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

项目概要/摘要 慢性感染B型肝炎病毒（HBV）是世界范围内肝癌的主要原因， 可被B e抗原（HBeAg）通过诱导免疫耐受而促进。以来 HBeAg丢失是一个治疗目标，识别负责HBeAg丢失的宿主酶至关重要。 其生产。而结构相关的核心蛋白（p21; 183 aa）组装成衣壳， 为基因组复制提供场所，HBeAg是一种分泌的可溶性蛋白。最初翻译 作为融合的前核心/核心蛋白（p25; 29+ 183 aa），N末端19 aa将蛋白质靶向至 分泌途径，随后是其分裂。所得p22在C-末端进一步裂解， trans-Golgi网络（TGN）生成成熟HBeAg。细胞系中HBeAg的产生可以 被前蛋白转化酶（PC）抑制剂阻断。存在于TGN中的PC包括弗林蛋白酶、PACE 4和 PC 7，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 顺序在p22的C-末端存在四个这样的基序，其中融合基序1和2 （151 RRGRSPR 157）和多元基序3和4（RRRR）。先前的突变分析确定 HBeAg作为基序1的裂解产物。HBV基因型A具有2-aa插入以将基序2与 motif 1（151 RRDRGRSPR 159），我们发现它产生三种大小的HBeAg。转染 在HepG 2和Huh 7人肝癌细胞系中的实验建立了小、中、 分别作为基序1、2和3（166 RRRR 169）的裂解产物的大形式HBeAg。在弗林- 缺陷LoVo细胞仅产生小的形式。此R21补助金申请的目的是 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。 目的1将确定弗林蛋白酶基因敲除对HepG 2和Huh 7产生HBeAg的影响 细胞亲本细胞和敲除克隆将用基因型A或非基因型B的HBV基因组转染。 A基因型，或感染HBV颗粒。目的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。可选择地， 将PC抑制剂dec-RVKR-cmk加入细胞培养物中。对HBeAg产生的影响， 将在HBV感染后测定基因组复制。考虑到p22可以抑制HBV， 通过与核心蛋白形成混合衣壳来复制DNA，我们还将研究是否被阻断 HBeAg成熟具有抑制HBV DNA复制的额外益处。验证宿主酶 HBeAg的形成和分泌应该为一种新的治疗方法提供一个具体的和不可改变的靶点。 作为有效的弗林蛋白酶/PC抑制剂，已经开发了针对慢性HBV感染的抗病毒方法。

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.