title: Expression of rotavirus NSP4 alters the actin network organization through the actin remodeling protein cofilin.
abstract: Rotavirus is a major cause of infantile gastroenteritis with a multifactorial pathogenesis. As with many other pathogens, rotavirus infection and replication leads to rearrangement of the cytoskeleton with disorganization of cytoskeletal elements such as actin and cytokeratin through a calcium-dependent process that has not been fully characterized. The rotavirus enterotoxin NSP4, shown previously to elevate intracellular calcium levels when added exogenously as well as when expressed intracellularly, is a key player in intracellular calcium regulation during rotavirus infection. Here, we investigated the role NSP4 may play in actin rearrangement. Expression of NSP4 fused to enhanced green fluorescent protein (NSP4-EGFP), but not expression of EGFP alone, caused stabilization of long cellular projections in fully confluent HEK 293 cells. Cells expressing NSP4-EGFP for 24 h were also resistant to cell rounding induced by cytochalasin D. Quantification of filamentous actin (F-actin) content by using rhodamine-conjugated phalloidin and flow cytometry showed an elevated F-actin content in NSP4-EGFP-expressing and rotavirus-infected cells in comparison with that in nonexpressing and noninfected cells. Normalization of intracellular calcium levels prevented alterations of F-actin content. Observed changes in F-actin amounts correlated with the increased activation of the actin-remodeling protein cofilin. These calcium-dependent actin rearrangements induced by intracellular NSP4 expression may contribute to rotavirus pathogenesis by interfering with cellular processes dependent on subcortical actin remodeling, including ion transport and viral release.
authors.affiliation: Department of Molecular Virology and Microbiology, Baylor College of Medicine, 1200 Moursund Street, Houston, TX 77030-3404, USA.
authors.forename: Zuzana
authors.initials: Z
authors.lastname: Berkova
authors.forename: Sue E
authors.initials: SE
authors.lastname: Crawford
authors.forename: Sarah E
authors.initials: SE
authors.lastname: Blutt
authors.forename: Andrew P
authors.initials: AP
authors.lastname: Morris
authors.forename: Mary K
authors.initials: MK
authors.lastname: Estes
mesh_terms: D051339:Actin Depolymerizing Factors / Q000378:metabolism*; D000199:Actins / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D015967:Gene Expression Regulation, Viral; D017930:Genes, Reporter / Q000235:genetics; D006023:Glycoproteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D010766:Phosphorylation; D012401:Rotavirus / Q000235:genetics / Q000378:metabolism*; D014118:Toxins, Biological / Q000235:genetics / Q000378:metabolism*; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D051339:Actin Depolymerizing Factors; D000199:Actins; D006023:Glycoproteins; C081369:NS28 protein, rotavirus; D014118:Toxins, Biological; D017361:Viral Nonstructural Proteins; D002118:Calcium