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Identification and analysis of MHV-A59 P28 cleavage site
S A Hughes1, P J Bonilla, S R Weiss
1Department of Microbiology, University of Pennsylvania School of Medicine, Philadelphia 94104-6076, USA.
Abstract:
During translation of Murine hepatitis virus (MHV-A59) ORF1a, p28, the N-terminal polypeptide is cleaved from the growing polypeptide chain. Amino terminal radiosequencing of the resulting downstream cleavage product demonstrated that cleavage occurs between Gly247 and Val248. Site directed mutagenesis of amino acids surrounding the p28 cleavage site revealed that substitutions of Arg246 (P2) and Gly247 (P1) nearly eliminated cleavage of p28. Single amino acid substitutions of other residues between P7 and P2' were generally permissive for cleavage although a few changes did greatly reduce proteolysis. The amino acids around the p28 cleavage site represent a new sequence recognized by a virus encoded papain-like proteinase.
Insights
Murine hepatitis virus (MHV-A59) proteolysis of the ORF1a polyprotein involves cleavage of p28. Specific amino acid substitutions at the cleavage site, particularly at P1 and P2, significantly inhibit this process, revealing a novel viral protease recognition sequence.
Area of Science:
- Virology
- Molecular Biology
- Protease Function
Background:
- Murine hepatitis virus (MHV-A59) is an important model for coronavirus research.
- The MHV-A59 ORF1a polyprotein undergoes proteolytic processing to generate functional viral proteins.
- Understanding viral protease specificity is crucial for developing antiviral strategies.
Purpose of the Study:
- To identify the precise cleavage site of the p28 polypeptide during MHV-A59 ORF1a translation.
- To investigate the role of specific amino acid residues in the p28 cleavage event.
- To characterize the recognition sequence of the viral papain-like proteinase.
Main Methods:
- Amino terminal radiosequencing to determine the exact cleavage point.
- Site-directed mutagenesis to alter amino acids surrounding the cleavage site.
- Analysis of proteolysis efficiency following mutagenesis.
Main Results:
- Cleavage of p28 occurs precisely between Gly247 and Val248.
- Mutations at Arg246 (P2) and Gly247 (P1) drastically reduced p28 cleavage.
- Most other amino acid substitutions (P7 to P2') were tolerated, but some reduced proteolysis.
Conclusions:
- The p28 cleavage site is defined by specific amino acid residues, with P1 and P2 being critical.
- These findings elucidate a novel recognition sequence for the MHV-A59 papain-like proteinase.
- This knowledge contributes to understanding coronavirus polyprotein processing and protease mechanisms.