Furin cleavage is required for swine acute diarrhea syndrome coronavirus spike protein-mediated cell – cell fusion

Figures & data

Table 1. Primers used for generating mutant furin cleavage site SADS-CoV S.

Primer name	Primer sequences
AYVA forward	5′ AGCCAGCTGGCCTATGTGGCCATCCTGGGC 3′
AYVA reverse	5′ GCCCAGGATGGCCACATAGGCCAGCTGGCT 3`
KSAA forward	5′ AAGAGCGCCGCCTTCGTGGAT 3′
KSAA reverse	5′ ATCCACGAAGGCGGCGCTCTT 3′
AVAA forward	5′ GTGGCCGTGGCCGCCATGACCTTT 3′
AVAA reverse	5′ AAAGGTCATGGCGGCCACGGCCAC 3′
AYVR forward	5′ AGCCAGCTGGCCTATGTGCGC 3′
AYVR reverse	5′ GCGCACATAGGCCAGCTGGCT 3′
AVAR forward	5′ GTGGCCGTGGCCAGAATGACC 3′
AVAR reverse	5′ GGTCATTCTGGCCACGGCCAC 3′
SADS S1 forward	5′ CATCATTTTGGCAAAGAATTCGCCACC 3′
SADS S1130 reverse	5′ AAAAAGATCTGCTAGCTCGAGTTATGC 3′

Figure 1. The swine acute diarrhea syndrome coronavirus (SADS-CoV) spike (S) protein induces cell – cell fusion in various cell types. (A) Schematic representation of the cell-cell fusion assay (see details in Materials and Methods). (B, C) Cells were transfected with SADS-CoV S-C9 or empty vector (control) and green fluorescent protein (GFP). After 48 h, cell – cell fusion was observed by fluorescence microscopy (B) and scored as 0, 1, 2, or 3, representing 0%, less than 33%, 33% to 66%, and more than 66% fuzed cells (C). Scale bars, 100 µm. (D) S expression was examined by western blot analysis using an anti-C9 antibody.

Figure 2. Furin activates SADS-CoV S-mediated cell – cell fusion and S cleavage. (A, B) HEK293T cells were transfected with SADS-CoV S and GFP. After 6 h, transfected cells were treated with the indicated protease inhibitors. Cell – cell fusion was observed by fluorescence microscopy (A). Scale bars, 100 µm. S expression was examined by western blotting (B). (C) Cells were transfected with SADS-CoV S and GFP in the presence or absence of human furin. After 48 h, cell nuclei were stained with DAPI and cell – cell fusion was assessed by fluorescence microscopy. Scale bars, 100 µm. (D) Nuclei in the fuzed cells were counted. Statistical significance was assessed by Student’s t test. ***, P < 0.001.

Figure 3. S cleavage at two furin cleavage sites is required for cell – cell fusion. (A) Schematic showing the SADS-CoV S protein containing a receptor binding domain (S1) and a fusion domain (S2). The amino acid (aa) residues of three putative furin cleavage sites and the S2` cleavage sites are indicated, and the mutations introduced into the cleavage sites are shown in bold. (B) HEK293T cells expressing wild type (WT) and mutant S proteins along with GFP. Cell – cell fusion was observed by fluorescence microscopy. Where indicated, cells were treated with 5 µg/mL trypsin. Scale bars, 100 µm. (C) S expression was examined by western blotting.

Figure 4. SADS-CoV S cleavage by furin is not required for viral entry. (A) S proteins in pseudotyped viruses (pv) were examined by western blotting. BALD pv indicates pseudotyped viruses lacking S proteins. (B) Huh7 cells were transduced with the indicated pseudotyped viruses. After 48 h, viral entry was quantified by measuring luciferase levels. Statistical significance was assessed by Student’s t test. *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant.