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Identification of the murine coronavirus p28 cleavage site
S A Hughes1, P J Bonilla, S R Weiss
1Department of Microbiology, University of Pennsylvania School of Medicine, Philadelphia 94104-6076.
Abstract:
Mouse hepatitis virus strain A59 encodes a papain-like cysteine proteinase (PLP-1) that, during translation of ORF1a, cleaves p28 from the amino terminus of the growing polypeptide chain. In order to determine the amino acid sequences surrounding the p28 cleavage site, the first 4.6 kb of murine hepatitis virus strain A59 ORF1a was expressed in a cell-free transcription-translation system. Amino-terminal radiosequencing of the resulting downstream cleavage product demonstrated that cleavage occurs between Gly-247 and Val-248. Site-directed mutagenesis of amino acids surrounding the p28 cleavage site revealed that substitutions of Arg-246 (P2) and Gly-247 (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 relationship between the p28 cleavage site and other viral and cellular papain proteinase cleavage sites is discussed.
Insights
Mouse hepatitis virus strain A59 uses a papain-like cysteine proteinase (PLP-1) to cleave p28. Key amino acids Arg-246 and Gly-247 are critical for this essential viral protein processing.
Area of Science:
- Virology
- Molecular Biology
- Biochemistry
Background:
- Mouse hepatitis virus (MHV) strain A59 encodes a papain-like cysteine proteinase (PLP-1).
- PLP-1 is responsible for cleaving the p28 protein from the N-terminus during ORF1a translation.
- Understanding viral protein processing is crucial for developing antiviral strategies.
Purpose of the Study:
- To determine the specific amino acid sequences surrounding the p28 cleavage site.
- To identify the key residues involved in PLP-1 mediated cleavage.
- To compare the MHV-A59 cleavage site with other papain proteinase sites.
Main Methods:
- Expression of the first 4.6 kb of MHV-A59 ORF1a in a cell-free transcription-translation system.
- Amino-terminal radiosequencing to identify the cleavage product.
- Site-directed mutagenesis to alter amino acids around the cleavage site.
Main Results:
- Cleavage by PLP-1 occurs precisely between Gly-247 (P1) and Val-248 (P1').
- Mutations at Arg-246 (P2) and Gly-247 (P1) significantly inhibited p28 cleavage.
- Substitutions at other positions (P7 to P2') had variable effects on proteolysis, with some greatly reducing it.
Conclusions:
- The study precisely mapped the p28 cleavage site within the MHV-A59 polyprotein.
- Specific residues, particularly at the P1 and P2 positions, are essential for PLP-1 activity.
- These findings contribute to understanding coronavirus polyprotein processing and protease function.