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Journal of Virology, April 2006, p. 3935-3946, Vol. 80, No. 8
0022-538X/06/$08.00+0     doi:10.1128/JVI.80.8.3935-3946.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Dissecting Rotavirus Particle-Raft Interaction with Small Interfering RNAs: Insights into Rotavirus Transit through the Secretory Pathway

Mariela A. Cuadras,1,2 Bruno B. Bordier,1,{dagger} Jose L. Zambrano,4 Juan E. Ludert,4 and Harry B. Greenberg1,2,3*

Division of Gastroenterology and Hepatology, Department of Medicine,1 Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California 94305,2 VA Palo Alto Health Care System, Palo Alto, California 94304,3 Laboratorio de Biología de Virus, Centro de Microbiología y Biología Celular, Instituto Venezolano de Investigaciones Científicas (IVIC), Caracas 1020-A, Venezuela4

Received 18 November 2005/ Accepted 3 February 2006

Studies of rotavirus morphogenesis, transport, and release have shown that although these viruses are released from the apical surface of polarized intestinal cells before cellular lysis, they do not follow the classic exocytic pathway. Furthermore, increasing evidence suggests that lipid rafts actively participate in the exit of rotavirus from the infected cell. In this study, we silenced the expression of VP4, VP7, and NSP4 by using small interfering RNAs (siRNAs) and evaluated the effect of shutting down the expression of these proteins on rotavirus-raft interactions. Silencing of VP4 and NSP4 reduced the association of rotavirus particles with rafts; in contrast, inhibition of VP7 synthesis slightly affected the migration of virions into rafts. We found that inhibition of rotavirus migration into lipid rafts, by either siRNAs or tunicamycin, also specifically blocked the targeting of VP4 to rafts, suggesting that the association of VP4 with rafts is mostly mediated by the formation of viral particles in the endoplasmic reticulum (ER). We showed that two populations of VP4 exist, one small population that is independently targeted to rafts and a second large pool of VP4 whose association with rafts is mediated by particle formation in the ER. We also present evidence to support the hypothesis that assembly of VP4 into mature virions takes place in the late stages of transit through the ER. Finally, we analyzed the progression of rotavirus proteins in the exocytic pathway and found that VP4 and virion-assembled VP7 colocalized with ERGIC-53, suggesting that rotavirus particles transit through the intermediate compartment between the ER and the Golgi complex.

* Corresponding author. Mailing address: VAPACHS, 3801 Miranda Ave., MC 154C, Palo Alto, CA 94304. Phone: (650) 493-5000, ext. 63121. Fax: (650) 852-3259. E-mail: hbgreen{at}stanford.edu.

{dagger} Present address: Biolumine SA, UMR CNRS 6144 GEPEA/ERTint 1052 CBAC, Campus de la Courtaisière, Institut Universitaire de Technologie, 85035 La Roche sur Yon, France.

Journal of Virology, April 2006, p. 3935-3946, Vol. 80, No. 8
0022-538X/06/$08.00+0     doi:10.1128/JVI.80.8.3935-3946.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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