FUSION-DEFECTIVE MUTANTS OF MOUSE HEPATITIS VIRUS-A59 CONTAIN A MUTATION IN THE SPIKE PROTEIN CLEAVAGE SIGNAL

Infection of primary mouse glial cell cultures with mouse hepatitis vi ms strain A59 results in a productive, persistent infection, but witho ut any obvious cytopathic effect. Mutant viruses isolated from infecte d glial cultures 16 to 18 weeks postinfection replicate with kinetics similar to those of wild-type vims but produce small plaques on fibrob lasts and cause only minimal levels of cell-to-cell fusion under condi tions in which wild type causes nearly complete cell fusion. However, since extensive fusion is present in mutant-infected cells at late tim es postinfection, the defect is actually a delay in kinetics rather th an an absolute block in activity. Addition of trypsin to mutant-infect ed fibroblast cultures enhanced cell fusion a small (two- to fivefold) but significant degree, indicating that the defect could be due to a lack of cleavage of the viral spike (fusion) protein. Sequencing of po rtions of the spike genes of six fusion-defective mutants revealed tha t all contained the same single nucleotide mutation resulting in a sub stitution of aspartic acid for histidine in the spike cleavage signal. Mutant virions contained only the 180-kDa form of spike protein, sugg esting that this mutation prevented the normal proteolytic cleavage of the 180-kDa protein into the 90-kDa subunits. Examination of revertan ts of the mutants supports this hypothesis. Acquisition of fusion comp etence correlates with the replacement of the negatively charged aspar tic acid with either the wild-type histidine or a nonpolar amino acid and the restoration of spike protein cleavage. These data confirm and extend previous reports concluding cleavage of S is required for effic ient cell-cell fusion by mouse hepatitis vims but not for virus-cell f usion (infectivity).