Among targets for antiviral therapy that arise during certain viral infections are the virus-coded proteinases. These enzymes are required to process virus-specific precursors involved in the maturation, assembly and replication of such pathogenic human viruses as poliovirus, encephalitis virus, hepatitis A virus, cytomegalovirus, and human immunodeficiency virus. These virus-coded proteinases are highly specific for their virus- coded substrates. Thus, if equally specific inhibitors can be developed and targeted to infected cells, they should interfere with virus replication and not with normal cellular metabolism. Human adenovirus 2 encodes a proteinase that processes six virion polypeptides during virus morphogenesis. In the absence of an active virus-coded proteinase, noninfectious virus is produced. Because much is known about the molecular biology of adenovirus 2 and about the interaction of proteinases and their inhibitors, adenovirus 2 is a good model system to test the efficacy of proteinase inhibitors as antiviral agents. The adenovirus 2 proteinase was cloned, expressed, purified and characterized during the first two and one half years of this grant. Now we propose to obtain mutants of bovine pancreatic trypsin inhibitor that will inhibit the adenovirus 2 proteinase. The inhibitors will be rationally (based upon substrate specificity) and randomly (with a mixed oligonucleotide) designed, synthesized, cloned, expressed, and selected for optimal properties. Bovine pancreatic trypsin inhibitor is chosen, because it has only 58 amino acids, has a high affinity for trypsin-like serine proteinases, and has been cloned and expressed in an active form in E. coli. These mutants of natural proteinase inhibitors should have more desirable antiviral properties compared to the noncleavable peptide inhibitors currently being synthesized by pharmaceutical companies for the HIV proteinase. After selecting for additional properties, mutant bovine pancreatic trypsin inhibitors will be tested for antiviral activity in the adenovirus 2 model system. If successful, the lessons learned from this model system can easily be extended to more medically relevant viruses.
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