Comprehensive Expression Profiling Analysis And Function Study Of LncRNAs In Cells With Porcine Delta Coronavirus Infection

Porcine delta coronavirus(PDCoV)is a newly discovered swine enteropathogenic coronavirus that causes acute vomiting,diarrhea,dehydration and even mortality in neonatal piglets.It was firstly identified in pig clinical samples by Woo et al.in 2012 and isolated in the United States in early 2014.In recent years,PDCoV poses a major threat to the swine industry,and is currently epidemic in several countries,such as the United States,Canada and Thailand.Several researchers have made outstanding contributions in PDCoV molecular epidemiological investigation,propagation and pathogenesis of PDCoV infection,as well as the interaction between PDCoV and the host.It has been reported that Long non-coding RNA(lncRNA)plays an important role in the regulation of virus replication and virulence.LncRNA is the member of noncoding RNAs family with consist of more than 200 nucleotides(nt)in length,yielding limited protein-coding abilities.With the development of high-throughput sequencing technology,lncRNA,as a new regulator molecule,has been attracted more and more attention and become one of the research focus in the field of life science.Studies have shown that lncRNA is involved in regulating gene expression includes various mechanisms,such as the epigenetic,transcription,and the post-transcriptional modification.In addition,LncRNA is also involved in many important.biological processes,such as cell growth,differentiation,metabolism,and apoptosis.In addition,some lncRNAs are also emerging as critical regulators of host immune responses during viral infection.However,the role of lncRNA in the PDCoV infection remains unclear.In order to explore the mechanism of functional lncRNAs induced by PDCoV and develop new drug therapeutic strategy,we performed transcriptome sequencing of PDCoV infected swine testicular(ST)cells using Illumina Hiseq4000 platform.In this study,we mainly focused on the function of two lncRNAs in PDCoV infection.?.The expression profile analysis of lncRNAs in PDCoV infected ST cellsIn this study,genome-wide profiling of lncRNAs in PDCoV infected ST cells was performed using RNA-seq.Based on the length of lncRNAs(>200nt),the sequence conservation and the coding potential,1190 novel lncRNAs candidates were identified by four coding potential prediction software(CPC,PFAM,phyloCSF and CNCI).Furthermore,we analyzed the differential expression of lncRNAs under the screening criteria of |Log2(fold change)|>0 and P value<0.05.The data analysis showed that 830 lncRNAs were significantly regulated,of which 477 were up-regulated and 353 were down-regulated,and it has been confirmed by RT-qPCR.Gene Ontology(GO)enrichment analysis revealed that the co-location and co-expression target genes of differentially expressed lncRNAs were all enriched in the three GO terms,biological process,cell component and molecular function.KEGG enrichment showed that the co-located target genes of up-regulated lncRNAs were mainly enriched in the four signaling pathways,which including Jak-STAT signaling pathway,HTLV-1 infection,Epstein-Barr virus infection and Cytokine-Cytokine receptor interaction.The most significant enrichment was found in the Jak-STAT signaling pathway and Cytokine-Cytokine receptor interaction.In addition,they were also enriched in the NF-?B signaling pathway and Cell cycle.The co-located target genes of down-regulated lncRNAs were mainly enriched in the five signaling pathways,Alcoholism,Insulin signaling pathway,Axon guidance,Vascular smooth muscle contraction and Adrenergic signaling in cardiomyocytes,of which Alcoholism and Insulin signaling pathway were the most significant enrichment pathways.Meanwhile,the mTOR signaling pathway,which regulates a variety of physiological functions such as cell growth and cell cycle,was also enriched.Both of the up-regulated and down-regulated lncRNAs were all mainly enriched in Metabolic pathways.At the same time,they were also enriched in TNF signaling pathway and Toll-like receptor signaling pathway,which can stimulate important biological effects.The analysis of network association between lncRNA and mRNA showed that many chemokines and inflammation factors,such as IL-6,CXCL2,CXCL10,CCL20 and CASP10,can be regulated by multiple lncRNAs,suggesting that lncRNA may regulate the process of PDCoV infection by participating in cell metabolism and immune response.?.The function analysis of ALDBSSCT0000007366 in PDCoV infectionALDBSSCT0000007366 with significant differential expression was firstly selected for function analysis.RNA-seq showed that ALDBSSCT0000007366 was 504nt in length,thus named as LNC504.PDCoV infection significantly increased the abundance of LNC504 in a dose-and time-dependent manner.The copy number calculation standard equation of LNC504 showed that the copy number of LNC504 in a single mock-infected or PDCoV-infected ST cell was 0.01 or 40 respectively.PDCoV infection could significantly increase the pre-mRNA levels of LNC504 co-located RNF144A,RSAD2,and CMPK2 genes.Overexpression of LNC504 could significantly inhibit the PDCoV replication.Further analysis shows that LNC504 had no significant effect on the pre-mRNA of RNF144A and CMPK2 genes,but had significant inhibitory effects on the pre-mRNA,mRNA and protein level of RSAD2 gene.PDCoV infection could significantly increase the RSAD2 mRNA and protein expression level,while the PDCoV viral protein M,N and NS7 but not NS6 could decreased the RSAD2 protein level.Overexpression of RSAD2 could significantly promote PDCoV and other porcine diarrhea viruses,PSV and PTV replication.In contrast,knockdown RSAD2 could significantly decrease the expression level of PDCoV N protein.Key domains identification showed that the ?-helix domain in N-terminal combined with the intermediate iron-sulfur cluster domain of RSAD2 played a key role in PDCoV replication.In addition,a transcript with a 3755nt full-length of ALDBSSCT0000007366 was obtained through RACE,which has a high identity with one of the transcripts of the RSAD2 gene,but the function of this full-length transcript has not been identified.In summary,LNC504 could significantly inhibit PDCoV replication,and its co-located RSAD2 gene could promote the PDCoV replication through its N-terminal?-helix and iron-sulfur cluster binding domain.The mechanism by which RSAD2 promotes PDCoV replication is unknown,but LNC504 may exert an antiviral effect by inhibiting the production of RSAD2 protein.?.The function analysis of LNC-001030 in PDCoV infectionPDCoV infection or poly(I:C)treatment could induce a significant increase in LNC-001030 abundance.Nuclear and cytoplasmic extraction assay showed that LNC-001030 was mainly located in the nucleus.The coding potential prediction tool CP AT analysis showed that the coding possibility of LNC-001030 is only 0.008,indicating that LNC-001030 had no protein coding ability.NCBI ORF Finder showed that there are four ORFs inside of LNC-001030,of which ORF3 and ORF4 are longer.Fluorescence observation and western blotting experiments showed that neither ORF3 nor ORF4 could be translated,indicating that LNC-001030 does not have the ability to encode small peptides.Overexpression of LNC-001030 could significantly inhibit the replication of PDCoV.In contrast,knockdown LNC-001030 could significantly promote the replication of PDCoV.Further analysis showed that LNC-001030 could obviously promote the apoptosis induced by PDCoV and Staurosporine,and it could also significantly promote the transcripition of poly(I:C)-induced interferon(IFN)and interferon-stimulated genes(ISGs).In summary,we demonstrated that LNC-001030 could exert antiviral effects by positively regulating the apoptosis and transcription of IFN and ISGs.