Identification Of Porcine Deltacoronavirus Accessory Protein And The Inhibitory Mechanism Of NS6 On IFN-? Production

Coronavirus(CoV)belongs to the family Coronaviridae of the Nidovirales,which could infect a wide range of hosts,including birds,humans and other mammals.According to the ninth report of the International Virus Classification Committee,the coronaviruses are divided into four genera(?,?,? and ?,respectively).These coronaviruses encode different amounts of accessory proteins between and within structural genes,in addition to encoding conventional nonstructural proteins and structural proteins.These accessory proteins are species-specific and have no homology or very low homology with other known proteins.Previous studies on the accessory proteins of SARS-CoV and other coronaviruses have found that coronavirus accessory proteins are not essential for viral replication,but mainly involved in the immune regulation and virulence of the virus.In 2014,the outbreak of porcine deltacoronavirus(PDCoV)in the United States was first reported and caused severe diarrhoea,vomiting and even death in newborn piglets.Subsequently,the occurrence and prevalence of the disease was also reported in many other countries,which causes the pig industry's attention.Based on the genomic sequence,it is predicted that PDCoV can encode two accessory proteins,NS6 and NS7,respectively.However,they have not been verified experimentally and their functions remain to be explored.In addition,it is unclear whether PDCoV encodes other accessory proteins,except for NS6 and NS7.Therefore,in this study,using PDCoV CHN-HN-2014 strain as a representative,we identify the expression of putative accessory proteins and explore their relationship with host innate immune responses.The specific research contents are as follows: 1.Identification of PDCo V accessory protein NS6 According to PDCoV CHN-HN-2014 genome sequences,two specific primers(Leader-F,NS6r)were designed,which are located at the 5' leader sequence of the genome and the 3' end of the NS6 gene,respectively.Total RNA was extracted from the PDCoV-infected cells,followed by RT-PCR and sequencing analysis.The results showed that NS6 was an independent subgenomic RNA(sgmRNA),containing a non-classical transcription regulating sequences(TRS)located to TRS upstream.Subsequently,two strains hybridoma cells stably secreting NS6-specific antibodies were obtained through preparation of monoclonal antibodies.Indirect immunofluorescence and western blot analysis revealed that the two monoclonal antibodies(2G3 and 4B9)specifically recognized LLC-PK1 cells overexpressing NS6 protein or infected with PDCoV,whcih confirmed that NS6 protein was expressed in the early of virus infection and localized in the cytoplasm.Further analysis found that NS6 protein mainly co-localizing with the endoplasmic reticulum(ER)and ER-Golgi intermediate compartments,as well as partially with the Golgi apparatus.These results for the first time demonstrate that NS6 protein is expressed in PDCoV-infected cells under the control of an independent subgenomic RNA with non-classical TRS(ACACCT).2.Identification of PDCo V accessory protein NS7 and the discovery of NS7 a Based on predicted sequence of PDCoV NS7 gene,we designed primers to clone the the putative NS7 gene into the pGEX-KG prokaryotic expression vector to obtain the recombinant plasmid pGEX-KG-NS7.Subsequently,the fusion protein GST-NS7 was achieved via induction with IPTG.Furthrtmore,four hybridomas cells stably secreting NS7 protein-specific antibodies were successfully obtained through preparation of monoclonal antibodies.IFA confirmed that all four monoclonal antibodies could specifically recognize LLC-PK1 cells with PDCoV infection or NS7 overexpression.However,western blot analysis revealed that not only NS7 protein-specific bands but also specific protein band of about 12 kDa could be detected by the four monoclonal antibodies in PDCoV-infected cells.However,the small protein band could not be detected in cells with ectopic expression of NS7.Subsequently,a new subgenomic RNA with a non-classical TRS was found via the analysis of viral sgmRNA in PDCoV-infected cells.The 3?end of the ORF in new sgmRNA is identical to 3?end of NS7 and can encode a polypeptide with 100 aa,suggesting a new accessory protein and designated as NS7 a.Further study found that overexpressed NS7 a was specifically recognized by four monoclonal antibodies aganinst NS7 and was similar in protein weight size to small proteins under viral infection.These results indicate that PDCoV exactly encodes a new accessory protein,NS7 a,which is an independent subgenome and uses a non-classical TRS(ACCCCA).3.PDCoV NS6 protein antagonizes IFN-? production Previous studies have confirmed that PDCoV encodes three accessory proteins(NS6,NS7,NS7a).However,whether these proteins involve in immune regulation function remains to be unclear.Therefore,we use the NS6 protein as a representative to further investigate the characteristic of PDCoV accessory proteins regulating natural immunity.NS6 gene was cloned into pCAGGS-HA vector to obtain eukaryotic expression plasmid pCAGGS-HA-NS6.We found that ectopic expression of accessory protein NS6 in cells significantly inhibits Sendai virus-induced interferon-?(IFN-?)production,as well as SeV-induced phosphorylation and nuclear translocation of the key transcription factors IRF3 and NF-?B.The results indicated that NS6 protein can antagonize IFN-? production.To our surprise,NS6 protein does not impede the IFN-? promoter activity mediated by the key molecules of RIG-I-like receptors signal pathway(RIG-I/RIG-IN,MDA5,MAVS,TBK1,IKK?,IRF3),suggesting that NS6 protein may targets at the levels of RIG-I/MDA5 or its upstream process.Meanwhile,NS6 interacted with RIG-I/MDA5 by co-immunoprecipitation assay(Co-IP)and IFA,which was not dependent on RNA.Further analysis showed that NS6 protein interacts with the CTD of RIG-I or Hel and CTD of MDA5.RNA pull-down analysis confirmed that NS6 is not an RNA-binding protein.Subsequent Co-IP competitive experiment assay also indicated that NS6 protein could significantly attenuate the binding of RIG-I/MDA5 to dsRNA.Taken together,these results suggest that a novel immunosuppressive strategy used by NS6 to inhibit RLRs-mediated IFN-? production.