Contribution of IL-32 gene expression to viral persistence

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Project Abstract A case is being made for the utility of MeV persistence in development of lifelong immunity. The thought is that persistent infection of epithelial cells, in addition to immune cells, may produce virus or viral antigens to promote affinity maturation of antibodies for a sufficient duration of time that antibodies of the highest avidity are produced. What governs establishment of a phenotype in an epithelial cell conducive to establishment of a persistent infection is not known. However, such a cell should be able to suppress but not eliminate virus multiplication, and virus release must occur by a non-lytic mechanism to avoid a state of chronic inflammation. Establishing the existence of such persistent- infection-competent epithelial cells and their gene expression, especially genes involved in control of virus multiplication, is an essential first step in understanding the molecular basis of viral persistence. Our single-cell analysis of poliovirus (PV) infection revealed a population of HeLa cells that were quite proficient at supporting PV multiplication without lysis for as long as 36 h post-infection, which was the duration of the experiment. We developed a strategy to isolate this sub-population of persistently- infected cells using standard cell-culture approaches by adding an inhibitor of PV entry to the culture media. We observed PV persistence for as long as six days post-infection, again the duration of the experiment. Gene-expression analysis of these persistently-infected cells revealed a near-complete suppression of sensors of viruses and other pathogens, including Toll-like receptors and “toll-free” (cytosolic) receptors18. The gene for IL-32 was induced 7 fold (P-value <0.001). IL-32 has been implicated in antiviral responses that are independent of type I and II interferons (IFNs); however, effectors of the IL-32-dependent, antiviral pathway have yet to be discovered. The overarching goal of the program that we will initiate under the auspices of this R21 grant is elucidation of genes, pathways, and mechanisms required for viral persistence. The first goal of this application is to identify genes that may contribute to establishment of persistence of CVB3 in human cardiomyocytes. The second goal of this application is to inspire hypotheses for the mechanism of IL-32 action. These goals will be pursued as indicated by the following aims: (1) Discover genes contributing to CVB3 persistence in human cardiomyocytes; and (2) Discover effectors of the IL-32-dependent antiviral state in HeLa cells.