Epitranscriptomic regulation of HBV gene expression

项目摘要/摘要 人类乙肝病毒(乙肝病毒引起慢性肝炎。慢性乙肝是慢性肝炎的主要危险因素 进展到肝细胞癌(HCC)的发病。尽管有一种有效的疫苗，目前正在使用 抗病毒药物，全球估计有3.5亿受感染的携带者。抗病毒药物不能消除体内的HBVDNA 原子核。更好地理解宿主和病毒因素在慢性肝炎中的作用 需要的。对乙肝病毒基因表达的表观转录调控提供了另一层调控机制 基因表达。在这项研究中，我们建议表征病毒RNA的转录后修饰 腺苷碱基的N6位称为m6A。我们在拨款中所描述的广泛的初步研究 应用表明，HBVRNA甲基化为m6A。利用沉默宿主甲基酶的策略 和去甲基酶参与表位转录动态平衡，我们提出证据表明，HBVRNAs 都是m6A基因修饰的。在缺乏酶的情况下，对病毒转录本的翻译有明显的影响 催化M6A修饰。我们的MERIP免疫沉淀和Tru-Seq分析的结果证实 富含m6A残基峰值的潜在序列范围。我们突变了大约13个‘A’残基 在乙肝病毒基因组中从1700到2000个核苷酸的区域。这些都被检查过了 乙肝病毒蛋白的翻译和逆转录功能。结果是令人震惊的，因为翻译是 M6A修饰负调控，反转录为正调控。基于这些 令人兴奋的数据，我们建议继续表征HBVRNA的m6A修饰，并确定 该修饰在乙肝病毒感染中的功能相关性。这项研究为研究乙肝病毒开辟了一条新的途径 研究独特的乙肝病毒生命周期的表位转录调控及其可能的作用。 进展为与感染相关的慢性化。

Project Summary/Abstract Human hepatitis B virus (HBV causes chronic hepatitis. Chronic hepatitis B is a major risk factor in the progression to the onset of hepatocellular carcinoma (HCC). In spite of an effective vaccine and currently used antivirals, there are estimated 350 million infected carriers worldwide. Antivirals do not eliminate HBV DNA in the nucleus. A better understanding of the role of host and viral factors contributing to chronic hepatitis is needed. Epitranscriptomic regulation of HBV gene expression provides another layer of regulatory schemes of gene expression. In this study, we propose to characterize the posttranscriptional modification of viral RNAs at the N6 position of the adenosine base termed m6A. Our extensive preliminary studies described in the grant application show that HBV RNAs are methylated as m6A. Using the strategy of silencing of host methylases and demethylase enzymes involved in epitranscriptomic homeostasis, we present evidence that HBV RNAs are m6A modified. There was clearly an effect on translation of viral transcripts in the absence of enzyme that catalyze m6A modification. Our results with MeRIP immunoprecipitation followed by Tru-seq analysis identified a potential range of sequences enriched in a peak of m6A residues. We mutated about 13 `A' residues in this region that spans from nucleotides 1700 to 2000 within the HBV genome. These were examined for translation of HBV proteins and reverse transcription function. Results were striking, in that, the translation was negatively regulated but reverse transcription was positively regulated by m6A modification. Based on these exciting data, we propose to continue characterization of m6A modification of HBV RNAs and identify the functional relevance of this modification in the HBV infection. This study opens a new avenue of HBV research to investigate epitranscriptomic regulation of the unique HBV life cycle and possible contribution to the progression to chronicity associated with infection.