The Roles of Lipid and RNA in Regulating Retroviral Gag Membrane Binding and Targeting.

[摘要] The HIV-1 matrix (MA) domain mediates proper Gag localization and membrane binding by interacting with phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2], a plasma membrane(PM)-specific phospholipid. HIV-1 MA also interacts with RNA, which prevents Gag from binding to membranes containing phosphatidylserine (PS), a prevalent negatively charged phospholipid. These results suggest that the MA-bound RNA promotes PM-specific localization of HIV-1 Gag by blocking non-specific interactions with membranes that do not contain PI(4,5)P2.In this thesis, I examined whether PI(4,5)P2 dependence and RNA-mediated inhibition collectively determine MA phenotypes across a broad range of retroviruses. By comparing a panel of Gag-leucine-zipper constructs (GagLZ) containing MA of different retroviruses, I found that membrane binding mediated by retroviral MA can be broadly divided into two categories: those that are PI(4,5)P2-dependent and RNase responsive, and those that are neither. I also found that the PM-localization and virus-like particles (VLP) release of the former group is sensitive to the overexpression of a PI(4,5)P2- depleting enzyme, polyphosphoinositide 5-phosphatase IV (5ptaseIV), while the latter group is much less sensitive to 5ptaseIV overexpression. Structural analyses further suggest that the basic patch size of the retroviral MA confer susceptibility to RNA-mediated membrane binding inhibition. In my thesis, I also provided in vitro and cell-based evidence supporting that RNA-mediated suppression occurs in cells and that RNA can inhibit membrane binding of HIV-1 Gag at a concentration that is much lower than the estimated RNA concentration in the cell. Hence, RNA-mediated suppression is a physiologically relevant mechanism that prevents Gag from binding promiscuously to prevalent PS-containing membranes.Finally, I examined the roles of PI(4,5)P2 and RNA in regulating the targeting of HIV-1 Gag to the site of assembly, the virus-containing compartments (VCC), in primary macrophages. I found that the VCC localization and virus release of HIV-1 are severely impaired upon 5ptaseIV overexpression. However, HIV-1 MA contributes only to membrane binding but not in Gag targeting to the VCC. I also determined that HIV-1 nucleocapsid (NC) is important for VCC-specific localization of HIV-1 Gag. This suggests that targeting of HIV-1 Gag to the VCC adopts a different mechanism than Gag targeting to the PM in HeLa and T cells.

[发布机构] University of Michigan

[效力级别] RNA

[关键词] Retroviruses;RNA;Phospholipids;Membrane binding;Microbiology and Immunology;Science;Microbiology and Immunology