This grant has supported the identification of bone marrow-derived cell types with characteristics of macrophage and dendritic cell progenitors as sites of CMV latency, provided evidence that the latent viral genome configuration circular similar to a plasmid, constructed a number of viral mutants to address the role of viral functions in latency and established the relationship between viral genome and latent transcript positive cells. We propose to continue investigations that seek to unravel the genetic and molecular basis of viral control of latency and reactivation, using granulocyte-macrophage progenitor cultures as the experimental system. The physiological role of viral genes in latency and reactivation will be investigated with a panel of mutant viruses and assays to test latency-associated gene functions. First, we will complete a function evaluation of previously characterized latent and immediate early (a) gene products in the establishment, maintenance and reactivation of latency in granulocyte-macrophage progenitor cultures. Second, we will investigate the cellular localization and function of ORFs that have been identified on ie1/ie2 region latent transcripts. Here we will investigate ORF94 which localizes to the nucleus and nucleolus and determine its role in nuclear shuffling, RNA binding and RNA splicing. Third, we will identify additional viral genes that are expressed during latency and reactivation using a set of PCR primers that have been made to all viral ORFs. This will complete the identification of latent genes on the viral genome. Here we will focus on UL112-113 region transcripts that have been detected in CMV strain Toledo infected GM-Ps. We will also undertake systematic RT-PCR amplification of all viral ORFs using a set of CMV ORF-specific PCR primers we have assembled. This work will continue to build an understanding of viral functions expressed during and involved in latency of this important pathogen and will continue to provide insights that can be evaluated in naturally infected individuals.
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