Assadian Farzaneh, Kamel Wael, Laurell Göran, Svensson Catharina, Punga Tanel, Akusjärvi Göran
Department of Medical Biochemistry and Microbiology, Uppsala Biomedical Center, Uppsala University, Uppsala, Sweden.
Department of Surgical Sciences, Otorhinolaryngology and Head and Neck Surgery, Uppsala University, Uppsala, Sweden.
PLoS One. 2017 May 25;12(5):e0177275. doi: 10.1371/journal.pone.0177275. eCollection 2017.
We have used high-throughput small RNA sequencing to characterize viral small RNA expression in purified tonsillar B and T lymphocytes isolated from patients tested positive for Epstein-Barr virus (EBV) or human adenovirus (HAdV) infections, respectively. In the small set of patients analyzed, the expression profile of EBV and HAdV miRNAs could not distinguish between patients diagnosed with tonsillar hypertrophy or chronic/recurrent tonsillitis. The EBV miR-BART expression profile among the patients diagnosed with tonsillar diseases resembles most closely the pattern seen in EBV+ tumors (Latency II/I). The miR-BARTs that appear to be absent in normal EBV infected cells are essentially all detectable in the diseased tonsillar B lymphocytes. In the EBV+ B cells we detected 44 EBV miR-BARTs derived from the proposed BART precursor hairpins whereof five are not annotated in miRBase v21. One previously undetected miRNA, BART16b-5p, originates from the miR-BART16 precursor hairpin as an alternative 5´ miR-BART16 located precisely upstream of the annotated miR-BART16-5p. Further, our analysis revealed an extensive sequence variation among the EBV miRNAs with isomiRs having a constant 5´ end but alternative 3´ ends. A range of small RNAs was also detected from the terminal stem of the EBER RNAs and the 3´ part of v-snoRNA1. During a lytic HAdV infection in established cell lines the terminal stem of the viral non-coding VA RNAs are processed to highly abundant viral miRNAs (mivaRNAs). In contrast, mivaRNA expression in HAdV positive tonsillar T lymphocytes was very low. The small RNA profile further showed that the 5´ mivaRNA from VA RNAI and the 3´ mivaRNA from VA RNAII were as predicted, whereas the 3´ mivaRNA from VA RNAI showed an aberrant processing upstream of the expected Dicer cleavage site.
我们利用高通量小RNA测序技术，分别对从感染爱泼斯坦-巴尔病毒（EBV）或人腺病毒（HAdV）呈阳性的患者中分离出的纯化扁桃体B淋巴细胞和T淋巴细胞中的病毒小RNA表达进行了表征。在所分析的一小部分患者中，EBV和HAdV miRNA的表达谱无法区分诊断为扁桃体肥大或慢性/复发性扁桃体炎的患者。在诊断为扁桃体疾病的患者中，EBV miR-BART的表达谱与EBV+肿瘤（潜伏期II/I）中所见模式最为相似。正常EBV感染细胞中似乎不存在的miR-BARTs在患病的扁桃体B淋巴细胞中基本上都能检测到。在EBV+B细胞中，我们检测到44种源自拟议的BART前体发夹结构的EBV miR-BARTs，其中5种在miRBase v21中未注释。一种先前未检测到的miRNA，BART16b-5p，源自miR-BART16前体发夹结构，是位于注释的miR-BART16-5p正上游的另一种5´ miR-BART16。此外，我们的分析揭示了EBV miRNA之间存在广泛的序列变异，异源miRNA具有恒定的5´端但3´端不同。还从EBER RNA的末端茎和v-snoRNA1的3´部分检测到一系列小RNA。在已建立的细胞系中进行溶细胞性HAdV感染期间，病毒非编码VA RNA的末端茎被加工成高度丰富的病毒miRNA（mivaRNA）。相比之下，HAdV阳性扁桃体T淋巴细胞中的mivaRNA表达非常低。小RNA谱进一步显示，来自VA RNAI的5´ mivaRNA和来自VA RNAII的3´ mivaRNA如预期的那样，而来自VA RNAI的3´ mivaRNA在预期的Dicer切割位点上游显示出异常加工。
