Molecular detection of proteolytic activity of human parechovirus ۲A protein by gene expression

Parechoviruses form one of the nine genera in the picornaviridae family, and include two human pathogens: Human parechovirus type۱ and ۲ (Hpev۱ and Hpev۲). The genome of picornaviruses encodes a single polyprotein, which undergoes a cleavage cascade performed by virus encoded proteases to give the final virus proteins. The primary cleavage occurs by ۲A protein and this step is critical for viral life cycle. Recent sequence analysis suggests that Hpev۱ is distinct from other picornaviruses and lacks the motifs believed to be involved in the protease function of ۲A. The aim of this study was to analyze proteolytic activity of ۲A protein in Hpev۱. For this purpose we made several recombinant plasmids contain ۲A region of parechovirus type۱ genome and expressed in prokaryotic and in vitro systems under T۷ promoter. Analyzing the expression products by SDS-PAGE revealed just a large single band (۹۰ KDa), the same size as primary translation product. Whereas with plasmids include ۳C gene several small bands were observed, indicating that processing had occurred. In conclusion: the results of this work show that Human parechovirus type۱ has a processing strategy different from the other members of picornaviruses and in this virus, as in hepatovirus, ۲A protein does not have a protease function.