Generation Mechanism And Biological Function Of A Noncoding RNA Produced By Avian Infectious Bronchitis Virus

IVA number of viruses employ different mechanism to produce noncoding RNA?nc RNA?,including nc RNAs transcribed by RNA polymerase II or III and subgenomic nc RNA produced by incomplete degradation of genomic RNA by the cellular 5' to 3'exonuclease XRN1.These virus-encoded nc RNAs play important roles in viral life cycle and virus-host interplay,including autoregulation of virus gene expression,alteration of the cell cycle,and avoidance of host defenses and so on.So far,the known nc RNAs are mainly produced by DNA viruses,few nc RNAs encoded by RNA viruses except Flaviviruses and a few of plant RNA viruses have been reported.There has been no report of nc RNAs encoded by coronaviruses.Infectious bronchitis virus?IBV?is a member of Gammacoronavirus within the Coronaviridae,with a positive-sense single-stranded RNA genome.In the IBV-infected cells,IBV produces 5 subgenomic RNAs via a discontinuous transcription mechanism mediated by a conserved core sequence?CU?U/G?AACAA?in transcription –regulating sequence?TRS?in the 5'-end of IBV genome and in the intergenic region preceding each m RNA body.These sg RNAs encode viral structural proteins and accessory proteins.In this study,a novel IBV-encoded nc RNA was identified and its generation mechanism was elucidated by reverse genetics and mutagenesis.Moreover,the biological function was preliminary studied.The experimental results are as follows:1.A small novel subgenomic RNA?sg RNA?was detected by RT-PCR in the IBV-infected Vero and chicken embryo.Subjected to cloning and sequencing,the results showed that this sg RNA consists of 563 nucleotides excluding a poly?A?tail,is mainly derived from the 3'-UTR?from nucleotides 27104 to 27608?of IBV genome,and contains a 63-nt-long of terminal leader sequence derived from the 5' end of the viral genome.In fact,this sg RNA is an nc RNA because it contains no AUG.2.In order to reveal the generation mechanism of this nc RNA and map the key nucleotides for nc RNA generation,a series of mutant viruses based on the genetic background of IBV-P65 were constructed and obtained by mutagenesis and reverse genetics,and their biological characteristics were analyzed.The results are as follows:?1?Insertion of an ORF encoding EGFP between 27149 and 27150 nt of IBV3'-UTR allowed the recovery of recombinant virus and the expression of EGFP in the virus-infected cells,suggesting that the nc RNA can function as an m RNA.?2?The recombinant viruses IBV-U27104 A,IBV-A27105 U,IBV-A27106 U,IBV-C27107 G,IBV-A27108 U,IBV-?27104-08 andIBV-A27110U/A27111U/G27113C/G27114 C were constructed and obtained by reverse genetics and mutagenesis.RT-PCR analysis showed that U27104 A and A27108 U had no significant effect on the synthesis of nc RNA,A27105 U and A27106 U reduced the synthesis of nc RNA,while C27107 G and ?27104-08 completely blocked the synthesis of positive and negative nc RNA,suggesting that at least three nucleotides?A27105/A27106/C27107?are involved in the effective nc RNA generation.In addition,A27100U/A27111U/G27113C/G27114 C located downstream of ₂₇₁₀₄UAACA₂₇₁₀₈ is identical to the downstream sequence of core sequence in transcription-regulating sequence in leader at 5'-end of viral genome.When cells were infected with the mutant virus IBV-A27110U/A27111U/G27113C/G27114 C containing four point mutations,the synthesis of nc RNA was not detected in infected cells,indicating that these four nucleotides are also critical for nc RNA synthesis.The above-mentioned results indicated that the IBV-encoded nc RNA is generated by a discontinuous transcription mechanism via template switch mediated by a noncanonical core sequence?UAACA?,at least three nucleotides are involved in the effective nc RNA generation.In addition,A27110/A27111/G27113/G27114 is also critical for nc RNA generation.3.The effects of nc RNAs on virus replication and pathogenicity?1?Plaque assay showed that the mean diameters of the CPE formed on Vero cells infected with r IBV and IBV-C27107 G viruses were 1.27±0.18 and 1.23±0.22 mm,respectively,suggesting that the pathogenicity of two viruses has no significant difference;?2?Quantitative Real-time PCR analysis indicated that the expression level of S gene and N gene in Vero cells infected with two viruses at 8h and 20h post infection had no significant differences;?3?Western blot analyzed the expression of S protein and N protein in the Vero cells infected with r IBV and IBV-C27107 G at 4h,8h,12 h,16h,20 h,24h and 28 h post infection,no significant differences were detected.These results suggested that nc RNA has little effects on viral replication and viral pathogenicity in Vero cells.4.nc RNA regulate the expression of host genes?1?Preliminary RNA microarray analysis revealed that nc RNAs play an important role in host gene expression.r IBV infection up-regulated expression of LBP and VNN3 genes in Huh7 by 2.50 and 3.29 times,respectively while IBV-C27107 G infection up-regulated the expression of LBP and VNN3 genes by 14.43 and 9.64-fold,respectively.The results showed that nc RNA is able to inhibit the up-regulation of LBPand VNN3 induced by IBV infection.?2?q PCR analysis revealed that r IBV infection up-regulated expression of Mda5 and IFN-? at 16 h post-infection in DF-1 cells by 7.97 and 20.11 times,respectively while IBV-C27107 G infection up-regulated the expression of Mda5 and IFN-? by 13.64 and 46.37-fold,respectively.All the results above indicate that nc RNA not only affects the transcription of certain host genes,but also function in the inherent antiviral response of the host.5.To further clarify the function of nc RNA,transcriptomic analysis was performed for RNAs extracted from IBV-C27107 G and r IBV infected with DF-1 cells at 24h post infection.The results are as follows:?1?A total of 1357 differentially expressed genes?DEG?were screened by transcriptome sequencing.Among which the expression of 740 were up-regulated while617 were down-regulated.1255 DEG were annotated in different databases: 466 DEG in COG,1140 DEG in GO,775 DEG in KEGG,823 DEG in KOG,1226 DEG in NR,1181 DEG in Swiss-Prot,and 1235 DEG in NOG.?2?1522 new genes were excavated,of which 770 were functionally annotated:122 in COG,224 in GO,340 in KEGG,518 in NR,252 in Swiss-Prot,and 738 in egg NOG.?3?Among 1357 DEG,775 were annotated in the KEGG data,of which 584 were located in six main KEGG biological reactions: 162 genes in 14 pathways involved in environmental information processing,116 genes in 9 pathways involved in cellular processes,101 genes in 11 pathways involved in the biological system,77 genes in 8pathways involved in metabolism,50 genes in 3 pathways involved in the classification of diseases and 48 genes in 5 pathways involved in the processing of genetic information.