Cleavage activity of the sapovirus 3C-like protease in Escherichia coli

Area of Science:

• Virology
• Molecular Biology
• Protease Function

Background:

• Human sapovirus (SaV) is a significant cause of gastroenteritis.
• Understanding viral polyprotein processing is key to viral replication.
• The ORF1 polyprotein of SaV encodes essential viral proteins.

Purpose of the Study:

• To determine the cleavage site within the human sapovirus (SaV) ORF1 polyprotein.
• To characterize the enzymatic activity of the SaV 3C-like protease.
• To identify the specific amino acid sequence targeted by the protease.

Main Methods:

• Expression of N-terminal GST and C-terminal 6xHis-tagged fusion proteins (p70 and p14-p70) in E. coli.
• Analysis of expressed proteins using SDS-PAGE and Western blotting.
• N-terminal amino acid sequencing to identify the cleavage site; site-directed mutagenesis of the protease active site.

Main Results:

• Proteolytic cleavage of SaV ORF1 polyprotein occurred between p14 (VPg) and p70 (Pro-Pol).
• The precise cleavage site was identified between glutamic acid (E) at position 1055 and alanine (A) at position 1056.
• The SaV protease demonstrated activity on a rhinovirus 3C protease recognition site, and mutagenesis of the GDCG motif abolished protease activity.

Conclusions:

• The study precisely identifies the ORF1 polyprotein cleavage site in human sapovirus (SaV) for the first time.
• The viral 3C-like protease is responsible for this specific cleavage event.
• The conserved GDCG motif is essential for the proteolytic activity of the SaV protease.