During the retroviral life cycle, reverse transcriptase (RT) processively synthesizes DNA using first the single-stranded retroviral RNA as the template and subsequently the complementary DNA strand as a template in order to produce a double-stranded DNA copy of the genome. Coupled to the synthetic activities is an RNase H activity that degrades the genomic RNA and allows RT to complete synthesis of the second strand of DNA. The double-stranded DNA copy of the retroviral genome is then integrated into the host's genome by integrase, another retroviral enzyme. Following successful integration into the host genome, the retrovirus then relies on the host's machinery in order to make transcripts of the retroviral genome, which are subsequently exported from the nucleus by taking advantage of an existing host nuclear export pathway. The nuclear export pathway that is used by type D retroviruses, which encode an RNA element referred to as the constitutive transport element (CTh), is a pathway that is normally used to export mRNA. The host protein that mediates nuclear export of mRNA and retroviral RNA including the CTh is Tap. The proposed studies include x-ray crystallographic studies and related functional studies that are directed toward understanding protein-nucleic acid interactions in atomic detail required for the following: (1) the initiation of retroviral replication and (2) nuclear export of unspliced retroviral RNA containing the CTh. These studies are related more generally to (1) the understanding of nuclear export of mRNA, which is mediated by the same host factor Tap, and (2) the understanding of nucleic acid interactions that are important during replication through comparative structural analyses with related polymerases. Structural studies have been proposed for biologically relevant and novel nucleic acid complexes with three proteins including the human protein Tap, Moloney murine leukemia virus reverse transcriptase (MMLV RI), and an N-terminal fragment of MMLV RT. The projects share a common goal of obtaining a complex with an RNA molecule derived from the CTE, which is of interest as a novel RNA molecule in addition to its biological role in mediating nuclear export of unspliced retroviral RNA. As the CTE is a retroviral element, it must be replicated by reverse transcriptase and therefore interact directly with this enzyme. These studies provide a unique opportunity to compare biologically relevant nucleic acid interactions of the CTh with different proteins.
