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J. Biol. Chem., Vol. 283, Issue 44, 29822-29830, October 31, 2008

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Avian Sarcoma Virus and Human Immunodeficiency Virus, Type 1 Use Different Subsets of ESCRT Proteins to Facilitate the Budding Process^*

Andrew Pincetic¹, Gisselle Medina², Carol Carter, and Jonathan Leis³

From the Department of Microbiology and Immunology, Northwestern University, Feinberg School of Medicine, Chicago, Illinois 60611 and the Department of Molecular Genetics and Microbiology, State University of New York at Stony Brook, Stony Brook, New York 11794-5222

Members of the Nedd4 family of E3 ubiquitin ligases bind the L domain in avian sarcoma virus (ASV) Gag and facilitate viral particle release. Translational fusion of ASV Gag with an L domain deletion (p2b) to proteins that comprise ESCRT-I, -II, and -III (the endocytic sorting complexes required for transport) rescued both Gag ubiquitination and particle release from cells. The ESCRT-I factors Vps37C or Tsg101 were more effective in rescue of Gag/p2b budding than the ESCRT-II factor Eap20 or the ESCRT-III component CHMP6. Thus ESCRT components can substitute for Nedd4 family members in ASV Gag release. Unlike wild type, ASV Gag/p2b -ESCRT chimeras failed to co-immunoprecipitate with co-expressed hemagglutinin-tagged Nedd4, indicating that Nedd4 was not stably associated with these Gag fusions. Release of the Gag-ESCRT-I or -II fusions was inhibited by a dominant negative mutant of Vps4 ATPase similar to wild type ASV Gag. In contrast to ASV Gag, HIV-1 Gag containing an L domain inactivating mutation (P7L) was efficiently rescued by fusion to a component of ESCRT-III (Chmp6) but not ESCRT-II (Eap20). Depletion of the endogenous pool of Eap20 (ESCRT-II) had little effect on HIV-1 Gag release but blocked ASV Gag release. In contrast, depletion of the endogenous pool of Vps37C (ESCRT-I) had little effect on ASV but blocked HIV-1 Gag release. Furthermore, an N-terminal fragment of Chmp6 inhibited both HIV-1 and ASV Gag release in a dominant negative manner. Taken together, these results indicate that ASV and HIV-1 Gag utilize different combinations of ESCRT proteins to facilitate the budding process, although they share some common elements.

Received for publication, May 30, 2008 , and in revised form, August 21, 2008.

^* This work was supported, in whole or in part, by National Institutes of Health Predoctoral Training Grant 5T32 CA-09176. This work was also supported by United States Public Health Service Grants AI054143 (to J. L.) and AI68463 (to C. C.).

¹ Supported in part by the Training Program in Viral Replication T32 AI060523.

² Supported in part by a W. Burghardt Turner Pre-doctoral Fellowship and by Grant 35583 from the National Science Foundation-Human Resource Development-funded SUNY AGEP Program at Stony Brook University.

³ To whom correspondence should be addressed: Dept. of Microbiology and Immunology, Northwestern University Feinberg School of Medicine, 303 E. Chicago Ave., Chicago, IL 60611. Tel.: 312-503-1166; Fax: 312-503-2790; E-mail: j-leis{at}northwestern.edu.

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