Fusogenic properties of uncleaved spike protein of murine coronavirus JHMV

Abstract

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.