Discovery of a novel accessory protein NS7a encoded by porcine deltacoronavirus.

Puxian Fang,Xiaorong Liu,Dang Wang,Shaobo Xiao,Panpan Ma,Jing Bi,Nan Dong,Yingying Hong,Liurong Fang

doi:10.1099/jgv.0.000690

Abstract

Porcine deltacoronavirus (PDCoV) is an emerging swine enteric coronavirus. Bioinformatics predicts that PDCoV encodes two accessory proteins (NS6 and NS7), the species-specific proteins for coronavirus. In this study, four mAbs against the predicted NS7 were prepared by using the purified recombinant NS7 protein. Indirect immunofluorescence assay demonstrated that all mAbs recognized cells transfected with an NS7 expression construct or infected with PDCoV. Western blot showed that NS7-specific mAbs recognized an additional protein band of about 12 kDa from PDCoV-infected cell lysates but not from cells with the ectopic expression of NS7. Detailed analysis suggested that this additional protein band represented a novel accessory protein, termed NS7a, a 100 amino acid polypeptide identical to the 3′ end of NS7. Moreover, NS7a is encoded by a separate subgenomic mRNA with a non-canonical transcription regulatory sequence. In summary, our results identified a third accessory protein encoded by PDCoV, which will enhance our understanding of PDCoV.

Highlights

Porcine deltacoronavirus (PDCoV) is an emerging swine enteric coronavirus
Previous reports indicated that accessory proteins contained in other coronavirus are non-essential for viral replication in vitro [18]; they are associated with immune modulation and viral pathogenesis in vivo [19,20,21,22]
Considering that no similar small protein band was detected in cells transfected with the NS7 expression construct pCAGGS-HA-NS7 (Fig. 1d), and at least two NS7 proteins (NS7a and NS7b) are predicted in deltacoronaviruses such as white-eye coronavirus, sparrow coronavirus and magpie robin coronavirus [4], we speculate that the observed smaller protein band may be a new unidentified PDCoV-encoded protein

Summary

Introduction

Porcine deltacoronavirus (PDCoV) is an emerging swine enteric coronavirus. Bioinformatics predicts that PDCoV encodes two accessory proteins (NS6 and NS7), the species-specific proteins for coronavirus. To determine the expression of NS7 in cells infected with PDCoV and analyse its biological function in the viral replication cycle, we prepared mAbs against the putative NS7 protein. To test the specific recognition of the four mAbs to the predicted NS7 protein, a eukaryotic expression plasmid containing NS7 was constructed by cloning the NS7 gene into the pCAGGS-HA vector with primers NS7-F and NS7-R, which was transfected into LLC-PK cells.

Results

Conclusion