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Identification of peplomer cleavage site mutations arising during persistence of MHV-A59
J L Gombold1, S T Hingley, S R Weiss
1Department of Microbiology, University of Pennsylvania School of Medicine, Philadelphia 19104-6076.
Abstract:
Primary mouse glial cell cultures were infected with mouse hepatitis virus strain A59 (MHV-A59) and maintained over an 18 week period. Viruses isolated from these cultures 16-18 weeks postinfection produce small plaques on fibroblasts and cause only minimal levels of cell-to-cell fusion at times when wild type causes nearly complete cell fusion. However, when mutant-infected cultures were examined 24-36 hours postinfection approximately 90% of the cells were in syncytia showing that the fusion defect is not absolute but rather delayed. Addition of trypsin to mutant-infected cultures enhanced cell fusion a small (2- to 5-fold) but significant degree. Sequencing of portions of the spike genes of six fusion-defective mutants revealed that all contained the same single nucleotide mutation resulting in a substitution of aspartic acid for histidine in the spike cleavage signal. Mutant virions contained only the 180 kDa form of spike protein suggesting that this mutation prevented the normal proteolytic cleavage of the 180 kDa protein into the 90 kDa subunits. Examination of revertants of the mutants supports this hypothesis. Replacement of the negatively-charged aspartic acid with either the wild type histidine or a non-polar amino acid was associated with the restoration of spike protein cleavage and cell fusion.
Insights
A mutation in the mouse hepatitis virus (MHV-A59) spike protein causes a delayed cell fusion defect. This defect is linked to impaired spike protein cleavage, affecting viral spread.
Area of Science:
- Virology
- Cell Biology
- Molecular Biology
Background:
- Mouse hepatitis virus (MHV-A59) is a significant pathogen.
- Viral fusion is critical for MHV-A59 pathogenesis.
- Spike protein processing is essential for MHV-A59 infectivity.
Purpose of the Study:
- To investigate the molecular basis of fusion defects in MHV-A59 mutants.
- To identify mutations affecting MHV-A59 spike protein cleavage and cell fusion.
- To understand the role of spike protein processing in viral pathogenesis.
Main Methods:
- Infection of primary mouse glial cell cultures with MHV-A59.
- Isolation and characterization of fusion-defective MHV-A59 mutants.
- Analysis of viral plaque formation and cell-to-cell fusion.
- Sequencing of MHV-A59 spike genes.
- Western blot analysis of spike protein cleavage.
Main Results:
- Fusion-defective MHV-A59 mutants exhibited delayed cell fusion and smaller plaques.
- A single nucleotide mutation in the spike cleavage signal was identified in all mutants.
- This mutation resulted in impaired proteolytic cleavage of the spike protein.
- Revertant viruses showed restored spike protein cleavage and cell fusion.
Conclusions:
- The identified mutation in the MHV-A59 spike protein is responsible for the fusion defect.
- Proper spike protein cleavage is essential for efficient cell-to-cell fusion and viral spread.
- This study elucidates a key mechanism of MHV-A59 pathogenesis.