Human cytomegalovirus (HCMV) is a frequent opportunistic pathogen, causing extensive morbidity and mortality. It is the most common known congenital virus infection in the-United States, sometimes with serious or fatal consequences; by one estimate 3000-4000 infants annually are born with symptomatic infection and suffer deafness, blindness, mental retardation, or death. In immunocompromised individuals HCMV infection or reactivation can be fatal. HCMV is a particular problem in AIDS patients, and may be a cofactor in HIV pathogenesis. Like other herpesviruses CMV exhibits both lytic and latent phases. An understanding of the molecular mechanisms of HCMV DNA replication and their regulation is needed to aid in developing effective antiviral strategies. Lytic-phase DNA replication requires both trans-acting factors such as the virus-specified DNA polymerase, and a recently identified cis-acting sequence, the origin of DNA replication, oriLyt. The experiments proposed will elucidate the structure of oriLyt, study its role in DNA replication, and examine its interaction with other elements of the DNA replication machinery.
The specific aims are: (i) to establish the boundaries, structure and environment of CMV oriLyt; (ii) to determine-which sequences within the established boundaries are required for, or contribute to,. origin function; (iii) to identify and characterize trans-acting factors which may interact specifically with oriLyt; and (iv) to study the mechanisms of oriLyt function., The methods to be used include: (i) the construction of extensive series of mutations and deletions in recombinant clones which will then be functionally tested in the transient assay that was used to locate oriLyt; (ii) band-shifting and footprinting studies to identify and characterize site-specific DNA-protein interactions, followed by column and affinity chromatography to purify the protein factors; (iii) various biochemical methods to examine the DNA structure, function, and conformation of DNA segments within oriLyt. The long term goal is to contribute to a comprehensive understanding of the molecular mechanisms and biological strategies of HCMV DNA replication, and to the development of an in vitro DNA replication system.
