Mechanisms of HBV cccDNA transcriptional regulation in persons with and without HIV

HIV感染者和未感染者的HBV cccDNA转录调控机制

• 批准号: 10882261
• 负责人: ASHWIN BALAGOPAL
• 金额: $ 81.1万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-08-14 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10882261
• 关键词: Anti-Retroviral Agents; Binding; Biological Assay; CD4 Positive T Lymphocytes; Candidate Disease Gene; Cause of Death; Cell Count; Cells; Cessation of life; Characteristics; Chronic; Chronic Hepatitis B; Circular DNA; Clinical; Complementary DNA; CpG Islands; CpG dinucleotide; Cytoplasm; DNA Methylation; Disease Outcome; Epigenetic Process; Funding; Gene Expression Profiling; Gene Silencing; Genes; Genetic; Genetic Transcription; Goals; HIV; HepG2; Hepatitis; Hepatitis B Therapy; Hepatitis B Virus; Hepatocyte; Human; Immune; Immune response; In Vitro; Individual; Integration Host Factors; Interruption; Knowledge; Liver; Liver diseases; Measurable; Measures; Messenger RNA; Methods; Modeling; Modification; Mutation; Nonsense Codon; Pathway interactions; Persons; Plasma; Primary carcinoma of the liver cells; Process; RNA; RNA Splicing; Resolution; Reverse Transcription; Reverse Transcription Inhibition; Sampling; T-Cell Depletion; Techniques; Testing; Therapeutic; Tissues; Transcript; Transcriptional Regulation; Use of New Techniques; Variant; Viral; Viral Genes; Viral hepatitis; Virion; Virus Diseases; Virus Replication; analog; analytical method; cDNA Library; circular RNA; co-infection; digital; end stage liver disease; immune activation; in vitro testing; indexing; insertion/deletion mutation; interest; intrahepatic; laser capture microdissection; multidisciplinary; nanopore; next generation sequencing; novel; response; therapy development; tool; transcriptome; transcriptome sequencing; viral DNA; viral RNA; viral rebound; virus genetics

Hepatitis B virus (HBV) infects >300 million people chronically and is the leading cause of end-stage liver disease and hepatocellular carcinoma (HCC), resulting in ~1 million deaths annually. HIV is a common co- infection in people with chronic hepatitis B (CHB) and worsens HBV and liver disease outcomes. Liver disease is a leading cause of death among PWH in the era of antiretrovirals, mostly due to viral hepatitis. Antiviral treatment with nucleos(t)ide analogues (NUCs) suppresses plasma HBV DNA but does not eliminate the covalently closed circular DNA (cccDNA) HBV template that resides in every infected cell. NUC interruption leads to HBV DNA rebound and clinical hepatitis. Therefore, finding an HBV cure is of major importance for HBV mono- and HIV/HBV co-infection. In the first funding cycle, we applied single-cell methods to HBV/HIV co-infected liver tissues and demonstrated that cccDNA is transcriptionally suppressed during NUCs. We now propose to understand the mechanism underlying this finding since permanently exploiting this mechanism therapeutically can lead to a functional cure. We propose an intensive study of NUC-associated cccDNA transcriptional suppression comparing people with HBV/HIV co- and HBV mono-infection. In aim one, we will quantify intrahepatic viral DNA and RNA quantities in hepatocytes from HBV mono- and HBV/HIV co-infected individuals using single-cell laser capture microdissection and our novel droplet digital PCR assays. We will quantify and compare the cccDNA transcriptional index (cccDNA TI), a measure of cccDNA transcription, in HBV mono- and HBV/HIV co- infection. In aim two we will use novel techniques to test whether cccDNA transcriptional suppression is due to genetic or epigenetic modifications of HBV using HBV mono- and HBV/HIV co-infected liver tissues from aim one. We will use Cas9-Nanopore sequencing to examine epigenetic modifications of CpG dinucleotides in cccDNA. We will use the NovaSeq platform to perform next-generation sequencing of cccDNA to look for mutations that may be associated with reduced transcription and to quantify global cccDNA transcriptional suppression of canonical viral genes and splice variants. In aim three we will test the hypothesis that the host response contributes to cccDNA transcriptional suppression and compare this response between HBV mono- and HBV/HIV co-infection. We will apply the novel 10X Visium spatial gene expression analysis (10x VSGEA) platform to liver tissue from Aim one to compare hepatocyte host transcriptomes in tissues with high vs. low cccDNA TI, directly interrogating HBV transcription using the same platform. We will confirm genes identified with 10X VSGEA using Tecan RNA sequencing, enriching for cells with high vs. low cccDNA TI. We will conduct in vitro studies to prove that genes of interest are associated with cccDNA transcriptional suppression, focusing on innate immune genes and HIV-associated immune activation. Our proposal fills knowledge gaps in understanding how to transform reversible cccDNA transcriptional suppression to a functional cure.

B型肝炎病毒（HBV）慢性感染超过3亿人，是终末期肝病的主要原因 疾病和肝细胞癌（HCC），每年导致约100万人死亡。艾滋病是一种常见的疾病， 慢性B型肝炎（CH B）患者的HBV感染和肝病结果。肝病 在抗逆转录病毒药物时代，是导致威尔斯亲王死亡的主要原因，主要是由于病毒性肝炎。抗病毒 核苷（酸）类似物（NUC）治疗可抑制血浆HBV DNA，但不能消除 共价闭合环状DNA（cccDNA）HBV模板存在于每个感染的细胞中。NUC中断 导致HBV DNA反弹和临床肝炎。因此，找到HBV治愈方法对于 HBV单一感染和HIV/HBV合并感染。 在第一个资助周期中，我们将单细胞方法应用于HBV/HIV共感染的肝组织，并证明了 cccDNA在NUC期间被转录抑制。我们现在建议理解 这是这一发现的基础，因为在治疗上永久地利用这一机制可以导致功能性 疗方我们建议对NUC相关cccDNA转录抑制进行深入研究， HBV/HIV合并感染和HBV单一感染者。在目标一，我们将定量肝内病毒DNA， 使用单细胞激光检测HBV单感染和HBV/HIV共感染个体肝细胞中的RNA量 捕获显微切割和我们的新型液滴数字PCR测定。我们将定量并比较cccDNA 在HBV单细胞和HBV/HIV共细胞中，cccDNA转录指数（cccDNA TI）是cccDNA转录的一种量度。 感染在第二个目标中，我们将使用新的技术来测试cccDNA转录抑制是否是由于 使用来自目标的HBV单感染和HBV/HIV共感染的肝组织进行HBV的遗传或表观遗传修饰 一个.我们将使用Cas9-纳米孔测序来检查CpG二核苷酸的表观遗传修饰， cccDNA。我们将使用NovaSeq平台进行cccDNA的下一代测序，以寻找 可能与转录减少相关的突变，并定量整体cccDNA转录 抑制典型病毒基因和剪接变体。在第三个目标中，我们将检验宿主 应答有助于cccDNA转录抑制，并比较HBV单- 和HBV/HIV混合感染。我们将应用新的10 x Visium空间基因表达分析（10 x VSGEA） 目的1的肝组织平台，以比较高与低表达的组织中的肝细胞宿主转录组 cccDNA TI，使用相同的平台直接询问HBV转录。我们将确认基因鉴定 使用Tecan RNA测序，用10 X VSGEA富集具有高相对于低cccDNA TI的细胞。我们将 进行体外研究以证明感兴趣的基因与cccDNA转录抑制相关， 专注于先天免疫基因和HIV相关免疫激活。我们的建议填补了知识空白， 了解如何将可逆的cccDNA转录抑制转化为功能性治疗。