Retroviruses encode a number of genes that are crucial for their replication in mammalian cells. Gag, Pol and Env are the main components of virions;during viral assembly, Gag and Gag-Pol polyproteins are targeted to the membrane of infected cells where they interact with Env and encapsidate genomic RNA to form a nascent viral particle. Gag molecules multimerize at the cell surface to form the shell of the virion. This step requires multiple domains in Gag: the N-terminal domain Matrix (MA) that targets Gag to the plasma membrane, an intact central capsid domain (CA) that assembles to form the retroviral central core, a nucleocapsid domain (NC) that binds genomic RNA, and a C-terminal p6 domain that carries highly conserved motifs that play essential roles during budding and release of virions. My laboratory investigates the mechanisms that control the late stages of viral morphogenesis, budding and release and the host cell proteins involved in these steps. The deletion of the p6 domain of Gag has been shown to arrest HIV-1 in late stages of viral release suggesting that the p6 domain contains cis-acting regions required for HIV-1 virions release and spread. A short motif located in the N-terminal end of p6, the PT/SAP motif, plays an essential role in late stages of budding and was then called Late domain or L domain. We and others have previously reported that HIV-1 p6 domain binds the cellular protein Tsg101 via direct interaction with the PT/SAP motif and this interaction is essential for the separation of the nascent HIV-1 virions from the surface of infected cells (Verplank, Bouamr et al., 2001, PNAS). Tsg101 binding to HIV-1 p6 helps to recruit a complex set of cellular factors that are normally used for endosomal protein sorting, membrane invagination, and fission at the MultiVesicular Body (MVB). The proteins involved in these cellular processes are organized in multi-protein complexes called Endosomal Complex Required for Transport (ESCRT). Other retroviruses also use this machinery to bud out from the cell;the Moloney Murine Leukemia Virus (MoMLV) and the Rous Sarcoma Virus (RSV) are both a type C Retrovirus that bud and release particles from the plasma membrane. To do so, they require a short motif within their Gag polyprotein, the PPPY motif. The host cell factor binding the latter motif is a member of the Nedd4-like E3 ubiquitin ligase family and was first reported by (Kikonyogo, Bouamr et al.,2001, PNAS). ESCRT complexes, I, II and III, are believed to be sequentially recruited to the surface of the endosome through interaction between the cellular protein Tsg101 and its natural partner in the cell, the Hrs protein. The current model hypothesizes that HIV-1 Gag mimics Hrs to bind Tsg101 and usurps the ESCRT machinery that facilitates HIV-1 virions release. Overexpression of fragments of the Hrs protein potently inhibited HIV-1 particle release. Yeast two hybrid assays were used to identify new and independent Tsg101 binding sites in Hrs. We identified mutants of Hrs that interfered with Tsg101 binding to HIV-1 Gag and consequently potently inhibited HIV-1 particle release. Scanning electron microscopy of cells expressing a mutant Hrs protein showed an accumulation of HIV-1 particles in abnormal structures at the cell surface. These findings are described in Bouamr et al., 2007 in JVI. Tsg101 functions in HIV-1 release as part of ESCRT-I. We found that the utilization of the latter complex in HIV-1 release requires a functional Nucleocapsid (NC) domain, suggesting NC and/or- its binding partner the viral genomic RNA, mediate interactions with the ESCRT pathway. The spacial and temporal involvement of these viral components in virus assembly and budding is currently under study in our laboratory. The p6 domain of HIV-1 Gag polyprotein was also described to bind a cellular protein called Alg-2 Interacting Protein or Alix. This direct interaction with Gag occurs via a conserved mootif, the LYPXnL motif, located in the C-terminal region of the p6 domain. Yeast two hybrid analysis showed that Alix binds both HIV-1 Gag and Tsg101. ALix-1 also binds a component of the ESCRT-III, the CHMP 4 protein, a member of ESCRT-III pathway that is critical for HIV-1 release. Overexpression of a dominant negative version of CHMP4 inhibited the release of HIV-1 virions. This indicated that Alix links HIV-1 Gag to components of the ESCRT machinery critical for virus release. We previously showed that the N-terminal Bro-1 domain of Alix interacts with the NC domain of Gag. Using a combination of capture assays and gain of function experiments, we identified key determinants NC-Bro1 interactions. These studies also revealed the involvement of RNA as a bridge between NC and Bro1 and provided a near complete picture of Alix dual interaction with HIV-1 Gag. We also study the role of the host ubiquitin machinery and ubiquitin ligases of the family of Nedd-like enzymes in virus budding. In particular, we examined the role of Itch in virus release. We found that Itch interacts with the model retrovirus MoMLV Gag and stimulates virus release in an L domain independent manner. We published these findings in Journal of Virology in 2009 (Jadwin et al., 2010). Additionally, we found that Nedd4-1 interacts and ubiquitinates the ESCRT-associated protein Alix. Remarkably, cellular Nedd4-1 is required for Alix mediated virus release. These data were published in Journal of Virology in 2010 (Sette et al., 2011). In the past fiscal year we reported our most recent findings regarding the role of ubiquitin in HIV-1 budding. Using a novel strategy we deubiquitinated HIV-1 budding sites by fusing the herpes virus UL36 to members of the ESCRT machinery or HIV-1 Gag. Remarkably, efficient interruption of ubiquitin conjugation to Gag assembly complexes halted virus budding. This is the first direct evidence for a natural role for ubiquitin in HIV-1 budding;which we reported in 2013 in Sette et al, in Retrovirology.

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Project End
Budget Start
Budget End
Support Year
9
Fiscal Year
2013
Total Cost
$792,018
Indirect Cost
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Sette, Paola; Nagashima, Kunio; Piper, Robert C et al. (2013) Ubiquitin conjugation to Gag is essential for ESCRT-mediated HIV-1 budding. Retrovirology 10:79
Dussupt, Vincent; Javid, Melodi P; Abou-Jaoude, Georges et al. (2009) The nucleocapsid region of HIV-1 Gag cooperates with the PTAP and LYPXnL late domains to recruit the cellular machinery necessary for viral budding. PLoS Pathog 5:e1000339