Departamento de Genética del Desarrollo y Fisiología Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Colonia Chamilpa, Cuernavaca, Morelos 62210, Mexico.
Proc Natl Acad Sci U S A. 2013 Jun 18;110(25):10270-5. doi: 10.1073/pnas.1304932110. Epub 2013 Jun 3.
Rotavirus (RV) is the major cause of childhood gastroenteritis worldwide. This study presents a functional genome-scale analysis of cellular proteins and pathways relevant for RV infection using RNAi. Among the 522 proteins selected in the screen for their ability to affect viral infectivity, an enriched group that participates in endocytic processes was identified. Within these proteins, subunits of the vacuolar ATPase, small GTPases, actinin 4, and, of special interest, components of the endosomal sorting complex required for transport (ESCRT) machinery were found. Here we provide evidence for a role of the ESCRT complex in the entry of simian and human RV strains in both monkey and human epithelial cells. In addition, the ESCRT-associated ATPase VPS4A and phospholipid lysobisphosphatidic acid, both crucial for the formation of intralumenal vesicles in multivesicular bodies, were also found to be required for cell entry. Interestingly, it seems that regardless of the molecules that rhesus RV and human RV strains use for cell-surface attachment and the distinct endocytic pathway used, all these viruses converge in early endosomes and use multivesicular bodies for cell entry. Furthermore, the small GTPases RHOA and CDC42, which regulate different types of clathrin-independent endocytosis, as well as early endosomal antigen 1 (EEA1), were found to be involved in this process. This work reports the direct involvement of the ESCRT machinery in the life cycle of a nonenveloped virus and highlights the complex mechanism that these viruses use to enter cells. It also illustrates the efficiency of high-throughput RNAi screenings as genetic tools for comprehensively studying the interaction between viruses and their host cells.
轮状病毒（RV）是全世界儿童肠胃炎的主要原因。本研究采用 RNAi 技术对与 RV 感染相关的细胞蛋白和途径进行了功能基因组规模的分析。在筛选出的 522 种能够影响病毒感染力的蛋白质中，发现了一组富含参与内吞作用的蛋白质。在这些蛋白质中，液泡 ATP 酶的亚基、小 GTP 酶、肌动蛋白 4，以及特别感兴趣的内体分选复合物所需的成分（ESCRT）机械被发现。在这里，我们提供了 ESCRT 复合物在灵长类和人类 RV 株在猴和人上皮细胞中进入的证据。此外，还发现 ESCRT 相关的 ATP 酶 VPS4A 和磷脂 Lysobisphosphatidic acid 对于多泡体中腔内小泡的形成至关重要，这对于细胞进入也是必需的。有趣的是，似乎无论恒河猴 RV 和人类 RV 株使用哪些分子进行细胞表面附着以及使用不同的内吞途径，所有这些病毒都在早期内体中汇聚，并利用多泡体进行细胞进入。此外，调节不同类型的网格蛋白非依赖性内吞作用的小 GTP 酶 RHOA 和 CDC42 以及早期内体抗原 1（EEA1）也被发现参与了这一过程。这项工作报告了非包膜病毒生命周期中 ESCRT 机械的直接参与，并强调了这些病毒进入细胞所使用的复杂机制。它还说明了高通量 RNAi 筛选作为全面研究病毒与其宿主细胞相互作用的遗传工具的效率。
