Mapping the functional domains of HAP95, a protein that binds RNA helicaseA and activates the constitutive transport element of type D retroviruses

The complex retroviruses such as human immunodeficiency virus, type 1, empl oy a virally encoded protein, Rev, to mediate the nuclear export of unsplic ed and partially spliced mRNA. In contrast, the simian type D retroviruses act through a cis-acting constitutive transport element (CTE) that presumab ly interacts directly with cellular export proteins. We first reported that RNA helicase A (RHA) is a shuttle protein that binds to functional CTE in vitro and in vivo. Recently, we isolated a novel protein, HAP95, that speci fically binds to the nuclear transport domain of RHA and up-regulates CTE-m ediated gene expression. Here, using truncation and deletion mutations, we mapped the domains of HAP95 that are important for RHA binding, transactiva tion of CTE, and nuclear cytoplasmic shuttling. We report evidence for a no vel nuclear export signal in HAP95 and showed that the domains involved in RHA binding and nuclear localization are required for CTE activation. Final ly, we showed that HAP95 synergizes significantly with RHA on CTE-mediated reporter gene expression and promotes nuclear export of unspliced mRNA in t ransfected cells. Taken together, these data support the proposal that HAP9 5 specifically facilitates CTE-mediated gene expression by directly binding to RHA.