This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.The long-term goal of this project is to develop an understanding of the cellular and molecular mechanisms of cytomegalovirus (CMV) persistence in the host. CMV is a species-specific virus that establishes a persistent/latent infection in the host after primary infection. This ability requires avoidance of immune detection and limitation of cytopathic effect on cells harboring virus. Herpesviruses achieve this by restricting expression of the majority of the viral genome, thereby reducing acute replication and minimizing the potential for presentation of antigenic proteins, while maintaining the ability to reactivate at a later stage. The process by which herpesviruses restrict their own gene expression, thereby promoting a persistent or latent state, is not well understood, but is likely to involve both cellular and viral factors. The recent discovery of RNA interference and the widespread expression of microRNAs has uncovered a new layer of post-transcriptional gene regulation that was previously unknown. A number of studies have suggested that a large percentage of transcripts may be targeted and regulated by miRNAs, indicating that regulation by small RNAs may represent a crucial mechanism in the control of gene expression. Studies by our own group and by others have identified over 70 miRNA genes encoded by multiple DNA viruses, the majority of which have been identified within the herpesvirus family. All three subgroups of herpesviruses, alpha, beta and gamma, have been shown to express miRNAs during both acute replication and during latency. Although relatively little is known about the function of virally encoded miRNAs, their potential ability to regulate multiple transcripts and the lack of an immunogenic response make miRNAs ideal candidates for the promotion and maintenance of a persistent or latent viral gene expression profile. Furthermore, preliminary data presented in this proposal suggests a possible role for at least one of the HCMV miRNAs in restricting acute replication of the virus. Following bioinformatics studies we have identified a number of potential viral target transcripts of the HCMV miRNA UL112-1. These targets include the uracil DNA glycosylase gene, which resides directly antisense to UL112-1 and is directly cleaved by the miRNA, and the major transactivating protein IE72, which is regulated post-transcriptionally via a target sequence within the 3' UTR of the messenger RNA. In this proposal we will extend the characterization of HCMV UL112-1 during acute infection and during persistent infection. In addition we will identify and characterize the HCMV gene targets of the other virally encoded miRNAs and analyze their contribution to regulation of the virus during acute infection. Lastly we will examine the expression and function of the HCMV miRNAs in monocyte macrophages and persistently infected endothelial cells. Elucidating the mechanisms of miRNA regulation of HCMV gene expression will provide an important contributions herpesviruses and miRNA field as a whole.
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