MicroRNA function in murine cytomegalovirus

The broad goal of this project is to determine the mechanism by which an animal virus utilizes and controls microRNA (miRNA) expression in order to establish an infection and persist in its host. MiRNAs are a class of small RNA molecules (~ 22 nucleotides long), discovered in the last decade, that regulate gene expression by targeting specific messenger RNA transcripts for degradation or inhibited translation. It is estimated that 30% of genes in humans could be regulated by miRNAs, implicating a fundamental role of these molecules in modulating the transcriptome. Viral-encoded miRNAs have been discovered recently in a number of different viruses, including all three herpesvirus subfamilies. Recent reports have demonstrated that viral miRNAs can target host genes involved in the immune response. This class of molecules, therefore, represents an important new class of potential drug targets. To date, no viral miRNA has been examined in vivo. Understanding the functional role of miRNAs in an intact physiological system is of vital importance; an in vivo analysis considers that both viral and cellular gene expression can be influenced by cell-context as well as the extent of the anti-viral immune response. We will examine viral miRNA function in murine cytomegalovirus (CMV). Cytomegalovirus, a member of the betaherpesvirus family, is a ubiquitous virus that is a major cause of morbidity in the clinical setting. CMV is highly species-specific; however, the pathogenesis of MCMV in mice is remarkably similar to that of HCMV in humans. Murine CMV is therefore an established model for studying human CMV and the MCMV genome can be modified to generate miRNA deletion mutants using bacterial artificial chromosome (BAC) technology. We will use both MCMV miRNA deletion mutants and antisense technology to examine viral miRNA function in vivo. Microarray technology will be used to examine the host genes that are targeted by MCMV miRNAs. This work is highly relevant to future analysis of miRNA inhibitors as anti-viral therapeutics. Finally we will establish a system for examining viral miRNA biogenesis in vitro. This is required to gain an in-depth understanding of how the virus controls and exploits this class of molecules and should complement the in vivo analysis. The tools and knowledge resulting from this work will be widely applicable in other viral systems. MiRNAs have also been shown to play a role in cancer formation and cardiovascular and metabolic diseases; expanding the understanding of miRNA function and mechanism, therefore, is of paramount importance to human health.

Viral-encoded microRNAs have been discovered in all three herpesvirus subfamilies, in polyomaviruses and retroviruses. Multiple reports have detailed potential functions of viral miRNAs, which can target both host and viral genes. In human cytomegalovirus, a viral miRNAs has been shown to target a host gene involved in the immune response. However, an in-depth of understanding of viral miRNA function requires an in vivo analysis, which considers that both viral and cellular gene expression can be influenced by cell-context as well as the extent of the anti-viral immune response. Here we will examine miRNA function in murine cytomegalovirus (MCMV); MCMV is an established model for studying human CMV and uniquely enables analysis of a natural infection in the natural host system. We previously identified and characterized viral miRNAs in MCMV and we will use MCMV miRNA deletion mutants and the latest antisense technologies to examine viral miRNA function in vitro and in vivo. Combined with microarray analysis, this will enable us to identify host genes and pathways targeted by MCMV miRNAs. This will also provide a basis for future work to evaluate the use of viral miRNA inhibitors as potential anti-viral therapeutics. We have preliminary data to suggest that one of the viral miRNAs may target a key gene in the host immune response, and may be distinctly regulated; we will use this viral miRNA as a focal point for the above studies. Ultimately, understanding viral miRNA function and mechanism in vivo will require further characterization of viral miRNAs in vitro. Therefore, the final aim of this project is to establish an in vitro assay for viral miRNA processing that can be used to identify viral and host proteins that modulate viral miRNAs.