Mouse mammary tumor virus (MMTV) has been classified as a simple retrovirus that encodes two accessory proteins, dUTPase (DU) and superantigen (Sag). The DU protein as well as Gag, protease (PR) and reverse transcriptase (RT) are encoded by unspliced viral RNA. Both simple and complex retroviruses require viral elements that facilitate the nuclear export of intron-containing mRNAs. Simple retroviruses have cis-acting elements that directly recruit cellular factors involved in nuclear export, whereas complex retroviruses encode adapter proteins, such as Rev. Rev binds to viral cis-acting sequences to facilitate cellular export factor recruitment. Our experiments indicate that MMTV encodes a third accessory protein that we have named Rem (regulator of export of MMTV mRNA). Rem is translated from a doubly spliced mRNA into a ca. 33 kDa protein, which is approximately two to three times larger than other retroviral export proteins. Mutations in the rem open reading frame within the 3' end of the MMTV genome inhibit gag-pol (unspliced) mRNA export from the nucleus and can be complemented by co-transfection of permissive cells with an infectious MMTV provirus or by rem complementary DNA. Moreover, the Rem C-terminus is not required for RNA export, but deletion of this domain increases export in transfection assays using an MMTV-based reporter vector. These data suggest that the C-terminus negatively regulates Rem-mediated RNA export to control MMTV structural protein production. Identification of the rem gene establishes MMTV as the only murine retrovirus that encodes an auto-regulatory export protein and challenges the idea that MMTV is a simple retrovirus. To further characterize this exciting finding, we have proposed three specific aims. In the first specific aim, we will determine if Rem has specific post-translational modifications, e.g., sumoylation or phosphorylation, which affect its RNA export activity. The cell type or differentiation-specificity of such modifications will be explored. In the second specific aim, both biochemical and genetic approaches have been proposed to determine additional functions of the Rem C-terminal domain. Mutants lacking the C-terminus will be characterized for their ability to affect MMTV RNA stability, splicing, or Gag localization, processing and assembly. The Rem C-terminus also will be used in yeast two-hybrid assays and mammalian tandem affinity purifications to identify cellular proteins that may elucidate Rem functions. In the third specific aim, MMTV proviruses that lack the C-terminus of Rem will be characterized for their ability to replicate in several cell types in vitro and in vivo. These experiments may provide valuable information about novel cellular pathways required for retroviral replication and the development of mouse models for complex human retroviruses, such as HIV. ? ? ?
