This project will investigate the in vivo synthesis and molecular biochemistry of a newly discovered group of human cytomegalovirus (HCMV) encoded RNA species and their related ribonucleoproteins (RNPs). Recent in vitro transcription studies of HCMV have revealed these RNA species to be a) HCMV gene specified, b) RNA polymerase III transcribed, c) encoded by gene clusters at three sites in the HCMV genome and d) remarkably diverse both in size and in number of species. In addition, our preliminary in vivo studies have demonstrated a) synthesis in HCMV infected cells, and not in mock-infected cells, of a variety of RNA species that species that specifically associate with at least the La protein (recently found to associate with other RNA polymerase III transcripts) recognized by the anti-La class of antibodies from certain patients having autoimmune disease (such as systemic lupus erythematosus, and b) within these HCMV infected-cell RNPs are RNA species homologous to HCMV gene sites transcribed by RNA polymerase III. The synthesis of these RNAs and related RNPs will be analyzed by time of appearance post-infection, turnover rate, and RNA precursor-product relationships to determine if correlations exist between their production in HCMV infected cells and phases of viral macromolecular synthesis. These studies will provide a clear indication of functional form (RNA or RNP) and time of functioning post-infection. Production of these species in permissively and non-permissively HCMV infected cells will be compared to detemine if synthesis and species produced may be modified or controlled by host cell factors. The subcellular localization of these RNA and RNP species following synthesis will be evaluated to provide additional focus on potential functions and RNA or RNP functional forms. The RNA species will be analyzed to determine and compare their nucleotide sequence and gene organization and to compare these features with other RNA polymerase III transcripts. Proteins of the RNP forms will be analyzed to determine if a variety of protein species in addition to the cell-specified La protein are associated, to reveal whether differences in protein composition exists among RNPs, to compare antigenic relatedness among different protein species, and to determine if associated proteins are virus or infected host cell derived. Results of this project will provide the focus essential for clearly directed functional analyses of these HCMV encoded, RNA polymerase III transcribed RNAs and their RNP forms.
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