| Avian infectious bronchitis virus(IBV)belongs to gamma coronavirus,the family Coronaviridae.Its genome is a positive single-stranded RNA,with a cap structure at the 5 '-end and a poly(A)tail at the 3'-end.IBV-5 'end encodes ORF1 a and ORF1 ab,and two polyproteins PP1 a and PP1 ab are synthesized through translation in the IBV-infected cells.Fifteen mature non-structural proteins(nsp2-16)are produced by cleavage of IBVencoded papain-like protease and 3CL protease,which participate in the virus replication and also play important roles in virus-host interaction.Among them,Nsp16 contains the conserved lysine-aspartic acid-lysine-glutamic acid(KDKE)motif in coronavirus and has the activity of 2 '-O-methyltransferase(2'-O-MTase)involved in the viral immune escape mechanism.However,the biological function of IBV-nsp16 has not been reported.Mutation of aspartic acid to alanine(A)in KDKE motif leads to loss of enzyme activity.We used reverse genetics to obtain an IBV mutant r IBV-D128 A by replacing an aspartic acid located at 128 amino acid in nsp16 with an alanine,resulting in the loss of 2 '-OMTase activity.We found that r IBV-D128 A had weaker pathogenicity than wild type r IBV,and nsp16 could regulate the expression of cell genes by transcriptome sequencing,but the data need experimental verification.Therefore,in this study,we employed fluorescence quantitative RT-PCR and/or Western blot to analyze the expression of the genes related to the cellular natural immunity in the IBV-infected A549 cells The results were as follows:(1)RSAD2(radical s-adenosyl methionine domain containing 2)gene could not be detected in A549 cells,and no significant difference in the expression of IFITM1(IFN-induced protein with tetratricopeptide repeats 1)gene in the normal and IBV-infected A549 cells was found.(2)Quantitative RT-PCR indicated that the expression levels of IFN-?,STAT1(Signal transductor and activator of transcription 1),OASL(2 '-5'-oligoadenylate synthetaselike)and SOCS3(suppressor of cytokine signaling)in r IBV-infected A549 cells were 1.59,1.10,7.04 and 7.72 times of those in normal A549 cells,respectiveiy;the expression levels of IFN-?,STAT1,OASL and SOCS3 in r IBV-D128 A infected A549 cells were 1.02,1.75,2.03 and 3.17 times of those in normal A549 cells,respectively.These results showed that r IBV infection up-regulated the expression of IFN-?,OASL and SOCS3 in A549 cells,and r IBV-D128 A infection up-regulated the expression of STAT1,OASL and SOCS3 in A549 cells.However,r IBV-D128 A infection inhibited the up-regulated expression of IFN?,OASL and SOCS3 induced by r IBV infection,which were 0.64,0.29 and 0.41 times of those in ribv-infected cells,but increased the expression of STAT1,which was 1.59 times of that in r IBV-infected cells.(3)Western blot analysis showed that the expression of SOCS3 protein in rIBV-D128 A infected A549 cells was less than that in ribv-infected cells,which was consistent with the results of RT-q PCR,confirming that r IBV-D128 A could reduce the upregulated expression of SOCS3 induced by r IBV infection.The above results indicated that IBV-nsp16 could significantly up-regulate the expression of OASL and SOCS3 in A549 cells and increase the expression of IFN-?,suggesting that the 2 '-O-methyltransferase of IBV-nsp16 is involved in the expression regulation of genes related to natural immunity in cells.The results proved for the first time that ibv-nsp16 can induce the up-regulated expression of SOCS3,which laid a foundation for further study on the biological function of IBV-nsp16 and the interaction between IBV and host. |
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