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Journal of Virology, June 2006, p. 6061-6071, Vol. 80, No. 12
0022-538X/06/$08.00+0     doi:10.1128/JVI.02167-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Rotavirus NSP4 Induces a Novel Vesicular Compartment Regulated by Calcium and Associated with Viroplasms

Z. Berkova,1,2 S. E. Crawford,1 G. Trugnan,3 T. Yoshimori,4 A. P. Morris,2* and M. K. Estes1*

Baylor College of Medicine, Department of Molecular Virology and Microbiology, One Baylor Plaza, Houston, Texas 77030,1 University of Texas, Health Science Center, Department of Integrative Biology and Pharmacology, 6431 Fannin, Houston, Texas 77030,2 INSERM UMR538, UPMC, CHU St. Antoine, 27 rue Chaligny, 75014 Paris, France,3 National Institute of Genetics, Department of Cell Genetics, 1111, Yata, Mishima, Shizuoka 411-8540, Japan4

Received 14 October 2005/ Accepted 22 March 2006

Rotavirus is a major cause of infantile viral gastroenteritis. Rotavirus nonstructural protein 4 (NSP4) has pleiotropic properties and functions in viral morphogenesis as well as pathogenesis. Recent reports show that the inhibition of NSP4 expression by small interfering RNAs leads to alteration of the production and distribution of other viral proteins and mRNA synthesis, suggesting that NSP4 also affects virus replication by unknown mechanisms. This report describes studies aimed at correlating the localization of intracellular NSP4 in cells with its functions. To be able to follow the localization of NSP4, we fused the C terminus of full-length NSP4 with the enhanced green fluorescent protein (EGFP) and expressed this fusion protein inducibly in a HEK 293-based cell line to avoid possible cytotoxicity. NSP4-EGFP was initially localized in the endoplasmic reticulum (ER) as documented by Endo H-sensitive glycosylation and colocalization with ER marker proteins. Only a small fraction of NSP4-EGFP colocalized with the ER-Golgi intermediate compartment (ERGIC) marker ERGIC-53. NSP4-EGFP did not enter the Golgi apparatus, in agreement with the Endo H sensitivity and a previous report that secretion of an NSP4 cleavage product generated in rotavirus-infected cells is not inhibited by brefeldin A. A significant population of expressed NSP4-EGFP was distributed in novel vesicular structures throughout the cytoplasm, not colocalizing with ER, ERGIC, Golgi, endosomal, or lysosomal markers, thus diverging from known biosynthetic pathways. The appearance of vesicular NSP4-EGFP was dependent on intracellular calcium levels, and vesicular NSP4-EGFP colocalized with the autophagosomal marker LC3. In rotavirus-infected cells, NSP4 colocalized with LC3 in cap-like structures associated with viroplasms, the site of nascent viral RNA replication, suggesting a possible new mechanism for the involvement of NSP4 in virus replication.

* Corresponding author. Mailing address for M. K. Estes: Baylor College of Medicine, Department of Molecular Virology and Microbiology, One Baylor Plaza, Houston, TX 77030-3404. Phone: (713) 798-3585. Fax: (713) 798-3586. E-mail: mestes{at}bcm.tmc.edu. Mailing address for A. P. Morris: UT-HSC Medical School, Department of Integrative Biology and Pharmacology, 6431 Fannin, Houston, TX 77030. Phone: (713) 500-6681. Fax: (713) 500-6699. E-mail: andrew.p.morris{at}uth.tmc.edu.

Journal of Virology, June 2006, p. 6061-6071, Vol. 80, No. 12
0022-538X/06/$08.00+0     doi:10.1128/JVI.02167-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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