Cloning and expression of Protease 2 from Coxsakievirus B3

Protease 2A [2Apro] of coxsackievirus B3 [CVB3] plays a major role in viral replication. In case of infection, viral proteins are being synthesized from viral mRNA using host biosynthesis machinery. 2Apro of virus, after being synthesized, exhibits two critical functions, cleavage of viral proteins and breaking eukaryotic initiation factor 4G. The enzyme plays an essential role in viral replication and cellular damage. To understand pathogenicity of infection and also developing potent and selective inhibitors against picornavirus infection, it is necessary to prepare pure 2Apro enzyme. cDNA of 2Apro was synthesized using in vitro infection of permissive host through reverse transcription process and was cloned in pET22b[+]. Since 2Apro is a toxic product, its expression will act on host before induction and damages the cells. For this reason, different hosts were checked and finally BLR[DE3]pLysS, which carries an extra-plasmid for lysozyme expression, that minimizes unwanted target protein products [leakage] was selected. By employing such expression system we could minimize the unwanted expression of 2Apro. Though it is not possible to avoide it, but seems negligible. Hence, this system is useful for expression of toxic proteins in sensitive hosts in order to prevent bacterial damage. The product was confirmed by SDS polyacrylamide gel electrophoresis and immunoblot analysis

Effect of activation and inhibition of cellular PKR on coxsackievirus B3 replication

The ds-RNA activated protein kinase [PKR] is a serine-threonine kinase with MW of 68 KDa. It belongs to a family of kinases that control one of the translational initiation factors, eIF2. PKR is produced at high level in response to viral infection. This protein by phosphorylating eIF2 inhibits cellular protein synthesis. In this study, the effect of gamma interferon [IFN- gamma], an activator, and 2-aminopurine [2AP], an inhibitor of PKR production on coxsackievirus B3 [CVB3] replication were studied. After addition of IFN- gamma and 2AP to Vero and HeLa cultures, cells were infected with CVB3. After 48 h and appearance of cytopathic effect [CPE], cells were collected and viral replication was assessed by standard method. For molecular detection of PKR, RT-PCR technique was used. The data show that interferon inhibited [or lowered] and 2AP promoted viral replication, though in all samples presence of PKR-mRNA could be detected. It seems that PKR plays a key role in CVB3 replication. Therefore, PKR can be considered as a promising and considerable target for designing small molecules and drugs against CVB3