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Further in vitro characterization of mouse hepatitis virus papain-like proteinase 1: cleavage sequence requirements

Henry Teng1, Susan R Weiss

  • 1Department of Microbiology, University of Pennsylvania School of Medicine, Philadelphia 19104-6076, USA.

Journal of Neurovirology
|April 6, 2002
PubMed

Insights

Mouse hepatitis virus (MHV) replicase processing involves specific cleavage sites. PLP-1 recognizes a proposed p50 cleavage site but not a predicted p72 site in vitro.

Area of Science:

  • Virology
  • Molecular Biology
  • Biochemistry

Background:

  • Mouse hepatitis virus (MHV) replicase gene product pp1a undergoes proteolytic processing.
  • This processing generates key polypeptides including p28, p65, p50, and p240 in infected cells.
  • Previous studies identified p28 and p65 cleavage sites, leading to a proposed p50 cleavage site.

Purpose of the Study:

  • To investigate the proteolytic processing of MHV-A59 replicase.
  • To confirm the proposed p50 cleavage site and explore a predicted p72 cleavage site.
  • To determine the substrate specificity of the viral protease PLP-1.

Main Methods:

  • Site-directed mutagenesis was employed to alter specific cleavage sequences.
  • In vitro cleavage assays were performed using modified substrates.
  • Analysis of polypeptide products from MHV-infected cells.

Main Results:

  • PLP-1 demonstrated in trans cleavage activity when the proposed p50 sequence replaced the p28 sequence.
  • No cleavage was detected in vitro for a substrate containing both the p65 and predicted p72 cleavage sequences.
  • The predicted p72 cleavage site was not recognized by PLP-1 under the tested in vitro conditions.

Conclusions:

  • PLP-1 can recognize and cleave at the proposed p50 site within the MHV replicase.
  • The predicted p72 cleavage site is not processed by PLP-1 in vitro.
  • These findings clarify specific proteolytic events in MHV replication.

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