Expression Profiling Analysis Of CircRNA In Mice Brain Infected With Rabies Virus And Research On The Function Of Circ-parp9

Rabies virus（RABV）causes acute fatal encephalitis in humans and other mammals.Previous reports revealed circular RNA（circ RNA）plays an important role in human neurological diseases and viral infections,but the expression profiling of circ RNAs altered by RABV infection and the mechanism of RABV infection regulated by circ RNA are still unknown.Based on the preliminary work of our laboratory,this experiment continued to screen andobtained the monoclonal antibodies（m Abs）3A6 and L-C,and identified the linear epitopes of m Abs 3A6,L-C and 3F3（anti-L m Abs prepared in the laboratory previously）.The¹⁴⁷⁹EIFSIP¹⁴⁸⁴,¹⁶⁵⁹RALSK¹⁶⁶³and ¹⁷²⁴VFNSL¹⁷²⁸ were identified as the minimal linear epitopes recognized by m Abs 3F3,3A6 and L-C,respectively.Amino acid alignment showed epitope ¹⁷²⁴VFNSL¹⁷²⁸ recognized by m Ab L-C is completely conserved among RABV strains,indicating that m Ab L-C could be used to detect all of the RABV strains.Epitope ¹⁴⁷⁹EIFSIP¹⁴⁸⁴ is highly conserved among RABV strains except for a P1484S substitution in a China I sub-lineage strain of Asian lineage,which eliminated the reactivity of the epitope with m Ab 3F3.However,the epitope ¹⁶⁵⁹RALSK¹⁶⁶³ was only completely conserved in the Africa-2 and Indian lineages,and a single A1660T substitution,mainly appeared in strains of the China I belonging to Asian lineage and a Cosmopolitan lineage strain,still retained the reactivity of the epitope with m Ab 3A6.While both A1660T and K1663R substitutions in a China I lineage strain,single K1663R/Q substitution in some China II strains of Asian lineage and some Arctic-like lineage strains and R1659Q mutation in a strain of Africa-3 lineage eliminated the reactivity of the epitope with m Ab 3A6,suggesting m Ab 3A6 could be used for differentiation of variable epitopes of some strains in different lineages.Thus,variability and conservation of the three epitopes of L protein showed the reactive difference of m Abs among RABV strains of different lineages.These results may facilitate future studies in development of detection methods for RABV infection,the structure and function of RABV L protein.The monoclonal antibody 3A6obtained in this experiment will be used for the detection of RABV in the following study.To analyze the changes of circ RNAs expression induced by RABV infection,mice brain with or without the RABV CVS-11 strain were subjected to RNA-sequencing and a total of30985 circ RNAs were identified.In total,636 circ RNAs were differentially expressed in RABV infection,of which 426 significantly up-regulated and 210 significantly down-regulated（p<0.05 and fold change≥2）.GO analysis revealed that the host genes of all differentially regulated circ RNAs were mainly associated with cellular components.Both KEGG and GSEA analysis showed that these host genes were involved in the c GMP-PKG and MAPK signaling pathway,and HTLV-I infection.Also,pathways related to glucose metabolism and synaptic functions were enriched in KEGG analysis.The circ RNA-mi RNA-m RNA network was constructed with 25 of 636 differentially expressed circ RNAs,264 m RNAs involved in RABV infection,and 29 mi RNAs.Several mi RNAs and many m RNAs in the network were reported to be related to viral infection and the immune response,suggesting that circ RNAs could regulate RABV infection via interacting with mi RNAs and m RNAs.Further,the expression of randomly selected 6 up-regulated and 6 down-regulated circ RNAs was validated by RT-q PCR,whilst Rnase R resistant assay and Sanger sequencing were conducted to verify the circularity of circ RNAs.The RT-q PCR results showed that the expression changes of 11 circ RNAs among the 12 randomly selected circ RNAs were consistent with the RNA-seq analysis results.In order to screen for circ RNAs that regulate RABV replication,we constructed 7 circ RNA overexpression plasmids（novel＿circ＿011422,novel＿circ＿015068,novel＿circ＿008143,novel＿circ＿015161,novel＿circ＿017745,novel＿circ＿010735 and novel＿circ＿026888）,and transfected them into N2a cells and we found ectopic expression novel＿circ＿011422 inhibited RABV replication.Novel＿circ＿011422（circ-Parp9）is derived from the Parp9 gene on mouse chromosome 16and is circularized from the exon at positions 35963975-35964364 on the genome.In this study,divergent and convergent primers were used to perform PCR with mouse brain c DNA and g DNA samples.As a result,divergent primer can only amplify in c DNA samples and there were purpose amplifications both in c DNA and g DNA samples with convergent primer,indicating that circ-Parp9 is resistant to RNase R digestion and has a circular structure.In addition,RABV infection can induce the up-regulation of circ-Parp9in N2a cells.Ectopic expression novel＿circ＿011422 inhibited RABV replication in N2a and SK-N-SH cells.RNAhybrid（v2.1.2）,Miranda（v3.3a）,and Target Scan（Version:7.0）were used to predict the mi RNA binding to circ-Parp9 and a total of 30 mi RNAs were obtained.Dual luciferase reporter gene assay found that circ-Parp9 can significantly inhibit the activity of mmu-mi R-28b,and mmu-mi R-6951-3p.Ectopic expression mmu-mi R-6951-3p promoted RABV replication.The dual luciferase reporter gene experiment showed that mmu-mi R-6951-3p targets the gene IIGP1;ectopic expression mmu-mi R-6951-3p inhibited expression of endogenous IIGP1 protein,indicating IIGP1 is the target of mmu-mi R-6951-3p.Overexpression of IIGP1 inhibits the replication of RABV.Therefore,circ-Parp9 promotes the expression of IIGP1 by inhibiting the activity of mmu-mi R-6951-3p,thereby inhibiting virus replication.