The Herpesviridae family of viruses includes several significant human pathogens, including herpes simplex virus, varicella-zoster virus, Epstein-Barr virus, Kaposi's sarcoma associated herpesvirus, and human cytomegalovirus (HCMV). DNA replication in these viruses produces large concatemeric intermediates, from which unit genomes are cleaved at specific sequences and packaged into viral particles. The halogenated benzimidazole ribonucleosides are a new class of antiviral drugs which block this process. Unfortunately, our understanding of herpesvirus cleavage and packing is scant and the mechanism of action of these drugs remains unknown. Using murine cytomegalovirus (MCMV), we have begun to define the cis elements that are recognized at the cleavage site. Using guinea pig cytomegalovirus (GPCMV), we have demonstrated that the mechanism of cleavage duplicates sequences at the viral termini. Recent findings in GPCMV and HCMV suggest that the benzimidazoles induce a premature cleavage to produce truncated genomes.
In aim 1 of this proposal, we will more accurately define the cis cleavage/packaging signals of MCMV and initiate efforts to analyze cis elements in HCMV.
In aim 2, experiments to elucidate the mechanisms of cleavage and packaging will define the phenotypes of cis mutations in MCMV with respect to formation of intracellular replicative DNAs and extracellular viral genomes. The benzimidazole-induced accumulation of truncated genomes will be investigated by further analysis of the novel ends formed, characterization of capsid composition and structure, and by electron microscopy. A model proposed for the mechanism of repeat duplication will be tested both by analysis of replicative intermediates and by construction of genetic mutations in cleavage/packaging cis elements.
In aim 3, viral cleavage/packaging proteins will be characterized both in vitro and in vivo using recombinant expression and their functions studied using either in vitro or permeabilized cell cleavage assays. As cleavage and packaging are highly conserved among herpesviruses, this information should be directly applicable to pathogenic human herpesviruses and may reveal the mechanism of action of the benzimidazoles or facilitate the discovery of novel compounds that target the cleavage and packaging processes.
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