| In the life cycle of all retroviruses, viral genomic RNA must be exported to the cytoplasm of cells, circumventing the host cells' natural processes of mRNA modification. Complex retroviruses have evolved to contain their own genes that promote the export of unspliced viral RNA from the nucleus to the cytoplasm. In HIV-1, for example, the viral Rev protein binds to the Rev Response Element sequence in viral RNA and directs the RNA to be exported from the nucleus. Simple retroviruses do not have their own gene products to serve this purpose. Host cell proteins, through interaction with viral RNA elements, are responsible for the export of unspliced viral RNA.; Our laboratory has previously identified RNA Helicase A (RHA) as a host protein that binds to the Constitutive Transport Element (CTE) of the Mason-Pfizer Monkey Virus, a simple retrovirus. RHA is a pleiotropic factor that interacts with numerous other nuclear and shuttle proteins and was shown to be a co-factor for CTE activity. Following up on this discovery, this dissertation presents efforts to identify and characterize RHA-binding proteins and ascertain what roles they may play in CTE function.; Using the yeast two-hybrid system, coding sequences belonging to three RHA-binding proteins were isolated. Study of Helicase-Associated Protein 95 (HAP95) began as the isolation of the partial coding sequence of a novel, unidentified protein. The full coding sequence of the Survival of Motor Neurons (SMN) protein and part of the coding sequence of human Poly (A) Binding Protein II (hPABP2) were also isolated.; Following their identification as RHA-binding proteins in the yeast two-hybrid system, binding to RHA was confirmed in vitro. HAP95 was found to associate with specifically with CTE RNA in vivo. Overexpression of these three proteins in cultured cells had significant effects on CTE function. HAP95 and hPABP2 were found to be positive factors, upregulating CTE function, while SMN overexpression by itself reduced the activity of the CTE. hPABP2 overexpression did not affect properties of the poly (A) tail on CTE or control RNAs, but did lead to a significant increase in cytoplasmic CTE RNA concentration. |
