MicroRNAs (miRNAs) are small RNAs that regulate gene expression by translational repression and/or mRNA degradation. miRNAs may also up-regulate translation under certain circumstances. There is increasing evidence indicating that miRNAs play a major role in fundamental cellular processes, the regulation of the immune system, as well as in the onset and progression of many diseases. The role of miRNAs in virus-host interaction is just starting to be addressed. miRNAs have been found in some viruses and miRNAs from the host cell may play a role in regulating viral genes and even determine viral tropism. We have investigated whether coronaviruses (CoVs), a group of emerging animal and human RNA viruses, may have the ability to modulate the expression of some cellular miRNAs, which may determine disease outcome. Using a reverse genetic system to manipulate the CoV genome, we have previously characterized the molecular determinants of CoV-induced hepatitis, showed that the ability of murine coronaviruses to induce hepatitis is virus strain specific, and identified cellular targets in the liver (hepatocytes, endothelial cells, and the resident macrophage Kupffer cells). In Preliminary Studies, we have used miRNA microarray analysis and validation by real-time RTPCR to provide evidence of the differential expression of selected cellular miRNAs in macrophages upon in vitro infection with various strains of murine CoVs that induce different hepatitis outcome in the mouse (from minimal inflammation to fulminant hepatitis). Changes in the macrophage miRNAs profiles after murine CoVs infection were overlapping but distinct, and were observed early after infection in the absence of type I IFNs, IFN-?, and pro-inflammatory cytokine secretion. Therefore, we hypothesized that early alteration of the miRNAs expression profile in the CoV-infected mouse liver is a consequence of virus infection, and that strain-specific changes in miRNAs expression contribute to the differences in hepatitis outcome. Thus, we propose the following Specific Aims: (1) Using microarray analysis and real time RT-PCR, we will determine alterations in miRNA profiles in the mouse liver and in primary liver cells (hepatocytes and Kupffer cells) freshly isolated from infected mice, which are different between murine CoVs that differ in hepatitis outcome (MHV-A59, acute moderate hepatitis;and MHV-3, fulminant hepatitis);(2) Based upon results in Specific Aim 1, we will subsequently identify potential cellular miRNAs targets in 3'UTRs by in silico and functional approaches, focusing on those miRNAs whose expression is differentially modulated (up or down) between the viruses listed above and might be involved in fulminant hepatitis;and, (3) We will define the biological effects of knocking-down selected miRNAs (identified in the previous aims) in Kupffer and regulatory T cells and determine the role that those selected miRNAs may have in CoV-induced hepatitis outcome by specific in vivo miRNA-silencing using antagomirs.
The study of the roles of microRNAs (miRNAs) in host-pathogen interactions is an emerging field with great potential for understanding pathogenesis and developing innovative therapeutic approaches. Virtually nothing is known regarding the potential role of cellular miRNAs in coronavirus pathogenesis. The murine coronavirus mouse hepatitis virus is an experimental model that will provide a distinctive opportunity to define the complexity of miRNA-mediated host-pathogen interactions in the liver and to explore innovative interventions for liver disease.
