Intracellular Localization of HIV-1 RNAs by Rev&Matrix

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DESCRIPTION (provided by applicant): Replication of human immunodeficiency virus type 1 (HIV-1) involves regulation of the intracellular localization of incompletely spliced viral mRNAs that encode structural proteins. This proposal seeks to understand this regulation in greater detail, focusing on two viral proteins: Rev and Matrix (MA). Rev is a sequence-specific RNA binding protein that facilitates the cytoplasmic accumulation of incompletely spliced viral mRNAs at early times. The basis for nuclear entrapment of these HIV-1 RNAs in the absence of Rev is poorly understood and experiments are proposed to study this phenomenon. Over the previous funding period we discovered that the HIV-1 MA moiety of the Pr55 Gag polyprotein has a previously undescribed nuclear export activity. Although MA lacks the canonical leucine-rich nuclear export signal (NES), nuclear export is mediated through the conserved Crm 1p pathway and functions in both mammalian cells and yeast. We will continue to study the mechanism and regulation of MA nuclear export, and its role in HIV-1 replication. We will analyze the functional relationship between Rev and Pr55 in regulating HIV-1 RNA intracellular localization, and in directing viral genomic RNA to translation or packaging pathways. Over the previous funding period we also found that a mutation that disrupts the MA NES (MA-M4) mislocalizes Pr55 and viral genomic RNA to the nucleus, severely compromising viral replication. The Pr55/MA-M4 mutant can act in a dominant-negative fashion to mislocalize genomic viral RNA even in the presence of wild type Pr55. Based upon these results, we will develop and test a new class of dominant-negative inhibitors of HIV-l replication. Finally, our results suggest a novel method for inhibiting the activity of any mRNA. This "mRNA nuclear entrapment" strategy will be developed and tested using HIV-1 replication as a model system.