The fusogenic properties of the uncleaved spike (S) protein of murine coronavirus JHMV variant cl-2 were studied by expressing the S protein with a deleted putative cleavage site. The amino acid sequence of the putative cleavage site, Arg-Arg-Ala-Arg-Arg, was replaced by Arg-Thr-Ala-Leu-Glu by in vitro mutagenesis of the cl-2 S protein cDNA. Recombinant vaccinia viruses containing the cl-2 S cDNA [RVV t(+)] or the mutated cDNA [RVV t(-)] were constructed and monitored for fusion formation and cleavage of the expressed S proteins. When cultured DBT cells were infected with RVV t(+) at a multiplicity of infection of 0.5, fusion formation was first observed at 10 to 12 h postinoculation and spread throughout the whole culture by 20 to 24 h postinoculation. In cells infected with RVV t(-) under the same conditions, fusion formation appeared by 12 to 14 h. This result represented a 2- to 4-h delay in the onset of fusion, compared with its appearance in cells expressing the wild-type S protein. By 25 to 30 h, most of the cells infected by RVV t(-) had fused. By immunoprecipitation and Western blotting (immunoblotting), the 170-kDa S protein was detected in DBT cells expressing the wild-type S protein and the mutated S protein. However, interestingly, the cleavage products of the S protein, S1 and S2, were not detected in RVV t(-)-infected cells, producing the mutated S protein, even though fusion was clearly visible. Both products were, of course, detected in RVV t(+)-infected DBT cells, producing the wild-type S protein. The same results concerning the fusion formation and cleavage properties of the S proteins were reproduced by the transiently expressed S proteins. These results suggest that the cleavage event in the S protein of murine coronavirus JHMV is not a prerequisite for fusion formation but that it does facilitate fusion formation.