Coronaviruses pp 157-163 | Cite as

Identification of Peplomer Cleavage Site Mutations Arising during Persistence of MHV-A59

  • James L. Gombold
  • Susan T. Hingley
  • Susan R. Weiss
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 342)

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.

Keywords

Titration Influenza Oligomerization Trypsin Methionine 

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Copyright information

© Springer Science+Business Media New York 1994

Authors and Affiliations

  • James L. Gombold
    • 1
  • Susan T. Hingley
    • 1
  • Susan R. Weiss
    • 1
  1. 1.Department of MicrobiologyUniversity of Pennsylvania School of MedicinePhiladelphiaUSA

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