Human adenoviruses are of major medical interest because they cause respiratory infections in children and because they are oncogenic viruses that can cause tumors in rats and other rodents. The initiation of adenovirus DNA replication is a model for the interactions of proteins with DNA ends: adenovirus DNA replication will not take place unless a DNA end is present. The development of an in vitro replication assay has allowed characterization of the virus proteins essential for adenovirus DNA replication at the molecular level. The in vitro assay system has also allowed identification of several host proteins that play important roles in adenovirus DNA replication. The focus of this proposal is on the structure and function of the preterminal protein (pTP) and the adenovirus polymerase (Adpol). In order to construct mutations in these genes to map functional domains of the polypeptides, an expression system to produce active Adpol and pTP proteins is essential. Using a transient expression assay, a 140 kd polypeptide precipitable by anti-Adpol antibodies can be detected. Continuing tests on this protein will show whether it has DNA replication or DNA polymerase activity. The structure of Adpol and pTP mRNA in these recombinant constructs will also be determined to precisely map the RNA splice points and to discover whether the amino-terminal segments of Adpol and pTP are shared within one exon encoded at genome coordinate 39. Finally, immunoprecipitation of pTP and Adpol by anti-peptide antibodies will be used to confirm whether these proteins share amino-terminal amino acid sequences. A second effort will focus on production of HeLa cell lines that express either pTP or Adpol. Retrovirus vectors will be used to express the DNA segments that encode Adpol and pTP. This approach is also an alternative method for determining the structure of the mRNAs that encode pTP and Adpol. As a long term goal, mutations in the pTP and Adpol polypeptides will be constructed and analyzed by a number of in vitro assays, to map structural and functional domains. These mutagenesis efforts will focus on the dCTP binding site in pTP, differences in the activities of pTP and the mature terminal protein, and on highly conserved regions of amino acid sequence in the C- terminus of Adpol, where its sequences has been conserved at the expense of an overlapping viral gene product.
Showing the most recent 10 out of 15 publications
