Identification And Functional Analysis Of Key MicroRNA During Newcastle Disease Virus Infection Of DF-1 Cells

Newcastle disease virus(NDV)is an enveloped virus with a single-stranded,negative-sense,non-segmented RNA genome.As one of the most devastating pathogens for poultry industry,velogenic strains of NDV are able to infect almost all species of birds and cause highly contagous Newcastle disease(ND).MicroRNAs(miRNAs),as a member of small non-coding RNA,are highly conserved and generally reside in eukaryotes.Previous studies have indicated that miRNAs play an important regulatory role in the growth and development of almost all cells.Emerging evidence demonstrates that miRNAs can also regulate virus replication and pathogenesis through targeting the viral genome directly or regulating host anti-viral response.However,the mechanism by which miRNAs regulate NDV replication in avian cells is still largely unknown.In this study,we identify miRNAs that regulate NDV replication in DF-1 cells(a chicken embryo fibroblast cell line)and investigate the mechanism by which they play a role,which will improve our understanding of the role of host miRNAs in regulating NDV replication.1.Screening and functional analysis of miRNAs which bind to NDV genome directlyTo investigate whether cellular miRNA can directly target NDV genome,NDV strains of different genotypes,including genotype ? to ?,were used to predict potential targeted miRNAs using RegRNA 2.0.The predicted miRNAs of different NDV strains were combined and the overlaps were used for the conservation analysis.After that,11 potential miRNAs were identified as the candidate miRNAs used for further research.To further verify the interaction between these miRNAs and NDV genome,a dual-luciferase assay was performed by co-transfecting indicated miRNA mimic or inhibitor or negative control and wild-type or miRNA binding site mutated reporter plasmids.It was found that gga-miR-1603 and gga-miR-1794 could negatively regulate the luciferase activities of cells transfected with wild-type reporter plasmids containing NDV L gene,and showed no effect on cells transfected with miRNA binding site mutated reporter plasmids.Those results indicated that gga-miR-1603 and gga-miR-1794 could directly bind to the L gene of NDV.Meanwhile,according to the results of real-time quantitative PCR(qPCR)and Western blotting assay,we found that these two miRNAs could negatively regulate the expression of NDV L gene in both RNA and protein levels,indicating that gga-miR-1603 and gga-miR-1794 inhibit the translation of NDV L gene and eventually lead to the degradation of L gene.Moreover,we found that in DF-1 cells overexpression of these two miRNAs significantly inhibited NDV replication,while knockdown of them significantly promoted NDV replication.In summary,our present results indicate that gga-miR-1603 and gga-miR-1794 can directly target the L gene of NDV and promote L gene degradation,which finally lead to the inhibition of NDV replication in DF-1 cells.2.MiRNA expression profiling in NDV-infected DF-1 cells by deep sequencingAccumulating evidences have revealed that cellular miRNAs can influence virus replication by controlling host-virus interaction.To identify miRNA expression profile and explore the roles of miRNA during NDV replication,small RNA deep sequencing was performed of non-inoculated DF-1 cells and JS 5/05-infected cells collected at 6 h and 12 h post infection(hereafter called mock,NDV-6 h,and NDV-12 h groups respectively).A total of 73 miRNAs of NDV-6 h group and 64 miRNAs of NDV-12 h group were significantly differentially expressed(SDE)when compared with those in mock group.Meanwhile,49 SDE miRNAs,including 47 up-regulated and 2 down-regulated,showed the same expression patterns in NDV-6 h and NDV-12 h groups.qPCR validation of 15 randomly selected miRNAs' expression patterns showed that upon NDV infection gga-miR-130a-5p and gga-miR-1434 were significantly down-regulated,while gga-miR-199-5p,gga-miR-301a-3p,gga-miR-451,gga-miR-1451-3p,gga-miR-29a-3p,gga-miR-130a-3p,gga-miR-181a-5p,gga-miR-140-5p,gga-miR-17-5p,gga-miR-107-3p,gga-miR-33-5p,gga-miR-19b-3p and gga-miR-18a-5p were significantly up-regulated.To investigate the roles of these SDE miRNAs in NDV replication,miRNA mimics and inhibitors were transfected into DF-1 cells followed by NDV infection.The results of qPCR,Western blotting and viral growth kinetics revealed that gga-miR-451 and gga-miR-199-5p promoted NDV replication while gga-miR-19b-3p and gga-miR-29a-3p inhibited NDV replication.Meanwhile,we found gga-miR-451 negatively regulated the expression of NDV-induced informatory cytokines.Previous studies have indicated that tyrosine3-monooxygenase/tryptophan5-monooxygenase activation protein zeta(YWHAZ)is a target of hsa-miR-451 in human dendritic cell.In our study,we found gga-miR-451 can negatively regulate the expression of YWHAZ in both mRNA and protein levels,indicating that YWHAZ is a target of gga-miR-451 in DF-1 cells.Further research of YWHAZ,we found that knockdown of YWHAZ in DF-1 cells by small interfering RNA(siRNA)suppressed NDV-induced informatory cytokines and promoted NDV infection,similar to the effect of overexpression of gga-miR-451.This suggested that via targeting YWHAZ gga-miR-451 inhibited the expression of inflammatory cytokines and consequently enhanced NDV replication.Overall,our study presented a global miRNA expression profile in DF-1 cells in response to NDV infection and identified the roles of some SDE miRNAs in NDV replication which would underpin further studies of miRNAs' roles between the host and the virus.3.The mechanism by which gga-miR-19b-3p regulates NDV replicationOur prior study indicated that gga-miR-19b-3p is up-regulated in NDV-infected DF-1 cells and functions to suppress NDV replication.In the present study,we used TargetScan and miRDB databases to predict the potential target genes of gga-miR-19b-3p.After enrichment of functions of the targeted genes by DAVID,we found that two potential target genes,ring finger protein 11(RNF11)and zinc-finger protein MYND-type containing 11(ZMYND11)play an important role in cellular immune response.Dual-luciferase and gene expression array analyses revealed that gga-miR-19b-3p inhibited the luciferase activity of the cells which were transfected with reporter plasmids containing the 3'untranslated region of RNF11 and ZMYND11,while no influence was showed on those transfected with binding sites mutated reporter plasmids,indicating that RNF11 and ZMYND11 are two targets of gga-miR-19b-3p in DF-1 cells.By further functional studies of RNF11 and ZMYND11,we found that these two genes positively regulated NDV-induced inflammatory cytokines and negatively regulated NDV replication,opposite to the effect of gga-miR-19b-3p,indicating that gga-miR-19b-3p enhances NDV-induced inflammatory response and inhibits NDV replication via targeting RNF11 and ZMYND11.Moreover,we found RNF11 and ZMYND11 inhibited the activity of nuclear factor ?B(NF-?B),promoted phosphorylation and degradation of inhibitor of NF-?B(I?B-?)and facilitated p65 nuclear translocation after NDV infection.These results indicated that RNF11 and ZMYND11 inhibit NDV-induced inflammatory cytokines production by negative regulation of NF-?B activity in DF-1 cells.Overall,our results indicate that NDV-induced gga-miR-19b-3p inhibits the expressions of RNF11 and ZMYND11 by directly binding to them,thereby enhancing NF-?B activity and increasing inflammatory cytokines production,which finally leads to the inhibition of NDV replication in DF-1 cells.