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 which play essential roles during budding and release of virions. My laboratory investigates the mechanisms that control the late stages of retroviral morphogenesis, budding and release and the host cell proteins involved in these steps.? ? 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 et al, 2001). Tsg101 binding to HIV-1 p6 helps to recruit a complex set of cellular machinery that is normally used for 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); they are essential for sorting of cargo proteins into the multivesicular bodies (MVB). Other retroviruses also used this machinery to bud out from the cell; the Moloney Murine Leukemia Virus (MOMLV) and the Rous Sarcoma Virus (RSV) are both a type 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, that binds E3 ubiquitin ligases of the family of Nedd4 (Kikonyogo et al,2001). ? ? ESCRT complexes, I, II and III, are 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. Mutants of Hrs that interfered with Tsg101 binding to HIV-1 Gag were identified and, as a consequence, 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., published in Journal of Virology 2007.? ? The p6 domain of HIV-1 Gag polyprotein was also described to bind a cellular protein called Alg-2 Interacting Protein or AIP-1 also called Alix. This direct interaction with Gag occurs via a conserved motif, the LYXPL 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; AIP-1 also binds a component of the ESCRT-III, the CHMP 4 protein. Overexpression of a dominant negative version of CHMP 4 inhibited the release of HIV-1 virions. This indicated that Alix links HIV-1 Gag to components of the ESCRT machinery that facilitate HIV-1 viral release. To understand the role of Alix in HIV-1 particle morphogenesis and release, we followed a dominant negative approach: fragments of AIP-1 containing the N-terminal or the C-terminal (two thirds of the protein exerted an inhibitory effect on the release). In contrast, overexpression of a fragment containing the central domain of Alix stimulated HIV-1 particle production. Electron microscopy analysis is being used to capture the phenotypes caused by over-expression of fragments of Alix; mutagenesis analysis is also currently used to map the regions in Alix responsible for these phenotypes. In addition, we made use of yeast two hybrid assays to identify Alix cellular partners that facilitate HIV-1 budding and release.
