Campbell Ashley M, Taylor Victoria C, Cohan Beata, Frappier Lori
Department of Molecular Genetics, University of Toronto, Toronto, Canada.
PLoS Pathog. 2025 Jul 17;21(7):e1013347. doi: 10.1371/journal.ppat.1013347. eCollection 2025 Jul.
Cellular and viral miRNAs are thought to play important roles in regulating Epstein-Barr virus (EBV) latent and lytic infections, however, to date, most studies have focussed on latent infections in B cells. To determine how cellular and viral miRNAs contribute to EBV lytic infection in epithelial cells, the main sites of lytic infection, we conducted miRNA-sequencing experiments in EBV-infected AGS gastric carcinoma cells, before and after reactivation to the lytic cycle, analysing both total miRNA and Ago2-associated miRNAs. We identified over 100 miRNAs whose association with Ago2 was affected upon EBV reactivation, most of which were due to changes in miRNA abundance. For EBV miRNAs, the most striking result was that the BHRF1 miRNAs, previously only reported to be expressed in B cells, were upregulated upon reactivation. The largest changes in cellular miRNAs upon EBV reactivation were increases in the abundance and Ago2-association of miR-409-3p, miR-381-3p and miR-370-3p, which appear to have pro-viral effects. In particular, inhibiting miR-409-3p reduced BZLF1 and other EBV lytic protein expression, at least in part through modulation of ZEB1. Interestingly, these miRNAs all originate from the DLK1-DIO3 locus (14q32.2 - 32.31), which encodes multiple lncRNAs. We showed that the lncRNAs MEG9, MIR381HG, and MEG8, from which miR-409-3p, miR-381-3p and miR-370-3p are derived, were also upregulated upon reactivation in AGS and nasopharyngeal carcinoma cells lines and occurred very early in the lytic cycle at the time of BZLF1 expression. In keeping with this timing, BZLF1 was sufficient to induce these lncRNAs dependent on its transactivation activity, and was detected at a key DLK1-DIO3 control element, consistent with a direct role in transcriptional activation. Therefore, we have identified a new role for BZFL1 in activating the expression of lncRNAs in the DLK1-DIO3 locus, resulting in induction of a subset of encoded miRNAs that promote lytic infection.
细胞和病毒的微小RNA（miRNA）被认为在调节爱泼斯坦-巴尔病毒（EBV）的潜伏感染和裂解感染中发挥重要作用，然而，迄今为止，大多数研究都集中在B细胞中的潜伏感染。为了确定细胞和病毒miRNA如何促进EBV在上皮细胞（裂解感染的主要部位）中的裂解感染，我们在EBV感染的AGS胃癌细胞重新激活进入裂解周期前后进行了miRNA测序实验，分析了总miRNA和与AGO2相关的miRNA。我们鉴定出100多种miRNA，其与AGO2的结合在EBV重新激活后受到影响，其中大多数是由于miRNA丰度的变化。对于EBV miRNA，最显著的结果是BHRF1 miRNA（以前仅报道在B细胞中表达）在重新激活后上调。EBV重新激活后细胞miRNA的最大变化是miR-409-3p、miR-381-3p和miR-370-3p的丰度和与AGO2的结合增加，这些miRNA似乎具有促病毒作用。特别是，抑制miR-409-3p可降低BZLF1和其他EBV裂解蛋白的表达，至少部分是通过调节ZEB1。有趣的是，这些miRNA都源自DLK1-DIO3基因座（14q32.2 - 32.31），该基因座编码多种长链非编码RNA（lncRNA）。我们发现，miR-409-3p、miR-381-3p和miR-370-3p所源自的lncRNA MEG9、MIR381HG和MEG8在AGS和鼻咽癌细胞系重新激活后也上调，并且在裂解周期早期BZLF1表达时就出现。与此时间一致，BZLF1足以依赖其反式激活活性诱导这些lncRNA，并且在一个关键的DLK1-DIO3控制元件处被检测到，这与转录激活中的直接作用一致。因此，我们确定了BZFL1在激活DLK1-DIO3基因座中lncRNA表达方面的新作用，导致诱导一组促进裂解感染的编码miRNA。
