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Journal of Virology, April 2007, p. 3545-3553, Vol. 81, No. 7
0022-538X/07/$08.00+0     doi:10.1128/JVI.01080-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Expression of Rotavirus NSP4 Alters the Actin Network Organization through the Actin Remodeling Protein Cofilin{triangledown}

Zuzana Berkova,1,2 Sue E. Crawford,1 Sarah E. Blutt,1 Andrew P. Morris,2* and Mary K. Estes1*

Baylor College of Medicine, Department of Molecular Virology and Microbiology, One Baylor Plaza,1 University of Texas, Health Science Center, Department of Integrative Biology and Pharmacology, 6341 Fannin, Houston, Texas 770302

Received 25 May 2006/ Accepted 19 December 2006

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.

* Corresponding author. Mailing address for Mary K. Estes: Baylor College of Medicine, Department of Molecular Virology and Microbiology, 1200 Moursund St., Houston, TX 77030-3404. Phone: (713) 798-3585. Fax: (713) 798-3586. E-mail: mestes{at}bcm.edu. Mailing address for Andrew P. Morris: University of Texas, Health Science Center, Department of Integrative Biology and Pharmacology, 6341 Fannin, Houston, TX 77030. Phone: (713) 500-6681. Fax: (713) 500- 7444. E-mail: Andrew.P.Morris{at}uth.tmc.edu.

{triangledown} Published ahead of print on 17 January 2007.

Journal of Virology, April 2007, p. 3545-3553, Vol. 81, No. 7
0022-538X/07/$08.00+0     doi:10.1128/JVI.01080-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.





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