The herpesviruses include several significant pathogens, including herpes simplex virus (HSV), varicella-zoster virus, Epstein-Barr virus, Kaposi's associated herpesvirus, and human cytomegalovirus (HCMV). These viruses replicate their genomes by synthesizing large concatemeric intermediates that are cleaved to form unit genomes packaged within progeny viral particles. Currently antivirals (aciclovir, ganciclovir, and foscarnet) target the DNA polymerase, but alternative therapeutics are needed to combat expanding resistance to these drugs. One class of drug that inhibits HCMV cleavage and packaging has demonstrated excellent antiviral potential; however, little is known about the mechanisms of cleavage and packaging or how they are blocked by these drugs. Data from HSV first suggested a link between cleavage and terminal repeat duplication, but the complexity of the HSV genome has made this hypothesis difficult to prove. Recent data obtained in our laboratory exploiting the simple genome structure of guinea pig cytomegalovirus (GPCMV) clearly demonstrate that two cleavage pathways exist: one that duplicates the terminal repeat (duplication-cleavage) and one that does not (staggered-cut cleavage). The proposed experiments will test three potential mechanism for duplication-cleavage: (1) repeats are taken from neighboring genomes in the concatemer; (2) a DNA polymerase primes at nicks formed on each side of the repeat, copies the repeat, and separates the strands; and (3), a DNA polymerase primes at nicks formed at a stem-loop and copies the repeat, leaving a hairpin end that is staggered-cut cleaved. To differentiate these mechanisms, analysis of GPCMV duplication-cleavage will determine: (i) the necessity for a DNA polymerase; (ii) the incorporation of labeled nucleotides specifically into terminal repeat sequences; (iii) the formation of blunt or 3 single base overhangs at DNA ends; and (iv) the importance of potential stem-loop forming sequences. As cleavage and packaging are highly conserved among herpesviruses, this information should be directly applicable to human pathogens such as HSV and HCMV and may reveal the mechanism of action of the current cleavage inhibitors or facilitate the discovery of novel compounds targeting these processes.
