The long-term objectives of this project are to develop antiviral drugs active against human cytomegalovirus (CMV) by targeting the CMV DNA polymerase (Pol) and its likely accessory protein, ICP36. CMV is an important human pathogen, especially in patients with AIDS, for which there is a critical need to develop safe, effective antiviral agents.
The specific aims of the project are: 1) To overexpress the CMV Pol, ICP36, and/or their functional domains. Baculovirus, vaccinia virus, and E coli expression systems will be used for expression, specific antisera will be generated, and the proteins will be purified by standard techniques and will be compared to material purified from CMV- infected human cells. 2) To investigate structural features of CMV Pol, ICP36, and their interactions with nucleic acid and substrate. Purified proteins will be provided to Dr. Hogle's laboratory who will solve their structures and the use molecular modeling methods to design inhibitors, which will serve as lead compounds for synthesis and testing. Limited proteolysis will help define functional domains and structural organization; mobility shift, footprinting, and methylation interference assays will examine protein-DNA interactions; and affinity labeling will identify protein-substrate contacts. Mutational studies will identify residues important for substrate recognition and exonuclease activities. 3) To map the interacting regions of Pol and ICP36. Each protein will be expressed by in vitro transcription and translation and an assay for complex formation will be developed. With this assay and standard mutagenesis approaches, small regions of each protein that are important for binding to the other and for related functions will be identified. 4) To synthesize peptides and other compounds to inhibit the CMV Pol-ICP36 interaction. The sequences of the interacting regions will be used as the basis for synthesizing peptides that interrupt the interaction and its functional consequences. As an alternative approach, RNA aptamers will be derived that interact with these regions. The information derived on these proteins in this project and in Project 1 will be used as the basis of the design of lead compounds for synthesis and testing by collaborators at Burroughs Wellcome.
