Transcriptomic Analysis Of Avian Reovirus-Infected Cells And The Regulation Of Stress Granule Formation By P17 Protein

Avian reovirus(ARV)infection is widespread in chickens,and some strains can cause severe diseases such as viral arthritis,causing great economic losses to the poultry industry.In order to fully elucidate the molecular mechanisms of ARV infection and pathogenesis,we took ARV GX/2010/1strain as the research object,and analyzed the transcriptome of ARV infected chicken embryo fibroblast cell line(DF-1 cell line)by high-throughput sequencing technology,and conducted subsequent studies according to the transcriptome results.1.Transcriptome analysis of ARV infected host cellsThe ARV strain GX/2010/1 was used to infect DF-1 cell line,and the cells in the infected group and the ARV uninfected control group were collected at 10 hpi and 18 hpi,respectively.RNA was extracted by Trizol method,and then high-throughput sequencing of cellular transcriptome was performed.The results showed that 168 differentially expressed genes(DEGs)were measured at 10 hpi in the ARV-infected group compared with the control group without ARV infection,of which 64 genes were up-regulated and 104 genes were down-regulated.At 18 hpi,48 DEGs were detected,of which 47 genes were up-regulated,and 31 of these up-regulated genes were consistent with those in the 10 hpi group.Only one gene with down-regulated expression level was measured.Analysis of the functions of the DEGs showed that,the up-regulated genes mainly included antiviral genes,endosomal vesicle fuion genes,nucleotide synthesis genes,transcriptional regulatory genes,etc.The down-regulated genes mainly included genes positively regulating cell proliferation,lipid metabolism-related genes,long non-coding RNA(lcRNA),etc.It was found that ARV infection upregulated the expression levels of Wnt14,a key gene in chicken synovial joint development,and the pro-inflammatory cytokine IL-1βrelated gene.The upstream kinases PKR and PERK of Stress granules(SGs)signaling pathway were up-regulated and down-regulated,respectively.2.ARV infection induced autophagy and up-regulation of IL-1βWnt14 gene expression in chickensIn order to further reveal the mechanism of ARV infection leading to viral arthritis,we examined the level of autophagy in various tissues and the expression of Wnt14 and IL-1β in the tarsal joint of ARV GX/2010/1 infected chickens(inoculated through paw pads).The results showed that ARV infection increased the level of autophagy in different tissues and significantly induced the formation of autophagosomes.ARV can persist in chicken tarsal joint for a long time and stimulate the expression levels of Wnt14 and IL-1β.The above changes could last for the whole cycle of ARV infection,which further confirmed that the occurrence of viral arthritis was related to the upregulation of autophagy and Wnt14/IL-1β gene expression.3.Regulation of stress granules in host cells by p17In this study,we found that SGs could be transiently induced by ARV infection through the PKR-eIF2α pathway.Unlike SGs induced by Mammalian Reovirus(MRV),ARV-induced SGs contained G3BP1 but did not contain TIAR,another common marker protein.Knockdown of G3BP1 promoted ARV replication,which was substantially reduced in the presence of SC-1 induced TIAR-containing SGs.Further studies showed that ARV inhibited the generation of TIAR-containing SGs induced by SC-1 at the late stage of infection,but not at the early stage.Treatment with puromycin showed that the blocking effect of ARV on TIAR was dependent on viral protein synthesis.This result suggested that TIAR-containing SGs could act as an important antiviral mechanism,and ARV could block the migration of TIAR into SGs by viral protein.In order to further study the blocking mechanism of TIAR by ARV,some structural and non-structural proteins encoded by ARV were fused with GFP.After transfection,it was found that non-structural protein p17 caused TIAR to accumulate in the nucleus,but could not migrate to the cytoplasm to form SGs.The nuclear import signal of the C terminus of p17 protein plays an important role,indicating that p17 can not function until it enters the nucleus.Co-immunoprecipitation assay showed that p17 and TIAR did not interact.As an RNA-binding protein,TIAR is closely related to the transcription,splicing and translation of cellular mRNA.Therefore,we hypothesized that p17 may interfere with the nucleoplasmic transport of cellular mRNA,thereby restricting TIAR from the nucleus.Further studies showed that p17 could interact with hnRNP A1,a cellular mRNA transporter.The N-terminal 40 amino acid(1-40 aa)of p17 was found to be the key peptide to block TIAR nuclear ejection by truncated expression,which coincided with p17(19-40aa)which could bind hnRNP A1.In summary,the mechanism by which p17 inhibits the generation of TIAR-containing SGs is proposed as following:p17 is imported into the nucleus through a nuclear localization signal and interacts with hnRNP A1 through the N-terminus of p1 7 to block the transport of mRNA(+TIAR)from the nucleus to the cytoplasm,thereby inhibiting the generation of TIAR-containing SGs in the cytoplasm.In conclusion,through transcriptome analysis and animal experiments,the present study showed that ARV infection induces autophagy and upregulates the expression of Wnt14 and IL-1β,two key upstream regulatory genes that probable be related to arthritis.The non-structural protein p17 of ARV could inhibit the formation of SGs by interacting with hnRNP A1 to inhibit TIAR transport.This study revealed the key host factors of ARV in the development of viral arthritis and elucidated the molecular mechanism by which ARV hijacks nucleocytoplasmic transport through the nonstructural protein p17.