Abstract:
Hepatitis B virus (HBV) infects hepatocytes that are in the G0/G1 phase with intact nuclear membrane and organized chromosome architecture. In the nucleus of the infected cells, HBV covalently closed circular (ccc) DNA, an episomal minichromosome, serves as the template for all viral transcripts and the reservoir of persistent infection. Nuclear positioning of cccDNA can be assessed by the spatial distance between viral DNA and host chromosomal DNA through Circular Chromosome Conformation Capture (4C) combined with high-throughput sequencing (4C-seq). The 4C-seq analysis relies on proximity ligation and is commonly used for mapping genomic DNA regions that communicate within a host chromosome. The method has been tailored for studying nuclear localization of HBV episomal cccDNA in relation to the host chromosomes. In this study, we present a step-by-step protocol for 4C-seq analysis of HBV infection, including sample collection and fixation, 4C DNA library preparation, sequence library preparation, and data analysis. Although limited by proximity ligation of DNA fragments, 4C-seq analysis provides useful information of HBV localization in 3D genome, and aids the understanding of viral transcription in light of host chromatin conformation.
摘要:
乙型肝炎病毒(HBV)感染处于G0/G1期的肝细胞,具有完整的核膜和有组织的染色体结构。在受感染细胞的细胞核中,HBV共价闭合环状(cc)DNA,附加型微小染色体,作为所有病毒转录本的模板和持续感染的库。cccDNA的核定位可以通过环状染色体构象捕获(4C)结合高通量测序(4C-seq)通过病毒DNA和宿主染色体DNA之间的空间距离来评估。4C-seq分析依赖于邻近连接,并且通常用于定位在宿主染色体内通信的基因组DNA区域。该方法已被定制用于研究与宿主染色体相关的HBV游离cccDNA的核定位。在这项研究中,我们提出了一个逐步的方案,用于HBV感染的4C-seq分析,包括样本收集和固定,4CDNA文库制备,序列库制备,和数据分析。尽管受到DNA片段的邻近连接的限制,4C-seq分析提供了在3D基因组HBV定位的有用信息,并有助于根据宿主染色质构象理解病毒转录。