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

• 批准号: 10495261
• 负责人: SHUPING TONG
• 金额: $ 20.5万
• 依托单位: RHODE ISLAND HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-24 至 2024-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10495261
• 关键词: Amino Acids; Antiviral Therapy; Applications Grants; Arginine; C-terminal; CRISPR/Cas technology; Capsid; Cell Culture Techniques; Cell Line; Cells; Chickens; Chronic; Chronic Hepatitis B; Codon Nucleotides; Core Protein; DNA biosynthesis; Development; Enzymes; Excision; Genes; Genome; Genotype; Goals; HBV Genotype; HepG2; Hepatitis B; Hepatitis B Core Antigen; Hepatitis B Infection; 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; Persons; Primary carcinoma of the liver cells; Production; Proprotein Convertases; Proteins; Risk; Series; Site; 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; knockout gene; novel; prevent; reconstitution; response; seroconversion; small hairpin RNA; stem cells; targeted treatment; therapeutic target; trans-Golgi Network

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.

项目摘要/摘要 慢性乙肝病毒感染是世界范围内导致肝癌的主要原因。 可由乙肝e抗原(HBeAg)通过诱导免疫耐受而促进。自.以来 HBeAg丢失是一个治疗目标，确定与HBeAg丢失有关的宿主酶至关重要 它的制作。而结构上相关的核心蛋白(p21；183aa)组装成衣壳 HBeAg是一种分泌型可溶性蛋白质，为基因组复制提供了场所。它最初是翻译的 作为融合的前C/核心蛋白(p25；29+183aa)，N-末端19AA将该蛋白靶向 分泌途径和它的裂解。得到的p22在C-末端被进一步切割 反式高尔基体网络(TGN)产生成熟的HBeAg。在细胞系中产生HBeAg可以是 被原蛋白转换酶(PC)抑制剂阻断。TGN中存在的PC包括Furin、PACE4和 PC7，大多数PC偏好多碱基序列，而Furin能够在RXXR之后切割 序列。在p22的C末端存在四个这样的基序，具有融合基序1和2 (151RRGRSPR157)和多碱基序3和4(RRRR)。先前的突变分析确定 HBeAg是基序1的切割产物。HBVA型有一个2-AA插入，以将基序2与 基序1(151RRDRGRSPR159)，我们发现它产生了三种大小的HBeAg。转染法 实验在HepG2和HuH7人肝癌细胞系中建立了小、中、小和 大形式的HBeAg分别作为基序1、2和3的切割产物(166RRRR169)。在弗林-- 缺陷型LoVo细胞仅产生小的形态。这项R21拨款申请的目标是 进一步评价呋喃西林作为HBeAg成熟的宿主因子和潜在的治疗靶点。 目标1将确定Furin基因敲除对HepG2和Huh7产生HBeAg的影响 细胞。亲本细胞和基因敲除克隆将被A型或非A型乙肝病毒基因组感染 A基因分型，或感染了乙肝病毒颗粒。目标2将确定呋喃类消音剂或 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Furin的shRNA将被输送到分化的HepaRG细胞和PHH。或者， PC抑制剂Dec-RVKR-CMK将加入到细胞培养中。对HBeAg生产和生产的影响 基因组复制将在乙肝病毒感染后确定。考虑到p22可以抑制乙肝病毒 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HBVDNA复制的额外好处。验证宿主酶 对于HBeAg的形成和分泌应该为新的 作为一种有效的Furin/PC抑制剂，已经开发出一种针对慢性乙肝病毒感染的抗病毒方法。