Explore furin as an antiviral target to block hepatitis B virus e antigen production

探索弗林蛋白酶作为抗病毒靶点来阻断乙型肝炎病毒 e 抗原的产生

基本信息

批准号：
10352854
负责人：
SHUPING TONG
金额：
$ 24.59万
依托单位：
RHODE ISLAND HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-09-24 至 2023-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10352854
关键词：
Amino Acids Antiviral Agents Antiviral Therapy Applications Grants Arginine C-terminal CRISPR/Cas technology Capsid Cell Culture Techniques Cell Line Cells Chickens Chronic Chronic Hepatitis B Cleaved cell Codon Nucleotides Core Protein DNA biosynthesis Development Enzymes Excision Genes Genome Genotype Goals HBV Genotype HepG2 Hepatitis B Hepatitis B Core Antigen Hepatitis B Virus Hepatitis B e Antigens Hepatocyte Human Human Herpesvirus 4 Immune Tolerance Immune response In Vitro Integration Host Factors Knock-out Lentivirus Vector Liver Malignant neoplasm of liver Mutation Analysis N-terminal Nucleosome Core Particle Nucleotides Pathway interactions Peptide Signal Sequences Primary carcinoma of the liver cells Production Proprotein Convertases Proteins Risk Series Site Structure Testing Therapeutic Therapeutic Intervention Transfection Translating Translation Initiation Viral Genome Virion Virus Diseases Virus Receptors Virus Replication adeno-associated viral vector anti-hepatitis B base chronic infection established cell line experimental study hepatoma cell immunogenic in vivo inhibitor/antagonist knockout gene novel prevent reconstitution response seroconversion small hairpin RNA stem cells targeted treatment therapeutic target trans-Golgi Network

项目摘要

Project Summary/Abstract Chronic infection by hepatitis B virus (HBV) is a leading cause of liver cancer worldwide, which can be promoted by hepatitis B e antigen (HBeAg) through induction of immune tolerance. Since HBeAg loss is a therapeutic goal, it is critically important to identify the host enzyme responsible for its production. While the structurally related core protein (p21; 183aa) assembles into capsids to provide the venue for genome replication, HBeAg is a secreted soluble protein. It is initially translated as fused precore/core protein (p25; 29+183aa), with the N-terminal 19aa targeting the protein to the secretory pathway followed by its cleavage. The resultant p22 is further cleaved at the C-terminus in the trans-Golgi network (TGN) to generate mature HBeAg. HBeAg production in cell lines can be blocked by a proprotein convertase (PC) inhibitor. PCs present in the TGN include furin, PACE4 and PC7, with most PCs preferring polybasic sequence while furin capable of cleaving after RXXR sequence. Four such motifs are present at the C-terminus of p22, with fused motifs 1 and 2 (151RRGRSPR157) and polybasic motifs 3 and 4 (RRRR). Previous mutational analysis identified HBeAg as cleavage product of motif 1. HBV genotype A has a 2-aa insertion to separate motif 2 from motif 1 (151RRDRGRSPR159), and we found it produced three size forms of HBeAg. Transfection experiments in the HepG2 and Huh7 human hepatoma cell lines established the small, middle, and large forms of HBeAg as cleavage products of motifs 1, 2, and 3 (166RRRR169), respectively. In furin- deficient LoVo cells only the small form was produced. The objective of this R21 grant application is to further evaluate furin as the host factor for HBeAg maturation and a potential therapeutic target. Aim 1 will establish the consequence of furin knockout on HBeAg production from HepG2 and Huh7 cells. Parental cells and knockout clones will be transfected with HBV genomes of genotype A or non- A genotypes, or infected with HBV particles. Aim 2 will establish the consequence of furin silencing or PC inhibition on HBeAg production from a liver progenitor cell line and primary human hepatocytes (PHH). shRNAs against furin will be delivered to differentiated HepaRG cells and PHH. Alternatively, PC inhibitor dec-RVKR-cmk will be added to cell culture. The impact on HBeAg production and genome replication will be determined following HBV infection. Considering that p22 can inhibit HBV DNA replication by forming mixed capsids with core protein, we will also examine whether blocked HBeAg maturation has the added benefit of inhibiting HBV DNA replication. Validating the host enzyme for HBeAg formation and secretion should provide a concrete and non-mutable target for a novel antiviral approach against chronic HBV infection, as potent furin/PC inhibitors have been developed.

项目概要/摘要乙型肝炎病毒（HBV）的慢性感染是全世界肝癌的主要原因，乙型肝炎e抗原（HBeAg）可通过诱导免疫耐受来促进。自从 HBeAg 消失是一个治疗目标，识别负责 HBeAg 的宿主酶至关重要它的生产。而结构相关的核心蛋白 (p21; 183aa) 组装成衣壳以 HBeAg 是一种分泌型可溶性蛋白，为基因组复制提供场所。最初是翻译的作为融合的前核心/核心蛋白 (p25; 29+183aa)，N 端 19aa 将蛋白质靶向分泌途径随后进行裂解。所得 p22 在 C 末端进一步裂解跨高尔基体网络 (TGN) 产生成熟的 HBeAg。细胞系中 HBeAg 的产生可以被前蛋白转化酶 (PC) 抑制剂阻断。 TGN 中存在的 PC 包括 Furin、PACE4 和 PC7，大多数 PC 更喜欢多碱基序列，而弗林蛋白酶能够在 RXXR 后裂解顺序。 p22 的 C 末端存在四个这样的基序，其中融合基序 1 和 2 (151RRGRSPR157) 和多元基序 3 和 4 (RRRR)。先前的突变分析已确定 HBeAg 作为基序 1 的裂解产物。HBV 基因型 A 有一个 2-aa 插入，将基序 2 与基序 1 (151RRDRGRSPR159)，我们发现它产生了三种大小形式的 HBeAg。转染在 HepG2 和 Huh7 人肝癌细胞系中进行的实验建立了小、中和大形式的 HBeAg 分别作为基序 1、2 和 3 (166RRRR169) 的裂解产物。在弗林- 有缺陷的 LoVo 细胞仅产生了较小的形式。此 R21 拨款申请的目标是进一步评估弗林蛋白酶作为 HBeAg 成熟的宿主因子和潜在的治疗靶点。目标 1 将确定弗林蛋白酶敲除对 HepG2 和 Huh7 产生 HBeAg 的影响细胞。亲本细胞和敲除克隆将被基因型 A 或非基因型的 HBV 基因组转染。 A基因型，或感染了HBV颗粒。目标 2 将确定弗林蛋白酶沉默或 PC 对肝祖细胞系和原代人肝细胞产生 HBeAg 的抑制作用（PHH）。针对弗林蛋白酶的 shRNA 将被递送至分化的 HepaRG 细胞和 PHH。或者， PC 抑制剂 dec-RVKR-cmk 将添加到细胞培养物中。对 HBeAg 产生的影响基因组复制将在乙型肝炎病毒感染后确定。考虑到p22可以抑制HBV DNA复制通过与核心蛋白形成混合衣壳，我们还将检查是否被阻断 HBeAg 成熟还有抑制 HBV DNA 复制的额外好处。验证宿主酶 HBeAg 形成和分泌的研究应为新型药物提供具体且不可改变的靶标随着有效的弗林蛋白酶/PC抑制剂的开发，针对慢性乙型肝炎病毒感染的抗病毒方法已被开发出来。