Characterizing the staufen 1 human immunodeficiency virus type 1 ribonucleoprotein

Human Immunodeficiency Virus type 1 (HIV-1) is a member of the Retrovirus family and is the causative agent of Acquired Immunodeficiency Syndrome (AIDS). As an obligate intracellular parasite, HIV-1 uses cellular proteins and machinery to ensure transmission to uninfected cells. One such cellular protein involved in the late stages of viral replication is Staufen1. It is a main component of mRNA ribonucleoproteins (RNPs) and plays distinct roles in mRNA transport, translation and decay. In the context of HIV-1, Staufen1 was initially found to be selectively encapsidated into the virions. Subsequent work determined its association with vRNA and precursor protein Gag within HIV-1 RNPs. Recently we demonstrated that Staufen1 regulates the viral assembly process by inducing Gag multimerization.In Chapter 2 of the present work, we used tandem affinity purification method followed by mass spectrometry to characterize the Staufen1-containing RNPs. We focused on the investigation of the proteins that associate with Staufen1 complexes, isolated from cells that either do or do not express HIV-1. We further performed a detailed comparison of the protein content between native Staufen1 and Staufen1 HIV-1 RNPs and defined the modulations caused from HIV-1 expression. In Chapter 3, we used bimolecular and trimolecular fluorescence complementation methods (BiFC and TriFC) that allow for direct visualization and localization of protein-protein and protein-protein and RNA interactions in living cells, to show that Gag and Staufen1 interact, which was demonstrated by small multi-fluorescent punctae or vesicles in cells. We demonstrated that these protein partners mainly associate in the cytoplasm. However, we also found that they interact at cholesterol-enriched GM-1-containing membrane microdomains. Importantly, Gag specifically recruited Staufen1 to these lipid raft membranes suggesting a key function for this host factor during Gag assembly. Notably in TriFC experiments, in which one protein partner was tethered to mRNA, Gag-Staufen1 interactions were observed demonstrating active recruitment of one protein when the other is bound to mRNA.Overall, we used both proteomics and live cell visualization to examine fundamental viral-host interactions. We have completely characterized the composition of Staufen1 RNPs and have demonstrated how HIV-1 uses these RNPs for its own purpose. These studies also enhance our understanding of the mechanisms and the specific dynamic features of the viral-host (Gag-Staufen1) interactions in living cells, as monitored by the modern fluorescence complementation assays. The findings presented here are significant help to advance research in the HIV-1 field because a better understanding of the mechanism of retrovirus assembly and budding will aid in the development of novel antiviral therapies targeting the critical late steps in the viral replication cycle.