title: Identification of Host Cell Factors Associated with Astrovirus Replication in Caco-2 Cells.
abstract: Astroviruses are small, nonenveloped viruses with a single-stranded positive-sense RNA genome causing acute gastroenteritis in children and immunocompromised patients. Since positive-sense RNA viruses have frequently been found to replicate in association with membranous structures, in this work we characterized the replication of the human astrovirus serotype 8 strain Yuc8 in Caco-2 cells, using density gradient centrifugation and free-flow zonal electrophoresis (FFZE) to fractionate cellular membranes. Structural and nonstructural viral proteins, positive- and negative-sense viral RNA, and infectious virus particles were found to be associated with a distinct population of membranes separated by FFZE. The cellular proteins associated with this membrane population in infected and mock-infected cells were identified by tandem mass spectrometry. The results indicated that membranes derived from multiple cell organelles were present in the population. Gene ontology and protein-protein interaction network analysis showed that groups of proteins with roles in fatty acid synthesis and ATP biosynthesis were highly enriched in the fractions of this population in infected cells. Based on this information, we investigated by RNA interference the role that some of the identified proteins might have in the replication cycle of the virus. Silencing of the expression of genes involved in cholesterol (DHCR7, CYP51A1) and fatty acid (FASN) synthesis, phosphatidylinositol (PI4KIIIβ) and inositol phosphate (ITPR3) metabolism, and RNA helicase activity (DDX23) significantly decreased the amounts of Yuc8 genomic and antigenomic RNA, synthesis of the structural protein VP90, and virus yield. These results strongly suggest that astrovirus RNA replication and particle assembly take place in association with modified membranes potentially derived from multiple cell organelles.
  
  
  OBJECTIVE
  Astroviruses are common etiological agents of acute gastroenteritis in children and immunocompromised patients. More recently, they have been associated with neurological diseases in mammals, including humans, and are also responsible for different pathologies in birds. In this work, we provide evidence that astrovirus RNA replication and virus assembly occur in contact with cell membranes potentially derived from multiple cell organelles and show that membrane-associated cellular proteins involved in lipid metabolism are required for efficient viral replication. Our findings provide information to enhance our knowledge of astrovirus biology and provide information that might be useful for the development of therapeutic interventions to prevent virus replication.
authors.affiliation: Instituto de Biotecnología, Universidad Nacional Autónoma de México, Colonia Chamilpa, Cuernavaca, Morelos, México.
authors.forename: Andrea
authors.initials: A
authors.lastname: Murillo
authors.affiliation: Instituto de Biotecnología, Universidad Nacional Autónoma de México, Colonia Chamilpa, Cuernavaca, Morelos, México.
authors.forename: Rosario
authors.initials: R
authors.lastname: Vera-Estrella
authors.affiliation: Southern Cross Plant Sciences, Southern Cross University, Lismore, NSW, Australia.
authors.forename: Bronwyn J
authors.initials: BJ
authors.lastname: Barkla
authors.affiliation: Instituto de Biotecnología, Universidad Nacional Autónoma de México, Colonia Chamilpa, Cuernavaca, Morelos, México.
authors.forename: Ernesto
authors.initials: E
authors.lastname: Méndez
authors.affiliation: Instituto de Biotecnología, Universidad Nacional Autónoma de México, Colonia Chamilpa, Cuernavaca, Morelos, México arias@ibt.unam.mx.
authors.forename: Carlos F
authors.initials: CF
authors.lastname: Arias
mesh_terms: D000255:Adenosine Triphosphate / Q000096:biosynthesis; D019200:Astroviridae / Q000235:genetics* / Q000378:metabolism; D018938:Caco-2 Cells; D002458:Cell Fractionation; D053487:DEAD-box RNA Helicases / Q000235:genetics / Q000378:metabolism; D054890:Fatty Acid Synthase, Type I / Q000235:genetics / Q000378:metabolism; D005227:Fatty Acids / Q000096:biosynthesis; D005786:Gene Expression Regulation; D054884:Host-Pathogen Interactions; D006801:Humans; D053496:Inositol 1,4,5-Trisphosphate Receptors / Q000235:genetics / Q000378:metabolism; D007425:Intracellular Membranes / Q000737:chemistry / Q000378:metabolism* / Q000821:virology; D058977:Molecular Sequence Annotation; D044925:Oxidoreductases Acting on CH-CH Group Donors / Q000235:genetics / Q000378:metabolism; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics / Q000378:metabolism; D025941:Protein Interaction Mapping; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D058886:Sterol 14-Demethylase / Q000235:genetics / Q000378:metabolism; D014764:Viral Proteins / Q000235:genetics* / Q000378:metabolism; D014779:Virus Replication / Q000235:genetics*
chemical_list: D000255:Adenosine Triphosphate; D053487:DEAD-box RNA Helicases; D054890:Fatty Acid Synthase, Type I; D005227:Fatty Acids; D053496:Inositol 1,4,5-Trisphosphate Receptors; D044925:Oxidoreductases Acting on CH-CH Group Donors; D017853:Phosphotransferases (Alcohol Group Acceptor); D034741:RNA, Small Interfering; D012367:RNA, Viral; D058886:Sterol 14-Demethylase; D014764:Viral Proteins; C548418:CYP51A1 protein, human; C506929:ITPR3 protein, human; C019995:7-dehydrocholesterol reductase; C518729:FASN protein, human; C581087:PI4KB protein, human; C498290:DDX23 protein, human