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.
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