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Characterization of the leader papain-like protease of MHV-A59
P J Bonilla1, J L Piñón, S Hughes
1Department of Microbiology, University of Pennsylvania, Philadelphia 19104-6076, USA.
Abstract:
Sequence analysis of the mouse hepatitis virus, strain A59 (MHV-A59) genome predicts the presence of two papain-like proteases encoded within the first open reading frame of the replicase gene. The more 5' of these domains, the leader papain-like protease, is responsible for the cleavage of the amino terminal protein, p28. We have defined the core of this protease to between amino acids 1075 and 1344 from the beginning of ORF 1a. Deletion analysis coupled with in vitro expression, was used to study p28 cleavage by this leader protease. Expression of a series of deletion mutants showed processing of p28, albeit at lower levels in some of them. Reduced p28 production resulting from a 0.4 kb deletion positioned between p28 and the protease domain suggests an involvement of this region in catalytic processing. Some mutants display cleavage patterns indicative of a second cleavage site. Interestingly, this newly identified cleavage site maps to a position similar to the expected cleavage site of a p65 polypeptide detected in MHV-A59 infected cells. Mutagenesis of the catalytic H1272 residue demonstrates that both cleavages observed are mediated by the leader papain-like protease encoded in ORF 1a.
Insights
Mouse hepatitis virus leader papain-like protease (PLP) cleaves the p28 protein. This study defines the core protease region and identifies a new cleavage site, implicating the leader PLP in multiple processing events.
Area of Science:
- Virology
- Molecular Biology
- Protease Function
Background:
- Mouse hepatitis virus (MHV-A59) genome encodes two papain-like proteases (PLPs) in its replicase gene.
- The leader PLP is responsible for cleaving the amino-terminal p28 protein.
Purpose of the Study:
- To define the core region of the MHV-A59 leader PLP.
- To investigate the mechanism of p28 cleavage and identify potential secondary cleavage sites.
Main Methods:
- Sequence analysis to predict protease domains.
- Deletion analysis of the leader PLP.
- In vitro expression and processing assays.
- Site-directed mutagenesis of the catalytic residue H1272.
Main Results:
- The core leader PLP was localized between amino acids 1075 and 1344 of ORF 1a.
- Deletion mutants showed altered p28 processing, with a 0.4 kb deletion impacting catalytic efficiency.
- A novel cleavage site was identified, mapping near the expected site for p65 polypeptide.
- Mutagenesis of H1272 confirmed its essential role in both observed cleavages.
Conclusions:
- The MHV-A59 leader PLP is crucial for p28 processing and mediates at least two distinct cleavage events.
- The identified catalytic residue H1272 is essential for the protease's activity.
- The study provides insights into the proteolytic processing of the MHV-A59 replicase polyprotein.