The Interaction of Human and Epstein-Barr Virus miRNAs with Multiple Sclerosis Risk Loci

Abstract

Although the causes of Multiple Sclerosis (MS) still remain largely unknown, multiple lines of evidence suggest that Epstein-Barr virus (EBV) infection may contribute to the development of MS. Here, we aimed to identify the potential contribution of EBV-encoded and host cellular miRNAs to MS pathogenesis. We identified differentially expressed host miRNAs in EBV infected B cells (LCLs) and putative host/EBV miRNA interactions with MS risk loci. We estimated the genotype effect of MS risk loci on the identified putative miRNA:mRNA interactions in silico. We found that the protective allele of MS risk SNP rs4808760 reduces the expression of hsa-mir-3188-3p. In addition, our analysis suggests that hsa-let-7b-5p may interact with ZC3HAV1 differently in LCLs compared to B cells. In vitro assays indicated that the protective allele of MS risk SNP rs10271373 increases ZC3HAV1 expression in LCLs, but not in B cells. The higher expression for the protective allele in LCLs is consistent with increased IFN response via ZC3HAV1 and so decreased immune evasion by EBV. Taken together, this provides evidence that EBV infection dysregulates the B cell miRNA machinery, including MS risk miRNAs, which may contribute to MS pathogenesis via interaction with MS risk genes either directly or indirectly.

Keywords: EBNA2; EBV; GWAS; MS; ZC3HAV1; miRNA.

Figure 1

The possible interactions between LCL miRNAs and host/EBV mRNAs which could be involved in MS pathogenesis: (I) Host MS risk miRNAs may target MS risk genes (II) host miRNA binding sites among MS risk genes may be affected by MS risk SNPs (III) EBV miRNAs may target MS risk genes (IV) EBV miRNA binding sites among host MS risk genes may be affected by MS risk SNP.

Figure 2

The eQTL effect and architecture of the LD block containing MS risk SNP rs4808760 LD block. rs4808760 is an MS risk SNP which is proximal to hsa-mir-3188-3p. Y and X axes in (A) demonstrate LD value and location of each SNP in rs4808760 LD block, respectively. Y and X axes in (B) demonstrate LD value and eQTL significance of each SNP in rs4808760 LD block, respectively. Blue circle represents a SNP which is targeted by EBNA2. The purple circle shows the MS risk SNP. Red circles show the other SNPs in this LD block which are were in eQTL with hsa-mir-3188-3p with FDR less than 5% In the Geuvadis Euro cohort (n = 373) [PMID: 24037378] of the Geuvadis study.

Figure 3

The over-representation of MS risk miRNAs differentially expressed (DE) between LCLs and B cells. We identified differentially expressed miRNAs in LCLs and B cells with a p value less than 0.05. MS risk miRNAs over-representation in DE miRNAs between LCLs and B cells.

Figure 4

EBV miRNAs’ targets among MS risk genes. (A) shows MS risk genes which are targeted by EBV miRNAs and the replication of these interactions in different LCL cell lines (blue fill). LCL cell lines EF3D-AGO2, LCL35 and LCL-BAC (transformed with the plasmid which contains EBV-B95-8 genome) contain complete EBV miRNA profile. LCL-BAC-D1, LCL-BAC-D2 and LCL-BAC-D3 cell lines lacking miR-BHRF1-1, miR-BHRF1-2, or miR-BHRF1-3, respectively. The miRNA:mRNA interactions were deemed to be most accurate if verified by PAR-Clip data in at least two of three LCL PAR-CLIP datasets with a full EBV 95.8 miRNA profile (EF3D, LCL35 and BAC). PAR-CLIP data for mutant EBV strain cell lines were used for further evaluation of BHRF1-1, BHRF1-2, and BHRF1-3 targets. (B) shows the significance of eQTL effect of MS risk SNPs on genes which targeted by EBV miRNAs. Red dots represent insignificant eQTLs at a cutoff p value of 0.05. Blue dots represent the MS risk genes that have an EBNA2 binding site collocated with the risk SNP. Green dots represent the significant eQTLs at a cutoff p value of 0.05 which have an EBNA2 binding site collocated with the risk SNP. Slope means the effect of risk allele relative to protective allele on expression.

Figure 5

SNPs which are located within or close to miRNA target sites on MS risk genes and the replication of these interactions in the different LCL cell lines used in the PAR-CLIP study (red fill). * shows the genes which are involved in LCL latency III specific MS risk transcriptome. ≡ shows genes where the proximal MS risk SNP, or SNPs in LD with them, are targeted by EBNA2.

Figure 6

Putative cellular miRNAs target sites on ZC3HAV1 and the genotype effect of MS risk SNP on ZC3HAV1 expression in LCLs and B cells. (A) The putative cellular miRNAs target sites on ZC3HAV1 which were located nearby the SNP rs7803490 in LD with MS risk SNP rs10271373. The consensus sequences obtained using pipeline B are highlighted red. The rs10271373 genotype effect on expression of ZC3HAV1 in LCLs (B) and B cells (C). AA is the risk genotype of rs10271373, and CC is the protective genotype. * indicates p ≤ 0.01.