Biological Function Of Avian Infectious Bronchitis Virusencoded Nonstructural Protein 16

Avian infectious bronchitis virus(IBV),a member of Coronavirus ? causes an acute and contagious disease in chickens with a significant impact on the poultry industry worldwide.IBV contains a 27.6 kb single-stranded,positive-sense RNA genome.Like other coronaviruses,IBV-encoded nonstructural protein 16(nsp16)has2'-O-methyltransferase(2'-O-Mtase)activity involving in viral immune escape.The active site of the known coronavirus-encode 2'-O-Mtase is consist of 4 conserved amino acid residues(lysine-aspartic acid-lysine-lysine-glutamic acid,KDKE).The replacement of D with alanine(A)can result in the loss of the activity of 2'-O-Mtase.This study aims to obtain the mutant virus containing point mutation D128 A in the active site of IBV-encoded 2'-O-Mtase using reverse genetics method and study the biological function of IBV-encoded nsp16 by analysis of its biological characteristics and transcriptomic analysis.The results are as follows:1.Using IBV reverse genetics technique,we obtained a mutant virus IBV-D128 A containing point mutation D128 A in the active site of IBV-encoded 2'-O-Mtase based on the genetic background of IBV-P65.2.The biological characteristics of IBV-D128 A were analyzed by plaque assay,viral growth curve,RT-PCR,and Western blotting compared to rIBV.The results are as follows:(1)Plaque assay showed that the mean diameter of the plaque formed in Vero cells infected by IBV-D128 A at 5d post infection was about 0.54±0.12 mm,while the mean diameter of the plaque formed in rIBV-infected Vero cells was about 1.20±0.23 mm,suggesting that the pathogenicity of IBV-D128 A was obviously lower than rIBV;(2)Both viruses displayed similar viral growth curve in Vero cells and their titers were up to peak at 24 h post infection,but the titer of IBV-D128 A was lower than rIBV at the same time-point;(3)Analysis of viral RNA replication by semi-quantitative RT-PCR demonstrated that compared to rIBV,the viral RNA synthesis of IBV-D128 A decreased at 24 h,36h and 48 h post infection;(4)Detection of viral spike protein expression by Western blot showed that compared to rIBV,the spike protein expression decreased at 24 h and 36 h and 48 h post infection;Above-mentioned results suggest that the enzyme activity of 2'-O-Mtase encode by IBV is not essential for viral replication,but the loss of the enzyme activity of2'-O-Mtase can lead to the decrease of IBV replication and the attenuation of viral virulence.3.To detect the regulation effects of IBV infection on the expression of host genes,RT-PCR was performed to detect the expression of cytokines or antiviral genes in H1299 cells and DF-1 cells after rIBV and IBV-D128 A infection respectively.Theresults indicated that the expression of IFN-?,IL-6 and IL-8 had no significant changes in the infected H1299 cells;However,in DF-1 cells infected with both viruses,both viruses,IFN ?,signal transducer and activator of transcription 1(STAT1),interferon-stimulated gene 12(ISG12),suppressor of cytokine signaling 3(SOCS3),2'-5'-oligoadenylate synthetase-like(OASL)and radical S-adenosyl methionine domain containing 2(RSAD2)were up-regulated,suggesting that IBV infection may activate IFN? signaling pathway and induce the expression of the relative antiviral genes such as SOCS3,OASL and RSAD2.4.To study the mechanism of attenuating virulence caused by IBV-D128 A and illuminate the biological function of IBV-encoded 2'-O-MTase,RNAs were extracted from IBV and IBV-D128 A infected DF-1 cells for transcriptome analysis.The results are as follows:(1)Compared to rIBV,883 differentially expressed genes(DEG)were selected in DF-1 cells infected with IBV-D128 A at 24 h post infection,among which the expression of 487 genes was up-regulated while expression of 396 genes was down-regulated;(2)799 DEGs were functionally annotated in different databases: 292 DEGs in COG,725 DEGs in GO,491 DEGs in KEGG,531 DEGs in KOG,777 DEGs in NR,749 DEGs in Swiss-prot,785 DEGs in eggNOG;(3)725 of 883 DEGs were annotated in Biological Process,Molecular Function and Cellular Component in GO classification;(4)292 of 883 DEGs were annotated in COG database.Among them,104 DEGs in General Function Prediction Only;42DEGs were related to replication,recombination and repair of DNA;32 DEGs were involved in signal transduction mechanism;(5)491 of 883 DEGs were annotated in 50 metabolism pathways in KEGG database.In detail,76 DEGs in 9 pathways in Cellular Processes,106 DEGs in 12 pathways in Environment Information Processing,11 DEGs in 2 pathways in Genetic Information Processing,43 DEGs in 3 pathways related to human diseases,11 DEGs in 2 pathways in Genetic Information Processing,64 DEGs in 9 pathways in Organismal System,and 102 DEGs in 14 pathways involved in metabolism;(6)151 of 883 DEGs were annotated in 18 immune-related signaling pathways in KEGG,including pattern recognition receptors,complement systems,cytokines and apoptosis,etc.KEGG classification revealed that Toll-like receptor 3,IFN?,interleukin 6 and 8 were up-regulated but interleukin 18 was down-regulated;(7)String analysis of key protein codes by differentially expressed genes showed that 2'-5'-oligoadenylate synthetase-like(OASL)(ENSGALG00000013723),an interferon-induced antiviral protein,is a key protein and can interact with 12 other protein.Identification and function of these proteins are being investigated.In conclusion,our research findings confirm that the enzyme activity of2'-O-Mtase of IBV-encoded nsp16 is not necessary for IBV replication but the loss of this enzyme activity results in the reduction of IBV replication and the attenuation of viral virulence;Moreover,IBV infection may activate IFN? signaling pathway and induce the expression of the relative antiviral genes such as SOCS3,OASL and RSAD2.In addition,transcriptome analysis shows that IBV-D128 A infection can up-regulate the expression of host genes associated with host innate immunity,suggesting that IBV-encoded nsp16 could function in viral immune escape.