Like other retroviruses, HIV-1 encodes a sequence within the Gag protein termed a late or 'L'-domain that is required for the scission of a nascent virion from the virus producing cell. L-domains are functionally interchangeable among different retroviruses and act by recruiting host-cell factors. In HIV-1, this activity is provided by a short peptide sequence (PTAP) that binds directly the host-cell protein, TsglOl, and recruits it to sites of particle assembly. TsglOl is one of a group of sixteen or more proteins, termed the class E vacuolar protein sorting (VPS) factors, that are normally required for the budding of vesicles into the endosomal lumen. As part of our long term objective to understand the mechanism by which viral L-domains mediate particle budding, this proposal aims to test the hypothesis HIV-1 L-domain activity requires the same cellular activities that mediate the topologically equivalent process of endosomal vesicle budding. TsglOl itself exhibits a homo-multimerization activity and is known to bind to a second class E VPS factor, hVPS28. The experiments described herein will first determine whether these interactions are required for the HIV-1 particle budding function of TsglOl . Subsequently, the identification other cellular proteins that are associated with Tsgl 01 will be undertaken. Using biochemical yeast 2-hybrid and microscopic analyses, it will be determined whether and how they and other, already identified, class E VPS factors are recruited to sites of HIV-1 budding. Moreover, RNA duplex mediated silencing will be used to test whether these additional factors are required for L-domain mediated HIV-1 particle release. Preliminary studies indicate that a second class of retroviral L-domains, that contain a PPXY rather than a PTAP sequence motif, act by a similar mechanism to the HIV-1 L-domain. While these L-domains do not bind directly to TsglOl, they are clearly able to recruit TsglOl and hVPS28. Therefore, in some experiments, a PPXY L-domain will be included to test the hypothesis that PPXY L-domains mediate viral budding by the same mechanism as does HIV-1 , but by binding to an alternative VPS factor. Finally, a determination of whether the HIV-1 L-domain recruits a deubiqutinating activity during viral particle assembly will be undertaken. Based on findings in yeast systems, this is anticipated to be a secondary consequence of VPS factor recruitment, and is likely to explain the apparent requirement for the cellular ubiqutination machinery in retroviral particle release. Overall this project will aim to advance our understanding to the molecular mechanisms used by HIV-1 to facilitate viral budding. These studies have the potential to reveal completely novel, host-cell targets for therapeutic blockade of the replication of HIV-1, and possibly other viruses. ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI052774-02
Application #
6640631
Study Section
Special Emphasis Panel (ZRG1-AARR-1 (01))
Program Officer
Wassef, Nabila M
Project Start
2002-05-15
Project End
2007-04-30
Budget Start
2003-05-01
Budget End
2004-04-30
Support Year
2
Fiscal Year
2003
Total Cost
$319,200
Indirect Cost
Name
Aaron Diamond AIDS Research Center
Department
Type
DUNS #
786658872
City
New York
State
NY
Country
United States
Zip Code
10016
Jouvenet, Nolwenn; Simon, Sanford M; Bieniasz, Paul D (2011) Visualizing HIV-1 assembly. J Mol Biol 410:501-11
Zhang, Fengwen; Zang, Trinity; Wilson, Sam J et al. (2011) Clathrin facilitates the morphogenesis of retrovirus particles. PLoS Pathog 7:e1002119
Jouvenet, Nolwenn; Zhadina, Maria; Bieniasz, Paul D et al. (2011) Dynamics of ESCRT protein recruitment during retroviral assembly. Nat Cell Biol 13:394-401
Zhadina, Maria; Bieniasz, Paul D (2010) Functional interchangeability of late domains, late domain cofactors and ubiquitin in viral budding. PLoS Pathog 6:e1001153
Eastman, Scott W; Yassaee, Mina; Bieniasz, Paul D (2009) A role for ubiquitin ligases and Spartin/SPG20 in lipid droplet turnover. J Cell Biol 184:881-94
Bieniasz, Paul D (2009) The cell biology of HIV-1 virion genesis. Cell Host Microbe 5:550-8
Zhadina, Maria; McClure, Myra O; Johnson, Marc C et al. (2007) Ubiquitin-dependent virus particle budding without viral protein ubiquitination. Proc Natl Acad Sci U S A 104:20031-6
Martin-Serrano, Juan; Eastman, Scott W; Chung, Wayne et al. (2005) HECT ubiquitin ligases link viral and cellular PPXY motifs to the vacuolar protein-sorting pathway. J Cell Biol 168:89-101
Eastman, Scott W; Martin-Serrano, Juan; Chung, Wayne et al. (2005) Identification of human VPS37C, a component of endosomal sorting complex required for transport-I important for viral budding. J Biol Chem 280:628-36
Martin-Serrano, Juan; Perez-Caballero, David; Bieniasz, Paul D (2004) Context-dependent effects of L domains and ubiquitination on viral budding. J Virol 78:5554-63

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