Molecular Cloning of Reovirus a3 Vaccinia E3L
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Preliminary data by Imani and colleagues support the hypothesis that viral infection commonly associated with asthma exacerbation leads to the formation of double-stranded RNA (ds-RNA) which induces pro inflammatory cytokines. The presence of dsRNA appears to trigger a cellular response to viral infection in animal cells. This activation is likely through dsRNA-dependent enzymes such as interferon-inducible protein-kinase (PKR) which in turn activate proinflammatory cytokines. In the present study, the reovirus a3 and vaccinia E3L genes, known to encode for proteins which inhibit PKR, were cloned for the purpose of transfection to human epithelial cells. The objectives of this experiment were to investigate the pathway by which proinflammatory cytokine activation can be inhibited via the inhibition of PKR. Because cytokines are recognized to be an important element of chronic inflammation and play a critical role in initiating the inflammatory response, discovery of a mechanism for cytokine inhibition may offer insight into the development of new treatment of asthmatic inflammation. Following successful cloning of the genes in to the cloning vector pCI-neo, the genes were transfected to human epithelial BEAS-2B cells. After permanent transfection, the western blot revealed that the protein <13 was not expressed in the BEAS-2B cell line. Though these results are negative, the hypothesis has not been rejected. Current experiments are aimed at finding a more suitable cell line for transfection. If expression is not detected using different human epithelial cells, it is possible that a different cloning vector may be more successful. Finally, if the hypothesis is incorrect, it is possible that the proteins are too toxic for the cells.