%0 Generic
%9 Doctoral Dissertation
%A Jin, Jing
%D 2008
%F pittir:9437
%K  assembly and budding; retrovirus; BiFC; trafficking
%T REGULATION OF GAG TRAFFICKING DURING RETROVIRUS ASSEMBLY AND BUDDING
%U http://d-scholarship-dev.library.pitt.edu/9437/
%X Retroviral Gag polyproteins are necessary and sufficient for virus budding, but little is known about how thousands of Gag polyproteins are transported to the budding sites. The actin cytoskeleton has long been speculated to take a role in retrovirus assembly and recent studies suggest that HIV-1 assembly is regulated as early as viral RNA nuclear export, however specific mechanisms for these regulations are unknown. In contrast to numerous studies of HIV-1 Gag assembly and budding, relatively little is reported for these fundamental pathways among animal lentiviruses. In this project, we used bimolecular fluorescence complementation (BiFC) (1) to reveal intimate (<15nm) and specific associations between EIAV Gag and actin, but not tubulin; (2) to characterize and compare assembly sites and budding efficiencies of EIAV and HIV-1 Gag in both human and rodent cells when the mRNA nuclear export context is altered to be Rev-dependent or Rev-independent; (3) to reveal co-assembly of Rev-dependent and Rev-independent HIV-1 Gag and rescued assembly of Rev-independent HIV-1 Gag in human cells by in cis provided membrane targeting signals. The results of these studies showed that (1) multimerization of EIAV Gag was required for association with filamentous actin and this association correlated with Gag budding efficiency, suggesting that association of Gag multimers with filamentous actin is important for efficient virion production; (2) HIV-1 and EIAV Gag assembled in different cellular at sites, and HIV-1 but not EIAV Gag assembly was affected by mRNA nuclear export pathways, suggesting that alternative cellular pathways can be adapted for lentiviral Gag assembly and budding; (3) Rev-independent HIV-1 Gag was deficient in lipid raft targeting and its assembly and budding could be restored by membrane targeting signals provided in trans or in cis, suggesting that raft association is critical for HIV-1 assembly and budding and is regulated as early as nuclear export of Gag-encoding mRNA. The findings presented in these studies are significant for public health because a better understanding of the mechanism of retrovirus assembly and budding increase the potential to develop novel antiviral therapies targeting this critical step in the viral life cycle.