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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.
Insights
Murine coronavirus spike protein cleavage is not essential for fusion but enhances it. This study investigated the role of spike protein cleavage in coronavirus-induced cell fusion.
Area of Science:
- Virology
- Molecular Biology
Background:
- Murine coronavirus (MCV) JHMV variant cl-2 utilizes its spike (S) protein for cell entry and fusion.
- The S protein undergoes proteolytic cleavage at a specific site, which is often crucial for viral infectivity.
Purpose of the Study:
- To investigate the role of S protein cleavage in the fusogenic properties of MCV JHMV.
- To determine if S protein cleavage is a prerequisite for, or merely enhances, cell fusion.
Main Methods:
- Recombinant vaccinia viruses (RVVs) were engineered to express either wild-type (cl-2 S gene, RVV t(+)) or mutated (cleavage site altered, RVV t(-)) MCV S protein in DBT cells.
- The ability of these RVVs to induce cell fusion and the cleavage status of the S protein were analyzed post-inoculation.
Main Results:
- Both wild-type and mutated S proteins induced cell fusion in DBT cells.
- Fusion occurred earlier and more rapidly with the mutated S protein (RVV t(-)) compared to the wild-type (RVV t(+)).
- Cleavage products of the S protein were detected only in cells expressing the wild-type S protein, not in cells expressing the mutated S protein.
Conclusions:
- Proteolytic cleavage of the JHMV-S protein is not essential for mediating cell fusion.
- S protein cleavage appears to enhance the efficiency or rate of fusion, rather than being a prerequisite event.