The aim of this renewal AIDS-FIRCA application is to further characterize the process of incorporation of the SIV envelope (Env) glycoprotein into budding particles, the step in SIV morphogenesis that confers infectivity to virions. In this regard, our mutagenesis studies have led to the identification of domains within the carboxy-terminal third of the SIV transmembrane (TM) cytoplasmic tail that are necessary for the incorporation of the SIV Env into particles and virus infectivity. Of note, some of these domains are partially conserved in the cytoplasmic tail of the HIV-1 Env glycoprotein. Due to the relatedness of SIV and HIV-1, a better understanding of the mechanism that underlies Env incorporation into budding particles may provide useful information for the rational design of alternative anti-retroviral strategies. In these studies we propose to: 1. Pinpoint the specific residues within the C-terminal third of the SIV TM cytoplasmic tail that mediate the association of the Env glycoprotein with virions. We will target for mutagenesis the C-terminal regions of the SIV TM protein that we have found to be involved in packaging of the Env protein into particles. 2. Engineer the Env mutations into a complete SIV provirus to isolate second site revertants of those mutants that are defective for Env incorporation. These results will allow us to pinpoint regions of interaction with the Gag precursor. They will be correlated with those from Aim #3 where mutations in MA that alter the capacity for Env incorporation will be studied. 3. Engineer into the matrix-coding region of an SIV provirus the mutations that we have found, by means of the vaccinia virus vector system, to affect the levels at which the Env glycoprotein is incorporated into virus particles. We will determine in the context of the provirus the effect of these mutations on Env incorporation and virus infectivity. 4. Establish in vitro association assays to quantitate the interaction between the SIV matrix and TM proteins. This system will let us determine whether the defect in Env incorporation imposed by mutations in the carboxyl terminus of the SIV TM protein reflects indeed a block in TM-matrix protein interaction. In addition, these assays will allow us to investigate whether those mutated Env glycoproteins that we have found to retain the ability to be incorporated into particles (despite the presence of mutations in the TM cytoplasmic domain) engage in specific interactions with the matrix protein, or are passively recruited into virions.
| Celma, Cristina C P; Manrique, Julieta M; Hunter, Eric et al. (2004) Second-site revertants of a simian immunodeficiency virus gp41 mutant defective in envelope glycoprotein incorporation. AIDS Res Hum Retroviruses 20:733-41 |
| Celma, C C; Manrique, J M; Affranchino, J L et al. (2001) Domains in the simian immunodeficiency virus gp41 cytoplasmic tail required for envelope incorporation into particles. Virology 283:253-61 |
