The Mechanism For LncRNA EDAL To Inhibit The Replication Of Rabies Virus

Rabies is caused by the rabies virus(RABV),a central nervous system(CNS)disease among human and animals.According to the statistics of the world health organization(WHO),there are about 59,000 people died for rabies every year in the worldwide.China is one of the countries with high incidence of rabies.Rabies virus mainly infects host CNS,and the mortality is nearly 100% once the clinical symptoms appear.Since there is still no efficient method of cure,rabies is still an important disease that threatens the safety to human life.However,the pathogenesis of RABV,especially how it evades the host immune response,is still not fully understood.Therefore,it is still very important to investigate the pathogenesis of RABV,which provides the basis for effective control of rabies.In recent years,a large number of reports have shown that long non-coding RNAs(lncRNAs)play extremely critical roles in the host's defense to viral infection.Therefore,this study use RABV infection model to screen the host lncRNAs suppressing RABV infection through large-scale RNA sequencing(RNA-seq).We intracranially infected the mice brains with WT RABV strain DRV-AH08(DRV)and lab-attenuated RABV strain CVS-B2c(B2c),respectively.The mice brains were collected when the two viruses have reached a similar viral load in the mice brain tissues at 6 d post infection and then RNA-seq was performed.Then the differentially expressed lncRNAs were analyzed.We also infected mouse neuronal cell line N2 a cells as the infection model with lab-attenuated RABV CVS-B2 c strain and performed RNA-seq analysis.Total 1434 differentially expressed lncRNAs were identified compared with uninfected N2 a cells.In these differentially expressed lncRNAs we found a significantly up-regulated lncRNA which can inhibit RABV proliferation via over-expression,we named it as EZH2 degradation associated lncRNA(EDAL).EDAL was significantly up-regulated post RABV infection,and can only be induced by the genomic RNA of RABV,which is an interferon-independent lncRNA.EDAL over-expression can significantly inhibit RABV replication both in neuronal cells and mice.Then we found that EDAL can specifically bind to epigenetic regulator EZH2(Enhancer of zest homolog 2,EZH2)by using western blot,RNA pull-down and RIP-q PCR assays.The binding of EDAL and EZH2 lead to the degradation of EZH2 via the lysosomal pathway,thus significantly down-regulating the histone modification marker H3K27me3.We then divided EDAL into multiple domains dependent on RNA secondary structure prediction.The 98-153 nt at the 5'end of EDAL was identified to play the antiviral function and the functional domain could be used as an independent antiviral element.We have confirmed the functional domain of EDAL,and then we continued to explore the mechanisms of EDAL induced EZH2 degradation.We mutated the key phosphorylation and O-Glc NAcylation modification sites of EZH2 which associated with its stability and found that the degradation of EZH2 induced by EDAL was eliminated after the T309 amino acid site mutation.Then we successfully predicted the interaction sites between the functional domain of EDAL and EZH2 by using the 3d RPC software,and confirmed the interaction sites between EDAL and EZH2 by point mutation and RNA pull-down assays.We found that the 125-142 nt of EDAL interacts with EZH2 and may shields the T309 O-Glc NAcylation site of EZH2 and affects its O-Glc NAcylation modification.Finally,we performed EZH2 O-Glc NAcylation analysis and confirmed that EDAL does affect the O-Glc NAcylation modification of EZH2 at T309 site,leading to the degradation of EZH2 via lysosomes.As a methyltransferase,the major function of EZH2 is to promote H3K27me3 modification to achieve the goal of silencing the expression of some genes.In order to find the antiviral genes which regulated by EDAL,RNA-seq was performed post EDAL over-expression,and Ch IP-seq was performed by using the antibody of H3K27me3.The genes which were significantly up-regulated post EDAL over-expression and modified with H3K27me3 are the candidate targets for further study.After testing such genes,we found that over-expression of the protein PCP4L1(Purkinje cell protein 4 like 1)can significantly inhibit the proliferation of RABV.As a novel antiviral protein,PCP4L1 has never been reported for its antiviral function and the clear mechanisms of PCP4L1 inhibits RABV proliferation remains to be further studied.