The Mechanism Of Long Non-coding RNA IRF1-AS Inhibiting The Replication Of Peste Des Petits Ruminants Virus In Host Cells

Peste des petits ruminants（PPR）is an acute,virulent,highly contagious disease caused by peste des petits ruminants virus（PPRV）infection,which mainly infects goats,sheep and other wild small ruminants.It is an important epidemic disease that has long plagued the breeding industry and is a legally reported animal disease by the Office international desépizooties（OIE）.Although numerous studies have shown that PPRV infection can cause severe immunosuppression in the host to promote its own proliferation.However,how the host enhances the innate immunity to inhibit the replication of the virus is rarely reported.Long non-coding RNAs（lncRNAs）are a class of noncoding RNAs in human and animal cells that are more than 200 nucleotides in length and have no or limited coding potential,which can regulate viral infection through variety of mechanisms.However,the role of lncRNA in PPRV infection is still unclear.Type I interferon and the interferon stimulated genes（ISGs）as the important components of innate immunity,are key immune molecules for the host to resist viral infections.Previous study showed that PPRV infection can cause differential expression of a large number of lncRNAs in host cells,from which the long non-coding RNA IRF1-AS,which is closely associated with innate immunity,was significantly up-regulated.To clarify how the host-derived lncRNA IRF1-AS regulates PPRV replication by influencing the cellular antiviral natural immune response,relevant studies were conducted,and the research results were as follows.（1）After Caprine endometrial epithelial cells（EEC）were infected with PPRV,the expression levels of IFN-βand ISGs were positiveiy correlated with the infectious dose and infection time of the virus.When EECs were infected with PPRV at different infection doses（MOI=0.5,3,10）,the m RNA levels of IFN-βand ISGs increased gradually with the increase of PPRV N protein expression,with the largest change at MOI=3;In different infection times,the expression levels of PPRV N protein gradually increased in a time-dependent manner,while the IFN-βand ISGs.The expression levels of IFN-βand ISGs reached a peak at 48 hpi.Importantly,the results of qRT-PCR showed that the expression of long non-coding IRF1-AS in EECs were gradually increased at 48 h during PPRV infection.（2）Interfering with IRF1-AS expression promotes PPRV replication and overexpression of IRF1-AS significantly inhibits virus replication.In order to evaluate the influence of IRF1-AS on PPRV replication,two different si RNAs were designed and synthesized to interfere with the expression of IRF1-AS in cells.QRT-PCR results showed that the IRF1-AS expression at the transcription level was significantly reduced after transfection with si RNAs.Western blot and TCID₅₀assays showed that when the expression of IRF1-AS was interfered,the expression levels of viral N protein were significantly increased,and the virus titers were also significantly increased compared with the control group,indicating that the IRF1-AS expression was interfered to promote viral replication.In addition,IRF1-AS was constructed into an overexpression vector and transfected into cells.The results of qRT-PCR showed that the expression of IRF1-AS was significantly increased in the overexpression vector transfected cells.Western blot and TCID₅₀assays showed that the viral N protein expression levels and viral titers of overexpression group were significantly decreased compared with the control group.The results indicated that overexpression of IRF1-AS inhibited the replication of the virus.These results show that IRF1-AS plays an important role in PPRV replication.（3）IRF1-AS significantly enhances the innate immune response and inhibits PPRV replication in cells.To investigate the mechanism of IRF1-AS regulating viral replication from the perspective of innate immune response,the expression levels of IFN-βand ISGs were detected after interfering with IRF1-AS expression.The results showed that interference with IRF1-AS expression significantly reduced the expression of IFN-βand ISGs,and inhibited the phosphorylation of IRF3,the key molecule of type I IFN signaling pathway;In contrast,when IRF1-AS was overexpressed in PPRV-infected cells,overexpression of IRF1-AS could significantly increase the expression levels of IFN-βand ISGs,and promote the phosphorylation of IRF3.These results suggest that IRF1-AS significantly enhances the innate immune response and inhibits viral replication during PPRV infection.（4）IRF1-AS regulates the expression of IFN-βand ISGs as well as the phosphorylation of IRF3 by up-regulating the expression of IRF1,which affects viral replication.In order to explore the mechanism of IRF1-AS regulating host innate immunity,we characterized the location of IRF1-AS in genome and found that IRF1 could be a target gene for IRF1-AS cis-regulation.The results of Western blot and qRT-PCR showed that after PPRV infection of cells,IRF1 were up-regulated at both the m RNA and protein levels,which was consistent with the expression trend of IRF1-AS during PPRV infection.The results also indicated that overexpression of IRF1-AS promoted the expression of IRF1,while interference of IRF1-AS inhibited the expression of IRF1.This indicated that IRF1-AS targeted and regulated the expression of IRF1.In addition,interfering with IRF1 expression not only reduced the expression levels of IFN-βand ISGs as well as the phosphorylation of IRF3,but also promoted the proliferation of the virus.Importantly,the results showed that IRF1-AS regulated the expression of IFN-βand ISGs as well as the phosphorylation of IRF3,and affected viral replication through regulating the expression of IRF1.In addition,we demonstrated that the interaction between IRF1 and IRF3 during PPRV infection.In conclusion,in this study,we demonstrated that PPRV infection activated the innate immune response in EECs,the host-derived long non-coding RNA IRF1-AS was up-regulated to promote the expression of IRF1 protein,which positively regulated type I IFN production and ISGs expression to inhibit PPRV replication.Our findings provide new evidence for revealing the mechanism of host lncRNA regulating PPRV infection.