HnRNP proteins in Rev mediated trafficking of HIV-1 mRNAs
Geza Ambrus-Aikelin, Scripps Research Institute
Human immunodeficiency virus (HIV)-1 Rev is essential in the late phase of viral gene expression and replication. Rev binds to its RNA target sequence, the Rev response element (RRE), and recruits the nuclear export factor CRM1 to transport unspliced and singly spliced HIV mRNAs out of the nucleus. Multiple host cell proteins have been shown to be necessary for efficient HIV replication and several were suggested to be involved in viral mRNA export. We have recently identified 11 hnRNP proteins in a proteomic study of host cell proteins that selectively interact in vitro with Rev and its RNA target sequence, the Rev response element (RRE). To our knowledge, this is the first systematic analysis of proteins that interact with Rev/RRE. We hypothesize that these proteins play an important role in Rev/RRE mediated mRNA trafficking, stability and translation. Our specific aims are the following:
- Identify the members of the group of 11 hnRNP proteins that interact with the Rev/RRE complex in vivo.
- Define the hnRNP proteins that are important for RRE-mediated reporter protein expression.
- Determine how silencing of selected hnRNP proteins influences the subcellular localization, transcript levels and translation of RRE-containing mRNAs.
First, we will confirm the interactions of the hnRNP proteins with Rev/RRE in vivo using immunoprecipitation of hnRNP proteins followed by quantification of RRE by quantitative RT-PCR. Then we proceed to use RNAi approaches to knock down individual hnRNP proteins and monitor the expression of a Rev/RRE dependent reporter protein. We will further will analyze how silencing of these proteins affect nuclear export, transcript level and translation of RRE-containing mRNAs using fluorescence in situ hybridization (FISH), Northern blotting and quantitative RT-PCR. The understanding how these hnRNP proteins influence the fate of late HIV mRNAs will enhance our knowledge about the molecular mechanism of HIV infection and might open up new therapeutic approaches.