Host LncRNAR-AS1 Regulates Interferon Stimulated Genes Against EV-A71 Infection

Hand,foot,and mouth disease(HFMD)is a common infectious disease in children caused by enteroviruses,and its morbidity and mortality ranks the first among Class C infectious diseases in China.Currently,enterovirus 71(EV-A71)is the dominant type of enterovirus in most regions of China.When EV-A71 infects a host,its nucleic acid or protein component could be recognized by the host pathogen recognition receptor(PRR),which in turn activates a series of transcription factors and initiates the expression of interferon-stimulated genes(ISGs)to activate the innate immune response against viral infection.Long non-codingRNA(lncRNA)is a type ofRNA molecule with a transcript length of more than 200 nt.Though most of them do not encode proteins,they partici-pate in transcriptional and post-transcriptional regulation,tumor metastasis,and antiviral innate immunity.Therefore,studying the role of lncRNA played in the host’s innate immunity would benefit the prevention and control of enterovirus infection.In the present study,RNA-Seq was used to detect the differential expression of lncRNAs in EV-A71-infected cells,and lncRNA R-AS1 was screened.Then,R-AS1 was found to be mainly located in the cytoplasm byRNA fluorescence in situ hybridization(RNA FISH),suggesting that R-AS1 may play a regulatory role in the cytoplasm.In addition,we successfully constructed the R-AS1 overexpression plasmid and R-AS1 CRISPR/Cas 9 plasmid,and packaged into adeno-associated virus(AAV)for in vitro and in vivo transduction experiments.In vitro,vero cells,which were previously transduced with a fixed dose of AAV to achieve the overexpression or knockdown of intracellular R-AS1,were infected with EV-A71,and then the replication of viralRNA was detected using real time PCR,and the expression levels of intracellular viral protein was detected using Western blot.In vivo,AAV was inoculated into newborn ICR mice at a certain dose to achieve the overexpression or knockdown of R-AS1,and then the suckling mice were challenged with the optimal dose of EV-A71.The body weight,survival rate,and clinical score of the mice were recorded daily.In addition,the viral loads and pathological changes of the experimental and control groups were compared.The results showed that the overexpression of R-AS1 in vitro could effectively promote the replication of viralRNA and increase the expression of viral VP1 protein.In vivo,weight loss of the mice in the R-AS1 overexpression group appeared earlier,the clinical symptoms increased rapidly,and the mortality rate was 40% higher than the R-AS1 knockout group(P(27)0.05).Furthermore,the viral load of each organ was significantly increased,and the pathological changes were more serious.In addition,high-throughput sequencing technology was used to screen the differentially expressed genes after EV-A71 infection,especially the ISGs associated with cellular innate immunity,which was confirmed by theRNA pull-down assay.Our results revealed that the up-regulated genes included Ifil6,Ifit2,Ifit1 and Isg20l1;the down-regulated genes included Mvp,Ifitm3 and Mxa.Notbaly,MVP was identified as an interaction protein of R-AS1 by pull-down assay and mass spectrometry,which may be paaticipated in the host’s natural immunity against EV71 infection.In summary,our study successfully screened a long non-codingRNA,lncRNA R-AS1,which is related to the host’s innate immunity against EV-A71 infection.R-AS1 is likely to be capable of promoting the replication of EV-A71 by down-regulating the expression of antiviral protein MVP,suggesting that R-AS1 can be used as a negative regulator of immune defense in EV-A71 infection.These findings shed light on the complex virus-host interaction and new targets for antiviral development.