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Proteolytic processing of the MHV polymerase polyprotein. Identification of the P28 cleavage site and the adjacent
1Department of Microbiology and Immunology, Loyola University Medical Center, Maywood, Illinois 60153, USA.
Abstract:
The polymerase gene of Mouse Hepatitis Virus strain JHM (MHV-JHM) encodes a polyprotein larger than 750 kilodaltons. This polyprotein is proposed to be processed by several viral proteinases into functional subunits. The amino-terminal subunit is p28, which is cleaved by the first viral papain-like proteinase domain. In this study, we identified the cleavage site of this papain-like cysteine proteinase by amino acid sequencing of radiolabeled polypeptide adjacent to p28. Proteolysis occurs between the glycine-247 and valine-248 dipeptide bond. To determine which amino acid residues are critical for proteolysis, we preformed site-directed mutagenesis on the coding sequences surrounding the cleavage site and assayed for the efficiency of cleavage of p28 in an in vitro transcription and translation system. We report that glycine-247 and arginine-246 are the most critical residues for efficient processing of p28.
Insights
Researchers identified critical residues for Mouse Hepatitis Virus polyprotein processing. Glycine-247 and arginine-246 are essential for efficient cleavage of the p28 subunit by the viral proteinase.
Area of Science:
- Virology
- Molecular Biology
- Biochemistry
Background:
- Mouse Hepatitis Virus (MHV) strain JHM encodes a large polyprotein (>750 kDa) from its polymerase gene.
- This polyprotein is processed into functional subunits by viral proteinases.
- The amino-terminal subunit, p28, is cleaved by the first papain-like proteinase domain.
Purpose of the Study:
- To identify the precise cleavage site of the MHV-JHM papain-like cysteine proteinase.
- To determine the specific amino acid residues critical for p28 subunit processing.
Main Methods:
- Amino acid sequencing of radiolabeled polypeptide adjacent to p28 to identify the cleavage site.
- Site-directed mutagenesis of coding sequences around the cleavage site.
- In vitro transcription and translation assays to measure cleavage efficiency.
Main Results:
- Proteolysis occurs between glycine-247 and valine-248.
- Glycine-247 and arginine-246 were identified as the most critical residues for efficient p28 processing.
Conclusions:
- The study precisely mapped the MHV-JHM papain-like proteinase cleavage site.
- Specific residues, G247 and R246, are crucial for the efficient processing of the p28 subunit, providing insights into viral polyprotein maturation.