Fusogenic properties of uncleaved spike protein of murine coronavirus JHMV.

We have tested the fusogenic properties of cleaved and uncleaved spike (S) protein of murine coronavirus (MCV) JHMV variant cl-2 by expressing the S protein by recombinant vaccinia viruses (RVVs). The amino acid sequence of the putative cleavage site of cl-2 S protein, Arg-Arg-Ala-Arg-Arg, was replaced by Arg-Thr-Ala-Leu-Glu by in vitro mutagenesis of cl-2 S gene. The RVVs having cl-2 S gene [RVV t(+)] or mutated cl-2 S gene [RVV t(-)] were tested for their ability to induce fusion as well as cleavability in DBT cells. After inoculation with RVV t(+) onto DBT cells, the fusion formation was first observed at 8 h postinoculation (p.i.) and spread throughout the whole culture by 24 h. In cells infected with RVV t(-), fusion appeared by 2 h and most of cells were fused by 30 h p.i. The S protein and its cleavage products were detected in DBT cells expressing wild type S protein. However, no cleavage products of the S protein were detected in RVV t(-) infected cells producing mutated S protein, even though fusion was clearly visible. These results suggest that the cleavage event of JHMV-S protein of MCV is not a prerequisite for fusion formation, but that it enhances fusion.