Mitrovich Margaret Dianne, Vahey Michael D
Department of Biomedical Engineering, Washington University in St. Louis, St. Louis, Missouri 63130, United States.
Center for Biomolecular Condensates, Washington University in St. Louis, St. Louis, Missouri 63130, United States.
ACS Infect Dis. 2025 Jan 10;11(1):95-103. doi: 10.1021/acsinfecdis.4c00321. Epub 2025 Jan 1.
RNA viruses possess small genomes encoding a limited repertoire of essential and often multifunctional proteins. Although genetically tagging viral proteins provides a powerful tool for dissecting mechanisms of viral replication and infection, it remains a challenge. Here, we leverage genetic code expansion to develop a recoded strain of respiratory syncytial virus (RSV) in which the multifunctional nucleoprotein is site-specifically modified with a noncanonical amino acid. The resulting virus replicates exclusively in cells capable of amber stop codon suppression and is amenable to labeling with tetrazine-modified fluorophores, achieving high signal to background. Virus with labeled nucleoprotein remains functional, retaining ∼70% infectivity relative to unlabeled controls. We leverage this tool to visualize RSV assembly, capturing the transfer of nucleoprotein complexes from cytoplasmic condensates directly to budding viral filaments at the cell surface and to cytoplasmic compartments containing viral surface proteins. Collectively, these results suggest multiple pathways for RSV assembly and establish a framework that may be extended to other viral nucleoproteins.
RNA病毒拥有小基因组，编码有限的一组必需且通常具有多种功能的蛋白质。尽管对病毒蛋白进行基因标记为剖析病毒复制和感染机制提供了强大工具，但这仍然是一项挑战。在此，我们利用遗传密码扩展技术开发了一种呼吸道合胞病毒（RSV）的重新编码毒株，其中多功能核蛋白被非天然氨基酸进行位点特异性修饰。产生的病毒仅在能够抑制琥珀色终止密码子的细胞中复制，并且适合用四嗪修饰的荧光团进行标记，实现了高信号背景比。带有标记核蛋白的病毒仍具功能，相对于未标记的对照保留约70%的感染力。我们利用这一工具可视化RSV组装过程，捕捉到核蛋白复合物从细胞质凝聚物直接转移到细胞表面正在出芽的病毒丝以及含有病毒表面蛋白的细胞质区室。总体而言，这些结果提示了RSV组装的多种途径，并建立了一个可扩展到其他病毒核蛋白的框架。
