Influence of integrant-derived HBV RNAs encoding the envelope proteins on HBV life cycle

编码包膜蛋白的整合体衍生的 HBV RNA 对 HBV 生命周期的影响

• 批准号: 10561639
• 负责人: Severin O Gudima
• 金额: $ 38.75万
• 依托单位: UNIVERSITY OF KANSAS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-02-04 至 2026-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10561639
• 关键词: Affect; Aftercare; Antiviral Agents; Automobile Driving; Binding; Biological Assay; Cells; Chronic; Chronic Hepatitis B; Code; Complement; DNA Integration; Data; FDA approved; Genome; Genotype; HBV Genotype; Harvest; Hepatitis B Infection; Hepatitis B Surface Antigens; Hepatitis B Virus; Hepatitis Delta Virus; Hepatocyte; Human; Individual; Intervention; Knowledge; Length; Life Cycle Stages; Light; Liver; Liver diseases; Maintenance; Messenger RNA; Modeling; Mutation; Open Reading Frames; Pathogenesis; Patients; Persons; Pharmaceutical Preparations; Poly(A)+ RNA; Polyadenylation; Primary carcinoma of the liver cells; Production; Property; RNA; RNA Stability; Research; Reverse Transcriptase Polymerase Chain Reaction; Role; Sampling; Serum; Signal Transduction; Source; Surface Antigens; Suspensions; Testing; Tissues; Untranslated Regions; Viral; Virion; Virus; Work; analog; design; env Gene Products; interferon therapy; liver biopsy; novel; patient subsets; prevent; single-cell RNA sequencing; transcriptome sequencing; vector

Abstract There are more than 250 million of chronic HBV carriers worldwide. Chronic HBV infection is the main cause of hepatocellular carcinoma (HCC). There is no cure for HBV. Interferon therapy gives temporary benefits only to a subset of patients. Regardless that FDA-approved nucleos(t)ide analogs can greatly inhibit HBV replication, chronic HBV infection is not well under control. None of the drugs works in all patients, and is able to achieve the loss of the surface antigen (HBsAg, or HBV envelope proteins) in most cases. HBV frequently rebounds after treatment is stopped. Mechanism of HBV infection is not fully understood, which affects treatment options. This proposal will analyze the relationship between HBV integrant-derived RNAs (id-RNAs) coding for HBsAg and HBV life cycle. HBV DNA integration into host genome is not considered important for HBV life cycle, and id-RNAs are not well studied. Analyzing matching liver/HCC tissues from chronic HBV carrier humans using RT-PCR, we found that regardless of HBV replication: id-RNAs accumulated in all tissues and represented 45- 100% of all HBV RNAs found in 60% of tissues (40% livers/80% HCCs); RNAs polyadenylated via cryptic HBV poly(A) signal (could be integrant-derived or produced by HBV replication) were found in 60% livers/40% HCCs, and were the most abundant HBV RNAs in 20% livers/20% HCCs; and replication-derived RNAs (rd- RNAs) polyadenylated using conventional HBV poly(A) signal were the least abundant species in 70% of tissues (40% livers/100% HCCs). Furthermore, analysis of selected tissues by RNA sequencing also showed id-RNAs' abundance in most of the analyzed samples. The data suggest that id-RNAs may produce significant fraction of HBsAg in cells bearing HBV integrants. If the same cells also support HBV replication, then a great fraction of formed HBV virions likely will bear id-RNA-derived HBsAg (id-HBsAg) as a major component of their envelopes. Thus, infectivity of these HBV virions, and their ability to support virus spread will mostly depend on the properties of id-HBsAg, and not on those of HBV replication-derived HBsAg (rd-HBsAg). Thus, id-RNAs could regulate the maintenance of chronic HBV infection, which may call for revision of the current model of HBV infection that suggests that HBV life cycle is independent of the integration. It also became apparent that anti-HBV drugs fail to achieve loss of serum HBsAg, because major amounts of HBsAg can be produced from id-RNAs regardless of HBV replication. In light of our data, Aim 1 using a large set of liver/HCC tissues will find if id-RNAs' abundance is common during chronic HBV infection. Aims 2 and 3 will (i) find if large fraction of id- RNAs do not bear alterations in HBsAg-coding sequences, and thus can serve as sizeable source of functional HBsAg regardless of HBV replication, (ii) analyze how 3'-end untranslated regions of id-RNAs (including the host inserts) affect HBsAg synthesis, and (iii) find if id-HBsAg can efficiently support assembly and infectivity of HBV virions. The proposal will advance our understanding on how id-RNAs and id-HBsAg can impact HBV life cycle and HBV pathogenesis, and may justify id-RNAs as important targets for novel antiviral interventions.

