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：
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
项目类别：
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

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

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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.

项目摘要/摘要乙型肝炎病毒（HBV）的慢性感染是全球肝癌的主要原因，这可以通过诱导免疫耐受性来促进丙型肝炎抗原（HBEAG）。自从 HBEAG损失是一个治疗目标，确定负责的宿主酶非常重要它的生产。而与结构相关的核心蛋白（p21; 183aA）聚集成帽膜提供基因组复制的场所，HBEAG是一种分泌的可溶性蛋白。它最初是翻译的作为融合的前蛋白/核心蛋白（p25; 29+183AA），n末端19AA将蛋白靶向分泌途径随后是乳沟。在C-末端进一步切割结果的P22 trans-golgi网络（TGN）生成成熟的HBEAG。细胞系中的HBEAG生产可以是被普洛蛋白转化酶（PC）抑制剂阻塞。 TGN中存在的PC包括Furin，Pace4和 PC7，大多数PC都偏爱多重序列，而FURIN能够在RXXR后切割顺序。 p22的C末端存在四个这样的基序，其中有融合的图案1和2 （151RRGRSPR157）和多重基主题3和4（rrrr）。以前的突变分析确定 HBEAG作为基序1的裂解产物。HBV基因型A具有2-AA的插入，可将基序2与图案1（151rrdrgrspr159），我们发现它产生了三种大小的hbeag。转染 HEPG2和HUH7人肝癌细胞系中的实验确定了小，中和大型的HBEAG作为基序1、2和3（166rrrrrrrrrrrrrrrr169）的裂解产物。在弗林 - 缺乏LOVO细胞仅产生小型形式。此R21赠款申请的目的是进一步评估Furin作为HBEAG成熟的宿主因素和潜在的治疗靶点。 AIM 1将确定Furin淘汰赛对HEPG2和HUH7的HBEAG生产的后果细胞。亲本细胞和敲除克隆将用基因型A或非基因型的HBV基因组转染一种基因型，或感染了HBV颗粒。 AIM 2将确定弹性沉默的后果或 PC抑制肝脏祖细胞系和原代人肝细胞的HBEAG产生（PHH）。针对纤维蛋白的shRNA将被输送到分化的HEPARG细胞和PHH中。或者， PC抑制剂DEC-RVKR-CMK将添加到细胞培养中。对HBEAG生产的影响 HBV感染后将确定基因组复制。考虑到P22可以抑制HBV DNA复制通过用核心蛋白形成混合的衣壳，我们还将检查是否阻塞 HBEAG成熟具有抑制HBV DNA复制的额外优势。验证宿主酶对于hbeag的形成和分泌，应为新颖的靶标提供一个具体的和不可刺激的目标由于已经开发了有效的FURIN/PC抑制剂，抗病毒方法针对慢性HBV感染。