IDENTIFICATION OF DDX1 AND REV/RRE COFACTORS

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Human immunodeficiency virus (HIV)-1 Rev is essential in the late phase of viral gene expression and replication. Rev binds to the Rev response element (RRE) that occurs in unspliced and singly spliced HIV mRNAs, and recruits the nuclear export receptor CRM1 to these transcripts to facilitate their transport out of the nucleus. In addition to its functions in viral mRNA export, Rev also has been suggested to stabilize and regulate translation of RRE-containing mRNAs, and potentially has a role in targeting the HIV genome to the plasma membrane for encapsulation in the budding virus. Rev-interacting host cell proteins that are required for Rev/RRE functions include the RNA helicases DDX1 and DDX3.Our aim is to identify DDX1 and Rev/RRE binding partners from nuclear and cytosolic extracts of HeLa cells by a series of pull-down assays, and subsequent analysis by multidimensional protein identification technology (MudPIT) followed by stringent data filtering.