Cellular SRC kinases and dsRNA dependent protein kinase (PKR) play key role in intracellular viral (CVB3) replication



SRC kinases and PKR are intracellular protein kinases, which play key roles in intracellular viral replication. In this research, the effect of SRC kinase inhibition and PKR activation and inhibition on replication of coxsakievirus (CVB3), an entrovirus of the family picornaviridae – causative agents of fatal myocarditis, was studied. Vero and Hela cells were cultured and infected with CVB3 in the presence of herbimycin A (specific tyrosin kinase inhibitor), added before and after infection in various time interval, gamma interferon (PKR activator) and 2-aminopurine (PKR inhibitor). Viral replication was monitored by molecular methods (RT-PCR) as well as plaque forming unit (PFU) count using a set of forward and reverse primer for viral gene, which could detect positive and negative strand mRNA of the virus, as well as PFU count. It was observed that SRC kinase inhibitor, when added before addition of CVB3 to the medium, can inhibit intracellular viral replication, whereas if herbimycin is added after infection, had no effect on viral proliferation. Using set of primers for conserved region of PKR gene, it was possible to observe the presence of PKR mRNA in all cultures either interferon treated, or 2Ap treated, or control. PFU studies showed that ?-INF has inhibitory effect on viral replication, where as 2Ap treatment enhanced viral replication. Relevance of the results towards development of new molecules for viral infection treatment will be discussed.