| Avian Influenza Virus(AIVs)is a negative strand RNA virus belongs to the family of Orthomyxoviridae,which has a broad host spectrum and rapid mutative ability.Therefore,continuous vaccine updates are needed for AIVs.Moreover,with the massive use of antiviral drugs in large-scale farms,the problem of drug resistance has become increasingly evident.Therefore,the development of potential broad-spectrum antiviral drugs becomes an urgent scientific problem to be solved.Long non-coding RNAs(LncRNAs)can regulate viral replication by modulating the expression pattern of genes and reshaping the cellular microenvironment.Our previous study has successfully screened a potentially functional LncRNA,NONMMUT011061(Lnc#61),by RNASeq technology and functional validation.Lnc#61 was found to have potential broad-spectrum antiviral effects,inhibiting the replication of multiple subtypes of AIVs,including H1N1,H5N1,H7N9,etc.Furthermore,we found that Lnc#61 can act its inhibitory effect at multiple stages of viral life cycle,including the entry,early and mid-late replication stages of the viral replication cycle.This study will further explore the mechanism of the broad antiviral ability of Lnc#61 against influenza virus,with a view to laying the foundation for the development of novel broadspectrum antivirals.1.Screening and validation of Lnc#61 and its functional domains-targeted host factorsTo preliminarily screen the crucial host factors involved in Lnc#61's broad antiviral activity,we first systematically analyzed the differential host transcriptomics responses induced by Lnc#61with CK10(A/Chicken/Jiangsu/k0402/2010,H5N1)virus infection or non-infection via RNA-seq technology.The results showed that overexpression of Lnc#61 significantly induced more differential expressed genes in the infected group than in the uninfected group,and there were only 127 shared genes between the two groups.In addition,without virus infection,overexpression of Lnc#61 significantly stimulated differential expression of genes related to host inflammatory response;while after virus infection,Lnc#61 significantly regulated some important biological processes,inculding arachidonic acid metabolic process,cell death,and inflammatory response.Furthermore,qRT-PCR validation revealed that Lnc#61 significantly promoted the expression of apoptosis-related genes(AEN,INPP5D,NLRP3),arachidonic acid metabolism-related genes(ALOX5,CYP4F2),and antiviral genes(IRGM,IKBKE,IFIT2)and significantly inhibited the expression of inflammatory cytokines(CXCL8,IL18,IL17C)during viral infection,suggesting the high correlation between the qRT-PCR results with the RNA-seq results.In addition,the functional area C and I of the Lnc#61 contributed to the antiviral activity of Lnc#61 and involved in regulation of Lnc#61-mediated expression of the cell death,inflamatroy and the metabolism-related host genes.2.Validation of Lnc#61-targeted host factor-associated cellular phenotypesTo further investigated the mechanism of Lnc#61 exerting broad-spectrum anti-AIVs activity,we next validated the relevant cellular phenotypes asscociated with the key host factors targeted by Lnc#61 that screened in the previous section.First,we examined the expression of IFN-? with the single luciferase reportering system and qRT-PCR assay,and we found that Lnc#61 did not significantly affect the expression of IFN-?,indicating that Lnc#61 may act its antiviral activity independent of IFN-? pathway.We then verified the regulation of cell death by Lnc#61and we found that Lnc#61 significantly promoted apoptosis at 12 h of virus infection,while significantly inhibited apoptosis at 24 h of virus infection.In addition,we found that Lnc#61 significantly regulated cellular pryoptosis in various cell lines,especially in MDCK cells,as evidenced by a significant increase in the release of LDH,IL-18,and IL-1? in the supernatant of MDCK cells at 24 h after virus infection,and the appearance of characteristic cellular ballooning.In addition,we found that inhibition of cell pryoptosis significantly promotes viral replication.We then examined the regulatory effect of Lnc#61 on arachidonic acid metabolism via targeted metabolomics techniques,and found that Lnc#61 significantly increased the expression of 9S-HODE,12SHETE,13S-HODE,15S-HETE,ARA,DHA and other products in the arachidonic and linoleic acid metabolic pathways.Taken together,we surmised that Lnc#61 may inhibit viral replication by regulating apoptosis,pyroptosis,and metabolic processes within host cells.3.Screening and functional validation of Lnc#61-targeted viral proteins and host proteins To screen which viral protein or proteins involved in Lnc#61's antiviral activity,we then transfected RLE-6TN cells with each nine plasmids of CK10 virus(PB2,PB1,PA,NP,HA,NA,M,NS,and PA-X),respectively.We found that PA-X significantly suppressed the endogenous expression of Lnc#61 in the absence of virus infection(Fold change<-1.5),while PA,NS,M,NP,NA,PB1,and PA-X significantly suppressed the endogenous expression of Lnc#61 in the presence of virus infection(Fold change<-1.5).We then cotransfected 293T cells with each viral plasmid and together with Lnc#61 plasmid,and we found that NA,M,PA,PB1,and PA-X(Fold change<-1.5)significantly inhibited the expression of ectopic Lnc#61 while HA significantly promoted the expression of ectopic Lnc#61(Fold change>1.5).Finally,we selected HA,PB1,and PA-X genes for the further exploration.We found that PA-X significantly elevated the antiviral effect of Lnc#61 and significantly affected the ability of Lnc#61 in regulating the cell death,inflammation,and arachidonic acid metabolism related genes.Moreover,PB1 protein significantly impaired the antiviral effect of Lnc#61 on viral replication and significantly affected the expression of Lnc#61-regulated apoptosis and inflammation-associated cytokines.In contrast,HA had no significant effect on Lnc#61-regulated viral replication and no significant effect on the expression of Lnc#6-regulated apoptosis(INPP5D)as well as inflammatory(IL-18)cytokine genes.In addition,both RIP and EMSA experiments demonstrated the interaction between Lnc61 and the PA-X protein.Furthermore,we found that PA-X significantly affected the ability of Lnc#61 in regulation of apoptosis and pyroptosis,suggesting that Lnc#61 may inhibit viral replication through regulating apoptosis and pyroptosis via interacting with PA-X protein.In addition,the results of RNA Pull-down,Mass spectrometry,and Western Blot experiments all suggest that Lnc#61 interacted with hnRNP Q host protein,suggesting the possible role of hnRNP Q in regulating Lnc#61's antiviral activity.In summary,this study preliminarily explored the potential mechanism for the broad antiviral activity of Lnc#61 agianst IAVs.Our findings lay the theoretical foundation for a more comprehensive understanding of the function and asscociated mechanism of LncRNA during influenza virus infection and also pave a way for screening of broad-spectrum antiviral drugs. |
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