Identification And Function Of Circ-udg And Circ-ORF^2A-3 Circular RNAs Derived From Cyprinid Herpesvirus 2

CyHV-2 is a kind of double-stranded DNA herpes virus that infects crucian carp and its variants,such as Carassius auratus gibelio.Gill hemorrhagic disease of C.auratus gibelio caused by CyHV-2 infection is highly contagious and mortality,which caused huge losses in aquaculture industry of C.auratus gibelio.At present,there have been some reports on the information of CyHV-2 genome,the response of C.auratus gibelio to CyHV-2,and the interaction between CyHV-2 and gibelio,but there are still a lot of questions to be answered.In recent years,with the rapid development of high-throughput sequencing technology,some circRNAs derived from DNA viruses have been identified,and it has been verified that some circRNAs play an important role in the regulation of virus replication and infection process.However,whether CyHV-2 could encode functional circRNAs has not been reported.In this study,ten kinds of circRNAs derived from CyHV-2 were identified by high-throughput sequencing.On this basis,the function of circ-udg derived from the CyHV-2 uracil-DNA glycosylase（udg）gene and circ-ORF2A-3 from the ORF2A locus were explored,respectively.The main findings are as follows.1.Identification of circRNAs derived from CyHV-2At present,most research focuses on the circRNA derived from cells,and there is little research on the circRNA encoded by virus.In this study,circRNA sequencing of CyHV-2 infected tissues was carried out and ten kinds of circRNAs derived from CyHV-2 were found by bioinformatics analysis.In order to verify the reliability of high-throughput sequencing data,the candidate circRNAs encoded by CyHV-2 were verified by PCR with divergent primer.Sanger sequencing results of PCR products showed the sequences of junction sites were consistent with the results obtained by high-throughput sequencing,indicating that these circRNAs are truly existed.The circRNA-miRNA-mRNA regulatory network showed that these circRNAs derived from CyHV-2 may play a role in regulation of insulin signaling pathway,wnt signaling pathway,endocytosis,necroptosis and other pathways.These results provide new clues for understanding the genomic information of CyHV-2 and exploring the interaction between CyHV-2 and the host.2.The function of circ-udg derived from CyHV-2The uracil-DNA glycosylase（udg）encoded by virus plays an important role in maintaining the stability of viral genomes.In the study,CyHV-2（NC₀19495.1）can also express udgwhich is encoded by ORF98（located in the 171218-172348 nt region）.The high-throughput sequencing result showed that circ-udg originated from partial sequence of exon 2 of udg gene,corresponding to the 171807₁72304 nt region of CyHV-2 genome.Firstly,to confirm the existence of circ-udg,divergent primers and convergent primers of circ-udg were used for PCR and the Sanger sequencing result of the PCR products showed that the junction site and complete sequence of circ-udg were consistent with the results of high-throughput sequencing.Northern blotting results showed that circ-udg is existent in the RNA extracted from CyHV-2 infected tissue mix.The qRT-PCR results showed that the expression level of circ-udg and its parent gene udg increased with the proliferation of the virus.But the expression level of circ-udg was lower than the udg.The experiments in vitro confirmed that overexpression of circ-udg could promote the proliferation of CyHV-2,and knockdown circ-udg using RNAi inhibited the proliferation of the virus.The cellular immunofluorescence showed that circ-udg was mainly located in the cytoplasm.On the basis of bioinformatics prediction,circ-udg encoded a circ-udg-P147 protein with 147 amino acid residues,a truncated UDG,was confirmed by Western blotting and immunofluorescence.And it was found that the translation of circ-udg-P147 was initiated by an internal ribosome enter site（IRES）in circ-udg.In vitro experiments confirmed that both UDG and circ-udg-P147 protein could promote virus proliferation and replication.Moreover,significant change in the transcription level of udg gene was not found when cells were co-transfected with UDG expression plasmid（pZsGreenl-C1-UDG）and circ-udg-P147 expression plasmid（pmCherry-Cl-circ-udg-P147）,but the UDG protein level increased with the transfection amount of pmCherry-C1-circ-udg-P147 suggesting that circ-udg-P147 could protect UDG from degradation.In addition,the promotion of circ-udg-P 147 on the virus proliferation was weakened after the initiation codon of circ-udg-P 147 was mutated.So circ-udg can promote CyHV-2 proliferation through other mechanisms.CircRNA-miRNA pull down result showed that circ-udg can interact with miRNA dre-let-7b.Bioinformatics analysis revealed that the target genes of miRNA dre-let-7b were ORF41,ORF71 and ORF79 of CyHV-2.Overexpression experiment indicated that upregulation of circ-udg could increase the ORF71 gene（helicase gene of CyHV-2）expression level,implying that circ-udg regulates viral gene expression by sponging miRNA dre-let-7b.3.The function of circ-ORF2A-3 derived from CyHV-2The sequence of circ-ORF2A-3 corresponds to the complementary sequence of 269457₂70345 nt region of the CyHV-2 genome（NC₀19495.1）,which is consist of a partial sequences of the ORF2A exon 1 and the complementary sequence of the ORF3 exon 1.Cell immunofluorescence results showed that circ-ORF2A-3 was mainly located in the cytoplasm.In the circRNA-protein pull down experiment,no specific protein interacting with circ-ORF2A-3 was found.Bioinformatics analysis showed that the target genes of miRNA which were targeted by circ-ORF2A-3 were involved in cell ubiquitination and nerve growth guidance.Overexpression of circ-ORF2A-3 promotes the expression of CyHV-2 ORF72 gene.These results laid a foundation for further investigation of the function of circ-ORF2A-3.