title: Genome-wide RNAi screen reveals a role for the ESCRT complex in rotavirus cell entry.
abstract: 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.
authors.affiliation: Departamento de Genética del Desarrollo y Fisiología Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Colonia Chamilpa, Cuernavaca, Morelos 62210, Mexico.
authors.forename: Daniela
authors.initials: D
authors.lastname: Silva-Ayala
authors.forename: Tomás
authors.initials: T
authors.lastname: López
authors.forename: Michelle
authors.initials: M
authors.lastname: Gutiérrez
authors.forename: Norbert
authors.initials: N
authors.lastname: Perrimon
authors.forename: Susana
authors.initials: S
authors.lastname: López
authors.forename: Carlos F
authors.initials: CF
authors.lastname: Arias
mesh_terms: D000074183:ATPases Associated with Diverse Cellular Activities; D000818:Animals; D018938:Caco-2 Cells; D002522:Chlorocebus aethiops; D056827:Endosomal Sorting Complexes Required for Transport / Q000235:genetics* / Q000378:metabolism*; D055106:Genome-Wide Association Study; D006801:Humans; D021381:Protein Transport / Q000502:physiology; D034622:RNA Interference; D012401:Rotavirus / Q000378:metabolism*; D012400:Rotavirus Infections / Q000378:metabolism* / Q000821:virology; D022161:Transport Vesicles / Q000378:metabolism* / Q000821:virology*; D025262:Vacuolar Proton-Translocating ATPases / Q000378:metabolism; D014709:Vero Cells; D033921:Vesicular Transport Proteins / Q000378:metabolism; D020764:cdc42 GTP-Binding Protein / Q000378:metabolism; D020691:rab GTP-Binding Proteins / Q000378:metabolism; D020696:rab5 GTP-Binding Proteins / Q000378:metabolism; D000090802:rab7 GTP-Binding Proteins; D020742:rhoA GTP-Binding Protein / Q000378:metabolism
chemical_list: D056827:Endosomal Sorting Complexes Required for Transport; D033921:Vesicular Transport Proteins; C093846:early endosome antigen 1; D000090802:rab7 GTP-Binding Proteins; D025262:Vacuolar Proton-Translocating ATPases; D000074183:ATPases Associated with Diverse Cellular Activities; C490305:VPS4A protein, human; D020764:cdc42 GTP-Binding Protein; D020691:rab GTP-Binding Proteins; D020696:rab5 GTP-Binding Proteins; D020742:rhoA GTP-Binding Protein