Previous studies have shown that proline mutations in the heptad repeat region stabilize the coronavirus spike (S) protein in a pre-fusion state. To understand the impact of proline substitutions on the fusogenicity of the S protein, we engineered the swine acute diarrhea syndrome coronavirus (SADS-CoV) S protein with two proline substitutions (S-PP) and examined its fusogenicity using dual-split-protein based cell-cell fusion assay. Unlike the wild-type S (S-WT), S-PP rarely formed syncytia. Additionally, protein expression of S-PP was impaired compared to S-WT, as previously reported. Our results indicate that pre-fusion stabilized S protein is unable to induce membrane fusion and provide a better understanding of SADS-CoV S and vaccine antigen design.

INTRODUCTION
MATERIALS AND METHODS
    Cells
    Plasmids
    Western blot analysis
    Immunofluorescence assay
    Dual split protein (DSP) based cell-cell fusion assay
RESULTS
    Expression of SADS S-PP in HEK293T cells
    Fusogenicity of SADS S-PP in HEK293T cells
DISCUSSION
ACKNOWLEDGEMENTS
REFERENCES

• Bac Tran Le(College of Veterinary Medicine, Chungnam National University, Daejeon 34134, Korea)
• Jung-Eun Park(College of Veterinary Medicine, Chungnam National University, Daejeon 34134, Korea) Corresponding author