Identification and subcellular localization of porcine deltacoronavirus accessory protein NS6

Abstract

Porcine deltacoronavirus (PDCoV) is an emerging swine enteric coronavirus. Accessory proteins are genus-specific for coronavirus, and two putative accessory proteins, NS6 and NS7, are predicted to be encoded by PDCoV; however, this remains to be confirmed experimentally. Here, we identified the leader-body junction sites of NS6 subgenomic RNA (sgRNA) and found that the actual transcription regulatory sequence (TRS) utilized by NS6 is non-canonical and is located upstream of the predicted TRS. Using the purified NS6 from an Escherichia coli expression system, we obtained two anti-NS6 monoclonal antibodies that could detect the predicted NS6 in cells infected with PDCoV or transfected with NS6-expressing plasmids. Further studies revealed that NS6 is always localized in the cytoplasm of PDCoV-infected cells, mainly co-localizing with the endoplasmic reticulum (ER) and ER-Golgi intermediate compartments, as well as partially with the Golgi apparatus. Together, our results identify the NS6 sgRNA and demonstrate its expression in PDCoV-infected cells.

Genetic manipulation of porcine deltacoronavirus reveals insights into NS6 and NS7 functions: a novel strategy for vaccine design

“…These results suggested that the NS6 protein is necessary for efficient viral replication in vivo, which may be attributable to the high pathogenicity of PDCoV. Recently, Fang et al demonstrated that the PDCoV NS6 predominantly localize to the ER complex and ER-Golgi intermediate compartment, which is the site of coronavirus assembly and packaging [29], suggesting that NS6 may be involved in virus assembly. Moreover, PDCoV NS6 was identified as an inhibitor of IFN-β expression by interacting with RIG-I and MDA5 to impede their association with doublestranded RNA, thus disturbing RLR-mediated IFN-β signal pathway [40].…”

“…Similarly, MHV and FIPV mutants bearing defective accessory protein genes were also dramatically attenuated when inoculated into their natural hosts [17,28]. Recently, PDCoV NS6, which is located between the M and N, and NS7, which is occupied within the N gene in an alternative ORF, were identified as the accessory proteins [29]. Moreover, NS7a, a 100-amino-acid-polypeptide identical to the C-terminus of NS7, has also been identified as a novel accessory protein [30].…”

Detection and spike gene characterization in porcine deltacoronavirus in China during 2016–2018

“…The genome of PDCoV is composed of approximately 25.4 kb nucleotides and has a genomic organization similar to other coronaviruses: a 5′ untranslated region (UTR), open reading frame 1a (ORF1a) and ORF1b encoding two overlapping polyprotein precursors; four structural protein genes encoding spike (S), envelope (E), membrane (M), and nucleocapsid (N); three accessory protein genes encoding NS6, NS7, and NS7a; and a 3' UTR and poly (A) tail (Chen et al, 2015a;Fang et al, 2017;Fang et al, 2016;Li et al, 2014;Woo et al, 2012). For coronaviruses, the function of the S protein is to recognize receptors and mediate viral entry into host cells.…”

Porcine Deltacoronavirus Accessory Protein NS6 Antagonizes Interferon Beta Production by Interfering with the Binding of RIG-I/MDA5 to Double-Stranded RNA

“…PDCoV is an enveloped, single-stranded, positive-sense RNA virus belonging to the genus Deltacoronavirus of the family Coronaviridae (4). The full-length genome of PDCoV is approximately 25.4 kb in length, with the essential genes occurring in the order 5= UTR-ORF1a/1b-S-E-M-NS6-N-NS7-NS7a-3= UTR and encoding a total of 15 mature nonstructural proteins, four structural proteins, and three accessory proteins (13,(19)(20)(21). Coronavirus accessory proteins are species specific, and each coronavirus encodes various amounts of accessory proteins interspaced between viral structural protein genes.…”