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

Abstract

Swine acute diarrhea syndrome coronavirus (SADS-CoV) was reported in China in 2017 and is a causative agent of porcine enteric disease. Recent studies indicate that cells from various hosts are susceptible to SADS-CoV, suggesting the zoonotic potential of this virus. However, little is known about the mechanisms through which this virus enters cells. In this study, we investigated the role of furin in SADS-CoV spike (S)-mediated cell – cell fusion and entry. We found that the SADS-CoV S protein induced the fusion of various cells. Cell – cell fusion was inhibited by the proprotein convertase inhibitor dec-RVKR-cmk, and between cells transfected with mutant S proteins resistant to furin cleavage. These findings revealed that furin-induced cleavage of the SADS-CoV S protein is required for cell – cell fusion. Using mutagenesis analysis, we demonstrated that furin cleaves the SADS-CoV S protein near the S1/S2 cleavage site, 446 RYVR 449 and 543 AVRR 546 . We used pseudotyped viruses to determine whether furin-induced S cleavage is also required for viral entry. Pseudotyped viruses expressing S proteins with a mutated furin cleavage site could be transduced into target cells, indicating that furin-induced cleavage is not required for pseudotyped virus entry. Our data indicate that S cleavage is critical for SADS-CoV S-mediated cell – cell fusion and suggest that furin might be a host target for SADS-CoV antivirals.

Detection and characterization of novel luchacoviruses, genusAlphacoronavirus, shed in saliva and feces of meso-carnivores in the northeastern United States

“…Comparative genetic analyses have shown that bat CoV HKU2 is closely related to SADS-CoV, suggesting that bat CoVs may also be involved in the emergence of pathogenic CoVs of veterinary importance (55). It has been previously shown that a substitution of a single residue in this cleavage site (residue 458 in Figure 3) is enough to abrogate the cleavage of S (11). In this study we show that several luchacoviruses, including those in groups 1 and 2 and divergent variants UMN2020/mouse/2018/USA and P83/plateau pika/China, have a cleavage motif in this region that differ only in this essential residue to that of SADS-CoV (R458S, Figure 3A) and that a single nucleotide substitution is sufficient to revert this residue (Figure 3B).…”

“…However, we show that unlike SADS-CoV and bat CoV HKU2, luchacoviruses do not have an S1/S2 cleavage site (Figure 3A). As cleavage in both S1 and S1/S2 sites is required for cleavage and cell fusion of SADS-CoV (11), more than a single non-synonymous change would be necessary for luchacoviruses to acquire a cleavage mechanism like the one of pathogenic SADS-CoV. In contrast to luchacoviruses, most rodent betacoronaviruses have a predicted S1/S2 cleavage site (78% of available S sequences, (22)), more than 13 times of what was predicted for bat betacoronaviruses (6% of available sequences, (22)).…”

“…These 17 partial S sequences were translated into their respective amino acid sequences and aligned with the homologous amino acid sequences of two alphacoronaviruses, SADS-CoV (MT294722), bat CoVs HKU2 (NC_009988), and three betacoronaviruses including SARS-CoV-2 (QQN72495), a rodent CoV (MT820632) and a rabbit CoV (JN874560) using the Clustal Omega algorithm (47). This alignment was used to compare regions in S in which known cleavage sites occur including the S1/S2 and S2’ regions of SARS-CoV-2 (53), and regions in the S1 domain in which Luchacovirus JC34 (22) and SADS-CoV (11) have predicted and characterized furin cleavage sites, respectively. We detected potential cathepsin cleavage sites by looking for specific amino acid motifs previously identified for human cathepsins L, V, K, S, F, B (14).…”

Detection and characterization of novel luchacoviruses, genus Alphacoronavirus , in saliva and feces of meso-carnivores in the northeastern United States

“…These 17 partial S sequences were translated into their respective amino acid sequences and aligned with the homologous amino acid sequences of two alphacoronaviruses, SADS-CoV (MT294722) and bat CoVs HKU2 (NC_009988), and three betacoronaviruses including SARS-CoV-2 (QQN72495), a rodent CoV (MT820632), and a rabbit CoV (JN874560) using the Clustal Omega algorithm ( 48 ). This alignment was used to compare regions in S in which known cleavage sites occur including the S1/S2 and S2′ regions of SARS-CoV-2 ( 54 ) and regions in the S1 domain in which Luchacovirus JC34 ( 22 ) and SADS-CoV ( 11 ) have predicted and characterized furin cleavage sites, respectively. We detected potential cathepsin cleavage sites by looking for specific amino acid motifs previously identified for human cathepsins L, V, K, S, F, and B ( 14 ).…”

“…In the Alphacoronavirus genera, CoVs like feline and canine CoV type 2 (FCoV-2 and CCoV-2, respectively), transmissible gastroenteritis virus and human CoV 229E do not have an identifiable S1/S2 cleavage site ( 9 ), while swine acute diarrhea syndrome CoV (SADS-CoV), an emergent and highly pathogenic CoV that possibly originated from bat CoV HKU2, does have an S1/S2 cleavage site ( 10 ). The S protein of SADS-CoV also has an additional cleavage site in the S1 domain whose cleavage together with that on the S1/S2 site is essential for cell-cell fusion ( 11 ). Overall, multiple cleavage sites may contribute to membrane fusion and virus entry, processes that are crucial determinants for an efficient infection and the disease outcome.…”