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Identification and characterization of a 65-kDa protein processed from the gene 1 polyprotein of the murine
M R Denison1, S A Hughes, S R Weiss
1Department of Pediatrics, Elizabeth B. Lamb Center for Pediatric Research, Vanderbilt University, Nashville, Tennessee 37232-2581.
Abstract:
A 65-kDa protein has been detected in mouse hepatitis virus A59 (MHV-A59)-infected DBT cells using polyclonal antibodies directed against polypeptides encoded by the 5' 1.8 kb of gene 1. The presence of this 65-kDa protein (p65) was previously predicted from immunoprecipitation studies of gene 1 expression in MHV-A59-infected DBT cells with other antisera (1). p65 was rapidly labeled in virus-infected cells at late times of infection; however, its cleavage from the polyprotein was significantly delayed compared to the amino-terminal gene 1 polyprotein cleavage product, p28. Similar to p28, p65 was cleaved from the growing polyprotein without detectable intermediate precursors. Kinetic analysis of p65 with specific antibodies indicates that p65 is immediately adjacent to p28 in the gene 1 polyprotein. The proteolytic activity responsible for the carboxy-terminal cleavage of p65, as well as the function of the p65 protein, remains to be determined.
Insights
Researchers identified a 65-kDa protein (p65) in mouse hepatitis virus A59-infected cells. This protein is processed late in infection, adjacent to p28, but its function and cleavage mechanism require further study.
Area of Science:
- Virology
- Molecular Biology
- Cellular Biology
Background:
- Mouse hepatitis virus A59 (MHV-A59) is a significant pathogen.
- Gene 1 encodes essential viral proteins, including polyproteins that undergo complex processing.
- Previous studies predicted the existence of a 65-kDa protein (p65) during MHV-A59 infection.
Purpose of the Study:
- To detect and characterize the 65-kDa protein (p65) encoded by MHV-A59 gene 1.
- To investigate the processing and kinetics of p65 during viral infection.
- To determine the relationship of p65 to other viral polyprotein cleavage products.
Main Methods:
- Infection of DBT cells with MHV-A59.
- Use of polyclonal antibodies against 5' gene 1 polypeptides.
- Immunoprecipitation and kinetic labeling studies.
- Analysis of polyprotein cleavage products.
Main Results:
- A 65-kDa protein (p65) was detected in MHV-A59-infected DBT cells.
- p65 was rapidly labeled at late infection times, indicating its synthesis.
- Cleavage of p65 from the polyprotein was delayed compared to p28, an amino-terminal cleavage product.
- p65 is located immediately adjacent to p28 within the gene 1 polyprotein.
- Cleavage occurred without detectable intermediate precursors.
Conclusions:
- The 65-kDa protein (p65) is a genuine MHV-A59 gene 1 product.
- p65 processing is temporally distinct from p28, suggesting complex regulation.
- The precise proteolytic activity and functional role of p65 remain to be elucidated.