The research proposed will identify cis- and trans-acting viral functions required for cytomegalovirus (CMV) DNA replication and inversion. Viral cis-acting replication functions, including origins of CMV DNA replication and DNA sequences required for inversion of genome components, will be isolated based on their function in virus-dependent trans-assay systems. Viral trans-acting replication functions will be characterized using two groups of temperature sensitive early viral mutants (having a DNA- phenotype at the non-permissive temperature). In the first series of proposed experiments, plasmid cloned DNA fragments carrying origins of CMV DNA replication will be isolated by their ability to replicate when introduced into permissive cells along with wild-type virus as helper. Replication will be detected by hybridization with a plasmid DNA probe. A DNA fragment found to carry an origin will be further subcloned to isolate a small (less than 1000 base pair) segment which will be subjected to nucleotide sequence analysis. The second series of experiments will establish whether CMV has a genomic region functionally homologous to the herpes simplex virus a sequence. Proposed experiments will demonstrate whether CMV DNA has a terminal direct repeat with inverted copies at the junction of the invertible L and S components of the viral genome. The nucleotide sequence of plasmid cloned derivatives of genomic termini and L-S junction fragments will provide structural information on this repeat sequence and reveal the site of cleavage used to generate unit length virion DNA from concatemers. Copies of the cloned L-S junction fragment will be linked to a cloned viral origin of DNA replication in a chimeric plasmid construct. This construct will be used to test the function of the repeat sequence as a site for inversion and for cleavage/packaging. Finally, studies on specific trans-acting viral replication functions will be initiated using two groups of temperature sensitive mutants. The phenotype, by infected cell polypeptide profile, and map position of the mutation, by marker rescue, will be determined for each of the temperature-sensitive mutants. The fundamental biochemical and molecular knowledge generated by the proposed research will allow future studies that identify host and viral trans-acting functions and that unravel the molecular details of the initiation, process and regulation of CMV DNA replication and inversion.
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