Expression of rotavirus NSP4 alters the actin network organization through the actin remodeling protein cofilin.
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.
Berkova
Zuzana
Z
Department of Molecular Virology and Microbiology, Baylor College of Medicine, 1200 Moursund Street, Houston, TX 77030-3404, USA.
Crawford
Sue E
SE
Blutt
Sarah E
SE
Morris
Andrew P
AP
Estes
Mary K
MK
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*
D051339:Actin Depolymerizing Factors; D000199:Actins; D006023:Glycoproteins; C081369:NS28 protein, rotavirus; D014118:Toxins, Biological; D017361:Viral Nonstructural Proteins; D002118:Calcium