Date

October 2010

Document Type

Dissertation

Degree Name

Ph.D.

Department

Dept. of Molecular Microbiology and Immunology

Institution

Oregon Health & Science University

Abstract

Cytomegalovirus (CMV) is a ubiquitous species-specific β-herpesvirus that establishes lifelong persistence and latency following primary infection. CMV persistence involves the continual production of low levels of virus, whereas viral latency is associated with a lack of virus production but both states maintain limited viral gene expression. While much is known about viral gene transcription during lytic infections in vitro, little is known about the specific gene transcription profiles that are associated with in vivo persistence, latency and reactivation. Persistence is likely to play a role in the development of chronic disease associated with human CMV (HCMV) infection including vascular diseases and chronic inflammatory diseases. Our laboratory has developed a rat heart transplant chronic rejection (CR) model that exhibits the hallmarks of transplant vascular sclerosis (TVS) in humans. In this transplant model, rat CMV (RCMV) accelerates the development of chronic allograft rejection by enhancing TVS formation. Using microarrays to analyze viral gene expression, we have shown that RCMV gene expression is highly restricted in tissues from infected allograft recipients. However, RCMV gene expression is also highly dynamic, changing in the same tissue over time. For instance, in the salivary glands the viral transcription profile significantly changes between 7 and 10 days post infection. Initially, RCMV expresses genes involved in viral replication but that quickly changes to a profile primarily involved in persistence (i.e. immune evasion and host manipulation). Identification of CMV genes expressed and the level of expression in specific cell types, tissue types and during different stages of the viral lifecycle is critical to our understanding of CMV persistence and pathogenesis. Our laboratory is actively determining the function of the viral genes expressed during persistence and identifying the control mechanisms involved in regulating viral gene expression in vivo. In this dissertation, I document the identification of RCMV microRNAs (miRNAs), which are a class of small non-coding RNAs involved in post-transcriptional regulation. Using a direct cloning/sequencing approach we discovered RCMV encodes 24 miRNAs and similar to RCMV mRNA expression, RCMV miRNA expression is dynamic and tissue specific in infected rat heart allograft recipients. We hypothesize that RCMV miRNAs are involved in regulating viral gene expression in vivo. During our study of RCMV gene expression in vivo we found a viral open reading frame (ORF) R116 with an unknown function highly expressed in infected salivary glands, the site of viral persistence. I characterized the RCMV gene R116 and show R116 is important for the production of infectious virus. We hypothesize that R116 plays a significant role in an entry step of the virus lifecycle. The expression, regulation and function of CMV genes involved in the establishment and maintenance of a persistent infection within host cells is critical to our understanding of CMV pathogenesis.

Identifier

doi:10.6083/M4416V2X

School

School of Medicine

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