REGULATION OF HIV RNA PROCESSING AND TRANSPORT

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The Human Immunodeficiency Virus (HIV) rev protein functions to facilitate nuclear export of unspliced and incompletely spliced HIV mRNAs. Rev may also play a role in translation of these mRNAs. Recent results in our laboratory indicate that rev binds to intron-containing HIV-RNA in cells in a complex that also contains splicing factors. Additional data suggest that rev binds directly to snRNP proteins involved in splicing. These results support the notion that rev interacts with components of the splicing machinery to resolve splicing factors from the rev regulated RNAs. This is a revised proposal that describes experiments designed to further clarify the role of rev in RNA processing and transport. To do this , we will characterize the complexes that form between rev and RRE-containing RNA in transfected cells and in in vitro splicing extracts. These experiments will include an analysis of the cellular proteins and RNAs in these complexes. We will further analyze interactions between rev and cellular proteins and RNA in the absence of RRE-containing RNA. These experiments will determine whether rev participates in the formation of a novel snRNP particle. In these studies we will also investigate whether rev acts as a shuttle protein between the nucleus and the cytoplasm and whether rev is present in polysome complexes. A novel yeast system will be used to identify cellular proteins that interact with rev. Recently, we have identified an element at the 3' end of the Mason Pfizer Monkey Virus (MPMV) genome that substitutes for rev and the RRE in HIV expression and replication. We hypothesize that this element interacts directly with constitutive cellular factors to effectuate transport of intron containing RNA. The MPMV element will be further characterized using random and site- specific mutagenesis. In addition, we will attempt to identify cellular proteins binding to this element. Genes for such proteins will be cloned and the properties of the proteins will be analyzed. Finally, we will analyze RNA processing and structural protein expression in cells infected with virus containing the MPMV element. The long term goal of this research is to further clarify the mechanism of action of the HIV rev protein. Further insight into how this protein promotes the expression of unspliced RNA, might enable the design of novel inhibitors of rev function. Since rev is absolutely essential for virus replication, this could lead to new drugs to efficiently combat AIDS.