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.

肝炎病毒（HBV）长期感染了3亿人，是终末期肝脏的主要原因 疾病和肝细胞癌（HCC），每年造成约100万人死亡。艾滋病毒是一个常见的共同 患有慢性肝炎的患者感染（CHB），并恶化HBV和肝病结局。肝病 在抗逆转录病毒时代，PWH是PWH中死亡的主要原因，主要是由于病毒肝炎。抗病毒物质 用核（T）IDE类似物（NUC）处理血浆HBV DNA，但不会消除血浆HBV DNA 共价闭合的圆形DNA（CCCDNA）HBV模板位于每个受感染的细胞中。 NUC中断 导致HBV DNA反弹和临床肝炎。因此，找到HBV治疗至关重要 HBV单和HIV/HBV共同感染。 在第一个资金周期中，我们将单细胞方法应用于HBV/HIV共感染的肝组织，并证明 在NUC中，该CCCDNA在转录中被抑制。我们现在建议了解机制 这一发现的基础 治愈。我们提出了一项针对NUC相关的CCCDNA转录抑制的深入研究，以比较 患有HBV/HIV共同和HBV单感染的人。在目标中，我们将量化肝内病毒DNA和 使用单细胞激光器的HBV单和HIV共感染的个体的肝细胞中的RNA量 捕获微分解和我们的新型液滴数字PCR分析。我们将量化和比较CCCDNA 转录指数（CCCDNA TI），HBV单和HBV/HIV共同的CCCDNA转录度量 感染。在目标两个中，我们将使用新技术来测试CCCDNA转录抑制是否是由于 使用HBV单和HBV/HIV共感染的肝组织对HBV的遗传或表观遗传修饰 一。我们将使用CAS9-NANANOPORE测序检查CpG二核苷酸的表观遗传修饰 CCCDNA。我们将使用Novaseq平台执行CCCDNA的下一代测序以寻找 可能与减少转录和量化全局CCCDNA转录的突变 抑制规范病毒基因和剪接变体。在目标三中，我们将检验主机的假设 响应有助于CCCDNA转录抑制，并比较HBV单声道之间的这种响应 和HBV/HIV共同感染。我们将应用新型的10倍visium空间基因表达分析（10x VSGEA） 从AIM ONE到肝组织的平台比较高与低的组织中的肝细胞宿主转录组 CCCDNA Ti，使用同一平台直接询问HBV转录。我们将确认所鉴定的基因 使用Tecan RNA测序使用10倍VSGEA，富含高与低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转录抑制有关， 专注于先天免疫基因和与HIV相关的免疫激活。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治疗。