Subcellular trafficking and early assembly of the retroviral Gag polyprotein

The Gag polyprotein, the main structural protein of all retroviruses, is both necessary and sufficient for virus particle assembly. The host and viral factors that regulate the dynamic trafficking of Gag through the nucleus and to the plasma membrane, as well as the role of vRNA binding in assembly and subcellular trafficking of Gag, are the focus of this dissertation.;The host import factors for the mature MA/NC proteins, transportin SR, importin 11 and importin alpha/beta, and the export receptor for p10, CRM1, were identified using a genetic screen in yeast 64. Through my studies, we found that Gag directly associates with importin 11 and importin alpha/beta in host cells and in vitro. Association of these importins with Gag is specifically competed by vRNA binding, indicating that Gag does not bind vRNA and import factors simultaneously. In contrast, direct binding of Gag to the CRM1-RanGTP nuclear export complex was stimulated by vRNA. These results suggest that vRNA interactions serve as a switch to regulate the association of Gag with host proteins that mediate nuclear trafficking and genomic-RNA packaging.;Detailed biochemical and biophysical analysis of the Gag:importin alpha complex revealed a 1:1 stoichiometry; however, previous studies show Gag oligomerizes in the nucleus 218, suggesting oligomerization first occurs after nuclear entry when Gag interacts with its vRNA. I became interested in how the Gag monomer that enters the nucleus associates with vRNA, multimerizes, and eventually forms the complete immature virus particle at the plasma membrane.;Initially, I showed that Gag, which appeared to be mostly monomeric in solution, underwent a conformational change when bound to micropsiRNA, the minimal vRNA packaging signal, leading to the cooperative formation of Gag dimers, tetramers and hexamers. These 6Gag:1vRNA units bound to other Gag hexamers, forming stable trimers and heptamers of hexamers. Estimatation of the binding affinities of Gag to psi-containing vRNAs revealed picomolar affinities, while other oligonucleotides, including a mutant-vRNA that alters the structure of the packaging signal, bound with nanomolar affinities. These studies suggest very tight, specific binding to the vRNA, with the high affinity interaction primarily due to the structure of the RNA packaging signal (psi).;Although these early assembly intermediates were formed using the Gag protein and vRNA, it appeared that complete virus particles could not form spontaneously at neutral pH. Because complete particles form at the plasma membrane in cells, we hypothesized that membrane lipids, specifically inositol phosphotidylinositol 4,5-biphosphate (PI(4,5)P2), previously implicated in membrane targeting of other retrovirus Gag proteins 68,76,178,329,374 , may trigger complete particle formation from early assembly intermediates.;I found that the addition of PI(4,5)P2 to preassembled Gag:vRNA complexes led to the formation of tubular structures, suggesting these inositol phosphates interact with Gag:vRNA complexes to promote polymerization. Characterization of the binding affinity of RSV Gag and MA proteins to PI(4,5)P2 showed that MA/Gag bound PI(4,5)P2 with high affinity, triggering a conformational change in MA/Gag, with implications in oligomerization. PI(4,5)P2 also competed with importin 11 for Gag/MA binding, suggesting PI(4,5)P2 may be involved in the regulation of both nuclear and plasma membrane trafficking of Gag. Examination of Gag:PI(4,5)P2 interactions in cells implicated this inositol phosphate in membrane targeting and budding of Gag during the virus life cycle.;Based on my studies, I formulated a model of early retroviral particle assembly. I propose, after synthesis, Gag binds to importin 11 and/or importin alpha/beta as a monomer. Once in the nucleus, Gag binds to the vRNA, inducing a conformational change in the Gag protein which not only increases its affinity for CRM1 but also nucleates Gag oligomerization. This assembly competent Gag:vRNA complex is exported from the nucleus, forming a hexameric lattice of Gag proteins enroute to the plasma membrane. At the membrane, this Gag:vRNA complex will interact tightly with PI(4,5)P2, resulting in another conformational change that induces oligomerization of Gag:vRNA complexes with other Gag proteins, culminating in complete particle production. (Abstract shortened by UMI.)...