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Journal of Virology, December 2004, p. 13943-13953, Vol. 78, No. 24
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.24.13943-13953.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Role of Nedd4 and Ubiquitination of Rous Sarcoma Virus Gag in Budding of Virus-Like Particles from Cells

Marcy L. Vana,1 Yi Tang,2 Aiping Chen,1 Gisselle Medina,3 Carol Carter,3 and Jonathan Leis1*

Department of Microbiology and Immunology, Feinberg School of Medicine, Northwestern University,1 Department of Pathology, Children's Memorial Hospital, Chicago, Illinois,2 Department of Molecular Genetics and Microbiology, State University of New York, Stony Brook, New York3

Received 24 May 2004/ Accepted 19 July 2004

Rous sarcoma virus (RSV) budding requires an interaction of the L domain within the p2b region of Gag with cellular Nedd4-family E3 ubiquitin protein ligases. Members of our laboratories previously demonstrated that overexpression of a fragment of the chicken Nedd4-like protein (LDI-1 WW) inhibits Gag release in a dominant-negative manner (A. Kikonyogo, F. Bouamr, M. L. Vana, Y. Xiang, A. Aiyar, C. Carter, and J. Leis, Proc. Natl. Acad. Sci. USA 98:11199-11204, 2001). We have now identified the complete 3' end of LDI-1 and determined that it has a C-terminal ubiquitin ligase HECT domain, similar to other Nedd4 family members. While overexpression of the full-length LDI-1 clone (LDI-1 FL) had little effect on Gag budding, an LDI-1 FL mutant with a substitution in the HECT domain catalytic site blocked Gag release, similar to LDI-1 WW. The coexpression of Gag and hemagglutinin-tagged ubiquitin (HA-Ub) resulted in the detection of mono- and polyubiquitinated forms of Gag in cells and mostly monoubiquitinated Gag in virus-like particles (VLPs). When the Nedd4-binding site (L domain) was deleted, ubiquitinated Gag was not detected. Interestingly, the release of Gag with ubiquitin covalently linked to the C terminus (Gag-Ub) was still blocked by LDI-1 WW. To understand the mechanism of this inhibition, we examined cells expressing Gag and LDI-1 WW by electron microscopy. In the presence of LDI-1 WW, VLPs were found in electron-dense inclusion bodies in the cytoplasm of transfected cells. In contrast, when cells that coexpressed Gag-Ub and LDI-1 WW were examined, inclusion bodies were detected but did not contain VLPs. These results indicate that the ubiquitination of Gag is dependent upon Nedd4 binding to the L domain and suggest that Nedd4 has additional functions during RSV release besides the ubiquitination of Gag.

* Corresponding author. Mailing address: Department of Microbiology and Immunology, Feinberg School of Medicine, Northwestern University, 303 East Chicago Ave., Chicago, IL 60611. Phone: (312) 503-0338. Fax: (312) 503-2790. E-mail: j-leis{at}northwestern.edu.

Journal of Virology, December 2004, p. 13943-13953, Vol. 78, No. 24
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.24.13943-13953.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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