Membrane Binding and Subcellular Localization of Retroviral Gag Proteins Are Differentially Regulated by MA Interactions with Phosphatidylinositol-(4,5)-Bisphosphate and RNA

The matrix (MA) domain of HIV-1 mediates proper Gag localization and membrane binding via interaction with a plasma-membrane (PM)-specific acidic phospholipid, phosphatidylinositol-(4,5)-bisphosphate (PI(4,5)P 2). HIV-1 MA also in- teracts with RNA, which prevents Gag from binding to membranes containing phosphatidylserine, a prevalent cellular acidic phospholipid. These results suggest that the MA-bound RNA promotes PM-specific localization of HIV-1 Gag by blocking non- specific interactions with cellular membranes that do not contain PI(4,5)P2. To examine whether PI(4,5)P2dependence and RNA-mediated inhibition collectively determine MA phenotypes across a broad range of retroviruses and elucidate the signifi- cance of their interrelationships, we compared a panel of Gag-leucine zipper constructs (GagLZ) containing MA of different ret- roviruses. We found that in vitro membrane binding of GagLZ via HIV-1 MA and Rous sarcoma virus (RSV) MA is both PI(4,5)P2dependent and susceptible to RNA-mediated inhibition. The PM-specific localization and virus-like particle (VLP) release of these GagLZ proteins are severely impaired by overexpression of a PI(4,5)P 2-depleting enzyme, polyphosphoinositide 5-phosphatase IV (5ptaseIV). In contrast, membrane binding of GagLZ constructs that contain human T-lymphotropic virus type 1 (HTLV-1) MA, murine leukemia virus (MLV) MA, and human endogenous retrovirus K (HERV-K) MA is PI(4,5)P 2inde- pendent and not blocked by RNA. The PM localization and VLP release of these GagLZ chimeras were much less sensitive to 5ptaseIV expression. Notably, single amino acid substitutions that confer a large basic patch rendered HTLV-1 MA susceptible to the RNA-mediated block, suggesting that RNA readily blocks MA containing a large basic patch, such as HIV-1 and RSV MA. Further analyses of these MA mutants suggest a possibility that HIV-1 and RSV MA acquired PI(4,5)P 2dependence to alleviate the membrane binding block imposed by RNA. IMPORTANCE MA basic residues in the HIV-1 structural protein Gag interact with phosphatidylinositol-(4,5)-bisphosphate (PI(4,5)P2) and RNA. RNA inhibits HIV-1 MA binding to non-PI(4,5)P2acidic lipids. This inhibition may promote PM specific- ity of Gag membrane binding, an early essential step in virus assembly. However, whether and how relationships between these interactions have developed among retroviruses are poorly understood. In this study, by comparing diverse retroviral MA do- mains, we elucidated a strong correlation among PI(4,5)P 2dependence, susceptibility to RNA-mediated inhibition, and cellular behaviors of Gag. Mutagenesis analyses suggest that a large basic patch on MA is sufficient to confer susceptibility to RNA- mediated inhibition but not for PI(4,5)P 2-dependent membrane binding. Ourfindings highlight RNA's role as a general blocker of large basic patches and suggest a possibility that some retroviruses, including HIV-1, have evolved to bind PI(4,5)P2, while others have adopted smaller basic patches on their MA domains, to overcome the RNA-mediated restriction of membrane bind- ing.