Tumor Virus RNA Biology Section, RNA Biology Laboratory, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, Maryland, USA.
Systems Biology Center, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland, USA.
J Virol. 2019 Jan 4;93(2). doi: 10.1128/JVI.01593-18. Print 2019 Jan 15.
Epstein-Barr virus (EBV) is a ubiquitous human pathogen associated with Burkitt's lymphoma and nasopharyngeal carcinoma. Although the EBV genome harbors more than a hundred genes, a full transcription map with EBV polyadenylation profiles remains unknown. To elucidate the 3' ends of all EBV transcripts genome-wide, we performed the first comprehensive analysis of viral polyadenylation sites (pA sites) using our previously reported polyadenylation sequencing (PA-seq) technology. We identified that EBV utilizes a total of 62 pA sites in JSC-1, 60 in Raji, and 53 in Akata cells for the expression of EBV genes from both plus and minus DNA strands; 42 of these pA sites are commonly used in all three cell lines. The majority of identified pA sites were mapped to the intergenic regions downstream of previously annotated EBV open reading frames (ORFs) and viral promoters. pA sites lacking an association with any known EBV genes were also identified, mostly for the minus DNA strand within the EBNA locus, a major locus responsible for maintenance of viral latency and cell transformation. The expression of these novel antisense transcripts to EBNA were verified by 3' rapid amplification of cDNA ends (RACE) and Northern blot analyses in several EBV-positive (EBV) cell lines. In contrast to EBNA RNA expressed during latency, expression of EBNA-antisense transcripts, which is restricted in latent cells, can be significantly induced by viral lytic infection, suggesting potential regulation of viral gene expression by EBNA-antisense transcription during lytic EBV infection. Our data provide the first evidence that EBV has an unrecognized mechanism that regulates EBV reactivation from latency. Epstein-Barr virus represents an important human pathogen with an etiological role in the development of several cancers. By elucidation of a genome-wide polyadenylation landscape of EBV in JSC-1, Raji, and Akata cells, we have redefined the EBV transcriptome and mapped individual polymerase II (Pol II) transcripts of viral genes to each one of the mapped pA sites at single-nucleotide resolution as well as the depth of expression. By unveiling a new class of viral lytic RNA transcripts antisense to latent EBNAs, we provide a novel mechanism of how EBV might control the expression of viral latent genes and lytic infection. Thus, this report takes another step closer to understanding EBV gene structure and expression and paves a new path for antiviral approaches.
爱泼斯坦-巴尔病毒（EBV）是一种普遍存在的人类病原体，与伯基特淋巴瘤和鼻咽癌有关。尽管 EBV 基因组含有一百多个基因，但完整的转录图谱及其聚腺苷酸化谱仍不清楚。为了阐明整个 EBV 基因组中转录物的 3' 末端，我们使用之前报道的聚腺苷酸化测序（PA-seq）技术首次全面分析了病毒聚腺苷酸化位点（pA 位点）。我们发现 EBV 在 JSC-1 中总共使用了 62 个 pA 位点，在 Raji 中使用了 60 个，在 Akata 细胞中使用了 53 个，用于表达正负 DNA 链上的 EBV 基因；其中 42 个 pA 位点在这三种细胞系中都被共同使用。大多数鉴定的 pA 位点被映射到先前注释的 EBV 开放阅读框（ORF）和病毒启动子下游的基因间区域。还鉴定了缺乏与任何已知 EBV 基因关联的 pA 位点，主要是 EBVNA 基因座中负 DNA 链上的 pA 位点，EBVNA 基因座是负责维持病毒潜伏和细胞转化的主要基因座。在几种 EBV 阳性（EBV）细胞系中，通过 3' 快速扩增 cDNA 末端（RACE）和 Northern blot 分析验证了这些新的 EBNA 反义转录本的表达。与潜伏期间表达的 EBNA RNA 相反，在潜伏细胞中受到限制的 EBNA-反义转录本的表达可以被病毒裂解感染显著诱导，这表明在 EBV 裂解感染期间，EBNA-反义转录可能通过调节病毒基因表达来控制病毒基因的表达。我们的数据首次提供了 EBV 具有一种未被识别的机制，可以调节 EBV 从潜伏状态中重新激活的证据。爱泼斯坦-巴尔病毒是一种重要的人类病原体，在几种癌症的发展中起病因作用。通过阐明 JSC-1、Raji 和 Akata 细胞中 EBV 的全基因组聚腺苷酸化图谱，我们重新定义了 EBV 转录组，并以单核苷酸分辨率和表达深度将每个病毒基因的单个聚合酶 II（Pol II）转录本映射到每个映射的 pA 位点。通过揭示一种新的病毒裂解 RNA 转录本反义潜伏 EBNAs，我们提供了 EBV 可能控制潜伏基因和裂解感染表达的新机制。因此，本报告在理解 EBV 基因结构和表达方面又迈进了一步，并为抗病毒方法开辟了新途径。