抽象的 全球有超过 2.5 亿慢性乙肝病毒携带者。慢性乙型肝炎病毒感染是导致乙型肝炎的主要原因 肝细胞癌（HCC）。乙型肝炎病毒无法治愈。干扰素治疗只能给患者带来暂时的好处 患者的一个子集。尽管 FDA 批准的核苷（酸）类似物可以极大地抑制 HBV 复制， 慢性乙型肝炎病毒感染尚未得到很好的控制。没有一种药物对所有患者都有效，并且能够实现 大多数情况下，表面抗原（HBsAg 或 HBV 包膜蛋白）丢失。乙肝病毒频繁反弹 治疗停止后。乙型肝炎病毒感染的机制尚不完全清楚，这影响了治疗选择。 该提案将分析编码 HBsAg 的 HBV 整合体衍生 RNA (id-RNA) 之间的关系 和乙型肝炎病毒的生命周期。 HBV DNA 整合到宿主基因组中对于 HBV 生命周期并不重要，并且 id-RNA 尚未得到充分研究。使用以下方法分析来自慢性 HBV 携带者的匹配肝脏/HCC 组织 RT-PCR，我们发现，无论 HBV 复制如何：id-RNA 在所有组织中积累，代表 45- 100% 的 HBV RNA 在 60% 的组织中发现（40% 肝脏/80% HCC）；通过隐性 HBV 进行多聚腺苷酸化的 RNA 在 60% 的肝脏/40% 的肝脏中发现了多聚 (A) 信号（可能是整合体衍生的或由 HBV 复制产生的） HCC，并且是 20% 肝脏/20% HCC 中最丰富的 HBV RNA；和复制衍生的 RNA（rd- 使用传统 HBV Poly(A) 信号进行聚腺苷酸化的 RNAs）是 70% 中最不丰富的物种。 组织（40% 肝脏/100% HCC）。此外，通过 RNA 测序对选定组织进行分析还表明 大多数分析样品中 id-RNA 的丰度。数据表明 id-RNA 可能产生显着的 携带 HBV 整合体的细胞中 HBsAg 的分数。如果相同的细胞也支持 HBV 复制，那么就很好 形成的 HBV 病毒粒子的一部分可能会携带 id-RNA 衍生的 HBsAg (id-HBsAg) 作为其主要成分 信封。因此，这些 HBV 病毒粒子的感染性及其支持病毒传播的能力将主要取决于 id-HBsAg 的特性，而不是 HBV 复制衍生的 HBsAg (rd-HBsAg) 的特性。因此，id-RNA 可以调节慢性乙型肝炎病毒感染的维持，这可能需要修订当前的模型 乙肝病毒感染表明乙肝病毒的生命周期与整合无关。还可以看出 抗 HBV 药物无法实现血清 HBsAg 的降低，因为大部分 HBsAg 可以从 id-RNA，与 HBV 复制无关。根据我们的数据，使用大量肝脏/HCC 组织的目标 1 将发现 如果 id-RNA 丰度在慢性 HBV 感染期间很常见。目标 2 和 3 将 (i) 找出大部分 id- RNA 不携带 HBsAg 编码序列的改变，因此可以作为功能性病毒的重要来源。 HBsAg，无论 HBV 复制如何，(ii) 分析 id-RNA 的 3' 端非翻译区（包括 宿主插入）影响 HBsAg 合成，以及 (iii) 找出 id-HBsAg 是否可以有效支持 HBsAg 的组装和感染性 乙型肝炎病毒病毒粒子。该提案将加深我们对 id-RNA 和 id-HBsAg 如何影响 HBV 寿命的理解 循环和 HBV 发病机制，并可能证明 id-RNA 作为新型抗病毒干预措施的重要靶标。