Human cytomegalovirus (HCMV) frequently causes disease in persons with immature or impaired immune systems, and often results from reactivation of latent virus. The monocyte lineage is important in the pathogenesis of HCMV disease. HCMV productively replicates in macrophages, but produces a latent infection in monocytes and their precursors. Viral major immediate-early (MIE) proteins are critical for productive HCMV replication. They are not expressed in viral latency, owing to a lack of transcription from the MIE promoter. Within the regulatory region of the MIE promoter is the UL126 promoter that is only active during viral latency. The UL126 gene is colinear with the MIE genes. Little is known about the mechanisms regulating the switch in usage of MIE and UL126 promoters, but they are likely influenced by state of cellular differentiation. These regulatory mechanisms may be vital to the HCMV-monocyte interaction. We hypothesize that HCMV latency in immature monocytic cells results from UL126 promoter activity and MIE promoter inactivity. We postulate that cis-acting regulatory mechanisms, such as the 21 bp repeats, Gf1-1 sites, and modulator, are responsible for this outcome. We propose that mobilized peripheral blood CD34+ hematopoietic progenitor/stem cells are natural sites of HCMV latency. As such, they will provide a convenient ex vivo model of HCMV latency in which to define mechanisms that regulate expression of the MIE and UL126 genes.
The first aim of the project is to determine the mechanisms regulating transcription from the MIE and UL126 promoters in infected cells of the monocyte lineage. This will be accomplished by site-directed mutagenesis of the viral genome; the modulator, 21 bp repeats, and Gfi-1 sites will be targeted for mutations.
Our second aim i s to characterize HCMV latency in mobilized CD34+ cells. Ex vivo-infected CD34+ cells will be evaluated as a model of HCMV latency. The mechanisms that regulate the MIE or UL126 genes in viral latency will be determined. Infected cells will be tracked throughout myelo-monocytic development.
