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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 P2 HIV-1 MA also interacts 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 nonspecific interactions with cellular membranes that do not contain PI 4 5 P2 To examine whether PI 4 5 P2 dependence and RNA-mediated inhibition collectively determine MA phenotypes across a broad range of retroviruses and elucidate the significance of their interrelationships we compared a panel of Gag-leucine zipper constructs GagLZ containing MA of different retroviruses We found that in vitro membrane binding of GagLZ via HIV-1 MA and Rous sarcoma virus RSV MA is both PI 4 5 P2 dependent 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 P2-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 P2 independent 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 P2 dependence 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 P2 acidic lipids This inhibition may promote PM specificity 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 domains we elucidated a strong correlation among PI 4 5 P2 dependence 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 P2-dependent membrane binding Our findings 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 binding

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Membrane Binding and Subcellular Localization of Retroviral Gag Proteins Are Differentially Regulated by MA Interactions...