MOLECULAR ASPECTS OF CYTOMEGALOVIRUS LATENCY

Human cytomegalovirus (HCMV) is now recognized as a significant pathogen
which can cause birth defects in congenitally infected infants,
interstitial pneumonia in immunologically compromised individuals, or
persist as a latent infection which can be reactivated at a later time.
The overall aim of this project is to identify viral-cellular interactions
which determine whether HCMV will productively infect a cell or remain
latent. Two different cell systems will be utilized to reach this goal.
To determine if lymphocytes can latently harbor HCMV, subpopulations of
peripheral blood lymphocytes, separated by the fluorescence activated cell
sorter, will be infected with laboratory strains and recent isolates. In
parallel with the lymphocyte experiments, studies will examine events
following HCMV infection of an established cell line of human
teratocarcinoma cells. Both cell systems will be examined for persisitence
of viral DNA, RNA, and proteins using Southern and Northern blotting
techniques and immunofluorescence with viral protein specific monoclonal
antibodies. Non-productively infected teratocarcinoma cells and
lymphocytes will be examined for expression of viral genes to determine at
what stage the block in replication occurs. Cellular processes such as
transcript processing, accumulation, transport, and association with
polysomes will also be studied as possible regulators of viral
replication. These studies will consist of examination of nuclear
cytoplasmic and polysome RNA, and in vitro run-off transcription of
specific HCMV genes. Active and inactive HCMV genes from these cells will
also be examined for DNaseI hypersensitivity and the degree of
methylation. Correlation of results from non-productively infected
lymphocytes and teratocarcinoma cells will enhance our knowledge of HCMV
latency.