AASLD 2013:HAPs衣壳蛋白抑制剂影响cccDNA的转录和稳定

StephenW

TITLE: HAPs hepatitis B virus (HBV) capsid inhibitors block core protein interaction with the viral minichromosome and host cell genes and affect cccDNA transcription and stability
AUTHORS (FIRST NAME, LAST NAME): Laura Belloni1, 2, Lichun Li4, Gianna Aurora Palumbo1, 3, Srinivas Reddy Chirapu 5, Ludovica Calvo1, 3, Mg Finn6, Uri Lopatin7, Adam Zlotnick4, 7, Massimo Levrero1, 2
Institutional Author(s):
INSTITUTIONS (ALL): 1. Dept. Internal Medicine (DMISM), Sapienza University Rome, Rome, Italy.
2. Life Nanosciences Laboratory, Sapienza University Rome, Rome, Italy.
3. EAL Inserm U785, Sapienza University Rome, Rome, Italy.
4. Department of Molecular & Cellular Biochemistry, Indiana University, Bloomington, IN 47405 , IN, United States.
5. Department of Chemistry and Biochemistry and The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA, United States.
6. Dept Chemistry, Georgia Institute of Technology, Atlanta, GA 30332, GA, United States.
7. Assembly Pharmaceuticals, Bloomington, IN 47401, IN, United States.
ABSTRACT BODY: Background and aim: The development of novel therapies for HBV infection requires new antivirals that target viral life cycle functions other than the viral polymerase. HBV Core protein (Cp) represents an attractive new therapeutic target. Cp capsid assembly is critical for viral RNA packaging, reverse transcription and intracellular trafficking. Core proteins have been shown to bind the nuclear cccDNA, possibly contributing to the regulation of its function and stability. Hetero-aryl-dihydropyrimidines (HAPs), a new class of antivirals inhibiting HBV replication in vitro and in vivo, enhance the rate and the extent of Cp assembly and, at high concentration, stabilize preferentially non-capsid polymers of Cp. Here we investigated the impact of HAP12 on cccDNA formation, levels and transcription as part of its antiviral activity against HBV.
Methods: Capsid-associated HBV-DNA (TaqMan real-time PCR), cccDNA (TaqMan real-time PCR) and pgRNA levels (quantitative real-time PCR with specific primers), were assessed in: a) HepG2 cells transfected with full length HBV genomes; b) the inducible HepAD38 stable HBV cell line, left untreated or treated with the hetero-aryl-dihydropyrimidine HAP12 at 1-5 microM. Recruitment of HBc and histone modifications on host genes and the viral minichromosome were assessed using standard ChIP and the cccDNA ChIP assay, respectively.
Results: HAP12 treatment of cells transfected with wild type linear HBV genomes showed a complete suppression of HBV replication at 72 and 96 hrs with a peak >50% reduction of pgRNA transcription at 96 hours. The strong HAP12 inhibitory effect on pgRNA transcription and HBV replication was confirmed in the HepAD38 HBV inducible cell line. Following induction of HBV from an integrated transgene, HepAD38 cells have been show to accumulate cccDNA. A sharp, time-dependent reduction of steady state cccDNA levels in HepAD38 cells was observed with HAP12. Additionally, HAP12 treatment both inhibited HBc occupancy of cccDNA in induced HepAD38 cells and reduced cccDNA-bound H3 histone acetylation. Interestingly, HAP12 treatment also reduced H3 histone acetylation and HBc occupancy of the host c-Src oncogene promoter region.
Conclusions: Targeting HBV Cp with HAPs results in inappropriate capsid assembly and function, presumably secondary to conformational changes in Cp oligomers. HAP12 treated cells demonstrate impaired functional capsid formation, reduced viral replication at both the DNA and pgRNA level, as well as altered Cp interaction with both host genes and the HBV cccDNA.

StephenW

背景和目的：乙肝病毒感染的新疗法的发展需要新的抗病毒药物，目标病毒的生命周期以外病毒聚合酶的功能。乙肝病毒核心蛋白（CP）是一个有吸引力的新的治疗目标。 CP衣壳的装配，是病毒RNA包装，反转录和细胞内贩运的关键。核心蛋白已被证明结合核的共价闭合环状DNA ，可能有助于其功能的调节和稳定性。杂芳基 - 二氢嘧啶（ HAPs）等，在体外和体内的抗病毒药物抑制HBV复制的一类新的，增强的速度和Cp的组件的程度，并在高浓度下，优先Cp的非衣壳聚合物的稳定。在这里，我们调查的HAP12 cccDNA的形成，其抗病毒活性，抗HBV的一部分的水平和转录的影响。
方法：衣壳相关HBV - DNA （ TaqMan探针实时荧光定量PCR ） ， cccDNA的（ TaqMan探针实时荧光定量PCR ）和前基因组水平（实时定量PCR特异性引物）进行了评估：1）转染细胞全长HBV基因组， b）该诱导HepAD38 HBV稳定细胞系，不及时治疗或治疗用的杂芳基 - 二氢嘧啶HAP12在1-5 MICROM 。招聘HBc和宿主基因和病毒的微小染色体组蛋白修饰进行了评估使用标准芯片的cccDNA的芯片检测，分别。
结果： HAP12治疗与野生型线性HBV基因组转染的细胞表现出一个完整的抑制HBV复制的峰值在72和96小时> 96小时前基因组转录减少50％ 。强HAP12前基因组的转录和HBV复制的抑制作用被证实在HepAD38 HBV诱导的细胞株。从一个集成诱导HBV转基因， HepAD38细胞已经显示cccDNA的积累。一个尖锐的，时间依赖性降低cccDNA水平在HepAD38细胞稳态观察HAP12 。此外， HAP12治疗既抑制HBC入住的cccDNA的在诱导HepAD38细胞的，降低cccDNA的绑定H3组蛋白乙酰化。有趣的是， HAP12治疗也减少H3组蛋白乙酰化和HBc占用主机C- Src癌基因的启动子区域。
结论：针对HBV CP与的HAPS结果在不适当的衣壳装配和功能，可能发生继发CP寡聚体的构象变化。 HAP12处理的细胞表明减值功能衣壳形成，减少病毒复制的DNA和前基因组水平，以及改变的Cp与宿主基因和HBV cccDNA的相互作用。