）糖蛋白的结构动力学介导细胞进入并促进免疫逃避。使用肽进行Env标记的单分子FRET揭示了Env的结构动力学，但使用肽可能会对结构完整性/动力学产生潜在影响。在通过琥珀色终止密码子抑制将非规范氨基酸（ncAA）掺入Env时，其次是点击化学，提供了一种微创方法，这已被证明对HIV-1具有技术挑战性.这里,我们开发了一个完整的无琥珀的HIV-1系统，克服了先前存在的病毒琥珀密码子的障碍。我们在无琥珀病毒体上实现了双ncAA并入Env，通过确认Env动态采样多个构象状态的内在倾向，实现了对点击标记的Env的单分子Förster共振能量转移（smFRET）研究，该研究验证了先前基于肽的标记方法。无琥珀色的点击标记Env还可以实时跟踪单个病毒体的内化和细胞中的贩运。因此，我们的系统允许蛋白质的病毒内生物正交标记，与病毒进入的研究兼容，贩运，从细胞中出去。
