Department of Cell Biology, Albert Einstein College of Medicine, Bronx, New York, USA.
Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, New York, USA Gruss Lipper Biophotonics Center, Albert Einstein College of Medicine, Bronx, New York, USA.
J Virol. 2014 Jun;88(12):6922-33. doi: 10.1128/JVI.00592-14. Epub 2014 Apr 2.
Alphaviruses are small enveloped RNA viruses with highly organized structures that exclude host cell proteins. They contain an internal nucleocapsid and an external lattice of the viral E2 and E1 transmembrane proteins. Alphaviruses bud from the plasma membrane (PM), but the process and dynamics of alphavirus assembly and budding are poorly understood. Here we generated Sindbis viruses (SINVs) with fluorescent protein labels on the E2 envelope protein and exploited them to characterize virus assembly and budding in living cells. During virus infection, E2 became enriched in localized patches on the PM and in filopodium-like extensions. These E2-labeled patches and extensions contained all of the viral structural proteins. Correlative light and electron microscopy studies established that the patches and extensions colocalized with virus budding structures, while light microscopy showed that they excluded a freely diffusing PM marker protein. Exclusion required the interaction of the E2 protein with the capsid protein, a critical step in virus budding, and was associated with the immobilization of the envelope proteins on the cell surface. Virus infection induced two distinct types of extensions: tubulin-negative extensions that were ∼2 to 4 μm in length and excluded the PM marker, and tubulin-positive extensions that were >10 μm long, contained the PM marker, and could transfer virus particles to noninfected cells. Tubulin-positive extensions were selectively reduced in cells infected with a nonbudding SINV mutant. Together, our data support a model in which alphavirus infection induces reorganization of the PM and cytoskeleton, leading to virus budding from specialized sites.
Alphaviruses are important and widely distributed human pathogens for which vaccines and antiviral therapies are urgently needed. These small highly organized viruses bud from the host cell PM. Virus assembly and budding are critical but little understood steps in the alphavirus life cycle. We developed alphaviruses with fluorescent protein tags on one of the viral membrane (envelope) proteins and used a variety of microscopy techniques to follow the envelope protein and a host cell PM protein during budding. We showed that alphavirus infection induced the formation of patches and extensions on the PM where the envelope proteins accumulate. These sites excluded other PM proteins and correlated with virus budding structures. Exclusion of PM proteins required specific interactions of the viral envelope proteins with the internal capsid protein. Together, our data indicate that alphaviruses extensively reorganize the cell surface and cytoskeleton to promote their assembly and budding.
甲病毒是一类具有高度组织化结构的小 RNA 包膜病毒，不包含宿主细胞蛋白。它们包含一个内部核衣壳和一个由病毒 E2 和 E1 跨膜蛋白构成的外部晶格。甲病毒从质膜（PM）出芽，但对甲病毒组装和出芽的过程和动力学知之甚少。在这里，我们在 E2 包膜蛋白上生成了带有荧光蛋白标签的辛德毕斯病毒（SINVs），并利用它们来描述活细胞中的病毒组装和出芽。在病毒感染过程中，E2 在 PM 上的局灶斑和丝状伪足样延伸处富集。这些带有 E2 标签的斑和延伸包含了所有的病毒结构蛋白。光镜和电子显微镜的相关研究表明，这些斑和延伸与病毒出芽结构共定位，而光镜显示它们排除了一种自由扩散的 PM 标记蛋白。这种排除需要 E2 蛋白与衣壳蛋白的相互作用，这是病毒出芽的关键步骤，并且与包膜蛋白在细胞表面的固定化有关。病毒感染诱导了两种不同类型的延伸：长度约为 2 到 4 微米的微管阴性延伸，排除了 PM 标记物；以及长度大于 10 微米的微管阳性延伸，包含 PM 标记物，并且可以将病毒颗粒转移到非感染细胞。在感染非出芽性辛德毕斯病毒突变体的细胞中，微管阳性延伸选择性减少。总的来说，我们的数据支持一种模型，即甲病毒感染诱导 PM 和细胞骨架的重排，导致病毒从特定位点出芽。
甲病毒是重要且广泛分布的人类病原体，迫切需要疫苗和抗病毒疗法。这些小而高度组织化的病毒从宿主细胞 PM 出芽。病毒组装和出芽是甲病毒生命周期中关键但知之甚少的步骤。我们开发了带有荧光蛋白标签的甲病毒，标签位于病毒膜（包膜）蛋白之一上，并使用各种显微镜技术在出芽过程中跟踪包膜蛋白和宿主细胞 PM 蛋白。我们表明，甲病毒感染诱导 PM 上形成斑块和延伸，包膜蛋白在这些部位聚集。这些部位排除了其他 PM 蛋白，并与病毒出芽结构相关。PM 蛋白的排除需要病毒包膜蛋白与内部衣壳蛋白的特定相互作用。总的来说，我们的数据表明，甲病毒广泛重组细胞膜和细胞骨架，以促进其组装和出芽。
