| Infectious bronchitis virus(IBV)is an enveloped,single-stranded RNA virus,which belongs to the genus Gammacoronavirus in the family Coronaviridae.After infection,IBV employes a discontinuous transcription mechanism to produce six viral m RNAs,including full-length genomic RNA1 and five sub-genomic RNA2-6(sg RNA2-6).The previous study has shown that IBV-Beaudette strain could generate a non-coding RNA(ncRNA)using a discontinuous transcription mechanism.The ncRNA consists of 563 nucleotides excluding the poly(A)tail,is mainly derived from the3’-untranslated region of IBV genome,and contains a 63-nt-long of terminal leader sequence derived from the 5’ end of the viral genome.Using reverse genetics,a mutant virus r IBV-C27107 G which is unable to produce ncRNA was obtained.It was found that it had no significant effect on the viral replication and pathogenicity in Vero cells.However,transcriptome sequencing analysis found that IBV-ncRNA could regulate the expression of the genes related to antiviral response via Toll-like receptor signaling pathway,Jak/Stat signaling pathway and other signaling pathways in H1299 cells.Many viruses employ different mechanisms to generate ncRNAs,which play important roles in the viral life cycle,but the biological function of IBV-ncRNA is still poorly understood.Therefore,to analyze the regulatory effect of the ncRNA on cellular factors,we selected some differentially expressed genes in H1299 cells infected with r IBV and r IBV-C27107 G and utilized fluorescence quantitative RT-PCR and Western blot to detect the expression levels of related genes in the two virus-infected cells.The main research results are as follows:(1)IBV-ncRNA inhibited the up-regulated expression of HSPA4 induced by r IBV-C27107 G but enhanced the expression of XRCC5.The expression levels of HSPA9,HSPA4,HSP90B1,XRCC5,PPIA and UCHL1 in r IBV-infected H1299 cells were 1.12,0.17,0.97,1.94,1.83 and 1.62 times of those in MOCK infection at 16 h post infection,respectively,while their expression levels in r IBV-C27107 G infected H1299 cells were 1.30,1.27,1.41,0.97,1.04 and 2.31 times of those in MOCK cells,respectively.The expression levels of HSPA9,HSPA4,HSP90B1,XRCC5,PPIA and UCHL1 in H1299 cells infected with r IBV-C27107 G were 1.16,7.47,1.45,0.50,0.57 and 1.43 times of those in r IBV-infected H1299 cells,respectively.(2)IBV-ncRNA can inhibit the expression of related genes in the TIRAP-TRAF6-NF-κB signaling pathway.The expression levels of HSPA4,TIRAP,TRAF6,NF-κB,ICAM-1 and MCP-1 in r IBV-infected H1299 cells were 0.44,0.59,0.41,0.62,0.47 and 0.53 times of those in MOCK cells at 12 h post infection,respectively.Correspondingly,their expression levels in r IBV-C27107 G infected H1299 cells were 1.23,1.74,1.02,1.77,1.40 and 2.64 times of those in MOCK cells,respectively.Comparative analysis showed that the expression levels of HSPA4,TIRAP,TRAF6,NF-κB,ICAM-1 and MCP-1 in r IBV-C27107 G infected cells were 3.24,2.95,2.49,2.85,2.98 and 4.98 times of those in r IBV-infected cells,respectively.(3)IBV-ncRNA inhibited the phosphorylation of JNK.The phosphorylation level of JNK increased gradually in 20-28 hours after infection.The ratio of p-JNK/JNK in r IBV-C27107 G infected cells was 0.16,0.34 and 0.58,respectively.The phosphorylation of JNK in r IBV-C27107 G infected cells was higher than that in r IBV-infected cells.The ratio of p-JNK/JNK in r IBV-infected H1299 cells was0.25,0.38 and 0.96,respectively.Compared to r IBV,r IBV-C27107 G infection increased the expression level of p-JNK by 1.66-fold.(4)IBV-ncRNA increased the expression of Cyclin D1.The expression of Cyclin D1 in r IBV-infected cells was significantly higher than that in r IBV-C27107 G infected cells at the same time point.The ratio of Cyclin D1 expression was 1.88,1.75,0.59,0.06 and 0.10 in H1299 cells infected with r IBV,respectively.The ratio of Cyclin D1 expression was 1.85,1.19,0.19,0.03 and 0.06 in r IBV-C27107 G infected cells,respectively.In comparison,in r IBV infection increased the expression of Cyclin D1 by 3.11-fold at 20 h post infection.(5)IBV-ncRNA inhibited the expression of c-Fos.The expression of c-Fos protein increased gradually in 16-24 h after infection and decreased in 24 hours after infection.The expression of c-Fos protein in r IBV-C27107 G infected cells was2.47 times of that in r IBV infected cells.(6)IBV-ncRNA had no significant effects in the SOCS3 expression and eIF2αphosphorylation.The expression level of SOCS3 protein increased gradually in8-16 hours after infection and reached the peak at 16 h after infection.The expression level of SOCS3 protein changed little at 20-28 hours after infection and at the same time point,the expression of SOCS3 in r IBV-C27107 G infected cells was not significantly different from that in r IBV-C27107 G infected cells;the expression ratios of p-eIF2α/eIF2α in r IBV infected cells were 0.50,0.60,0.37,0.76,0.99,1.29 and 1.48,respectively.The expression ratios of p-eIF2α/eIF2α in r IBV-C27107 G infected cells were 0.48,0.60,0.42,0.80,1.03,1.31 and 1.46,respectively,suggesting no significant difference between r IBV and r IBV-C27107 G infection.Based on these results,we speculate that IBV-ncRNA inhibited the activation of TIRAP-TRAF6-NF-κB pathway and caused the down-expression of downstream genes,such as ICAM-1,MCP-1,etc.In addition,inhibition of JNK phosphorylation in TIRAP-TRAF6-JNK pathway leads to decreased expression of downstream c Fos protein,both of which may further affect cell survival and virus replication;IBV-ncRNA induces an increase in cyclin D1 expression and upregulates the expression of XRCC5,which may increase cells arrested in G1 phase and affect cell cycle. |
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