Single-Nucleotide Resolution Mapping of Hepatitis B Virus Promoters in Infected Human Livers and Hepatocellular Carcinoma.
Single-Nucleotide Resolution Mapping of Hepatitis B Virus Promoters in Infected Human Livers and Hepatocellular Carcinoma.
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DOI：
10.1128/jvi.01625-16
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发表时间：
2016-12-01
影响因子：
5.4
通讯作者：
Carninci P
中科院分区：
文献类型：
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作者：
Altinel K;Hashimoto K;Wei Y;Neuveut C;Gupta I;Suzuki AM;Dos Santos A;Moreau P;Xia T;Kojima S;Kato S;Takikawa Y;Hidaka I;Shimizu M;Matsuura T;Tsubota A;Ikeda H;Nagoshi S;Suzuki H;Michel ML;Samuel D;Buendia MA;Faivre J;Carninci P
乙型肝炎病毒 (HBV) 是包括肝细胞癌 (HCC) 在内的肝脏疾病的主要原因，每年有超过 65 万人因 HBV 相关的肝功能衰竭而死亡。对各个启动子的广泛研究表明，异质 RNA 5' 末端导致 HBV 转录组和蛋白质组的复杂性。在这里，我们以单核苷酸分辨率提供了人类肝脏、HCC 和血液中 HBV 转录起始位点 (TSS) 的综合图谱，以及几个实验复制系统。通过对 16 个 HCC/非肿瘤肝脏对进行 CAGE（基因表达上限分析）分析，我们鉴定了 17 个稳健的 TSS，其中包括位于基因体中部的 X 基因的新型启动子，该启动子可能会产生从保守的第二起始密码子翻译而来的较短的 X 蛋白，以及两个可能代表病毒非编码 RNA 的次要反义转录物。有趣的是，HCC 和非肿瘤肝脏中的转录谱相似，尽管定量分析揭示了临床样本中 TSS 使用模式的高度可变，反映了每个启动子处 HBV 转录起始的精确调节。与肝脏和肝癌中发现的各种 TSS 不同，在 HBV 阳性血液样本中检测到的绝大多数转录本都是前基因组 RNA，很可能是由肝脏产生和释放的。我们使用 CAGE 技术进行的定量 TSS 绘图将有助于在旨在根除慢性携带者中的 HBV 的进一步研究中更好地了解 HBV 转录反应。重要性 尽管已经有了安全有效的疫苗，乙型肝炎病毒感染仍然是一个全球性的健康问题，目前的抗病毒方案无法消除慢性携带者中的病毒。先前关于 HBV 转录调控的研究描述了四个主要启动子和两个增强子，但对其在人类肝脏和 HCC 中的活性知之甚少。我们使用一种新的、灵敏的定量方法，称为基因表达帽分析 (CAGE)，对临床人体样本和实验模型中的 HBV RNA 5' 末端进行了深度测序。我们的数据提供了人类肝脏中整个 HBV 基因组的全球 TSS 分布的第一个全面图谱，验证了已知的启动子并确定了新的位置。更好地了解临床环境中的 HBV 转录活性对于评估针对 HBV 复制的治疗方法具有重要意义。
Hepatitis B virus (HBV) is a major cause of liver diseases, including hepatocellular carcinoma (HCC), and more than 650,000 people die annually due to HBV-associated liver failure. Extensive studies of individual promoters have revealed that heterogeneous RNA 5′ ends contribute to the complexity of HBV transcriptome and proteome. Here, we provide a comprehensive map of HBV transcription start sites (TSSs) in human liver, HCC, and blood, as well as several experimental replication systems, at a single-nucleotide resolution. Using CAGE (cap analysis of gene expression) analysis of 16 HCC/nontumor liver pairs, we identify 17 robust TSSs, including a novel promoter for the X gene located in the middle of the gene body, which potentially produces a shorter X protein translated from the conserved second start codon, and two minor antisense transcripts that might represent viral noncoding RNAs. Interestingly, transcription profiles were similar in HCC and nontumor livers, although quantitative analysis revealed highly variable patterns of TSS usage among clinical samples, reflecting precise regulation of HBV transcription initiation at each promoter. Unlike the variety of TSSs found in liver and HCC, the vast majority of transcripts detected in HBV-positive blood samples are pregenomic RNA, most likely generated and released from liver. Our quantitative TSS mapping using the CAGE technology will allow better understanding of HBV transcriptional responses in further studies aimed at eradicating HBV in chronic carriers. IMPORTANCE Despite the availability of a safe and effective vaccine, HBV infection remains a global health problem, and current antiviral protocols are not able to eliminate the virus in chronic carriers. Previous studies of the regulation of HBV transcription have described four major promoters and two enhancers, but little is known about their activity in human livers and HCC. We deeply sequenced the HBV RNA 5′ ends in clinical human samples and experimental models by using a new, sensitive and quantitative method termed cap analysis of gene expression (CAGE). Our data provide the first comprehensive map of global TSS distribution over the entire HBV genome in the human liver, validating already known promoters and identifying novel locations. Better knowledge of HBV transcriptional activity in the clinical setting has critical implications in the evaluation of therapeutic approaches that target HBV replication.