Molecular Analysis of Proteolytic Activity of 3C Protein in Human Parechovirus Type 1

Human parechovirus is a genus of picornaviruses which includes a number of important viruses of clinical significance. Recent sequence analysis suggests that human parechovirus type 1(HPEV-1) is distinct from other picornaviruses and lacks the motives believed to be involved in the protease function of 2A. The aim of this study was to analyse proteolytic activity of 3C protein in human parechovirus type 1. The region of HPEV-1 cDNA that was coded for 3C protein was inserted into plasmid pUBS by Ligase enzyme and pFG3 recombinant plasmid was prepared. After transformation and replication of this plasmid in E.coli MC 10.22, DNA was isolated by phenol extraction and then expressed in prokaryotic(E.coli BL-21) and in vitro systems under T7 promoter. The results were detected by SDS-PAGE and analyzed. The products of expression of recombinant plasmid(not included 3C gene) in both prokaryotic and in vitro systems were analyzed. Just one large band(90 KD) was observed, but with plasmid including 3C gene, several small bands were detected. These results indicated proteolytic activity of 3C protein. After addition of anti protease to the products of plasmid with 3C gene the result was the same as the plasmid without 3C gene. The results showed that HPEV-1 has a processing strategy different from other members of picornaviruses, and 3C protein seems to be the only virus encoded protease that can catalyze cleavages of all sites in the parechoviruses primery polyprotein.