Worldwide, ~250 million people are chronically infected with the hepatitis B virus (HBV) and are at increased risk of cirrhosis and hepatocellular carcinoma. The HBV persists as covalently closed circular DNA (cccDNA), which acts as the template for all HBV mRNA transcripts. Nucleos(t)ide analogs do not directly target the HBV cccDNA and cannot eradicate the HBV. We have discovered a unique structural motif, a G-quadruplex in HBVs pre-core promoter region that is conserved amongst nearly all genotypes, and is central to critical steps in the viral life-cycle including the production of pre-genomic RNA, core and polymerase proteins, and encapsidation. Thus, an increased understanding of the HBV pre-core may lead to the development of novel anti-HBV cccDNA targets. We utilized biophysical methods to characterize the presence of the G-quadruplex, employed assays using a known quadruplex-binding protein (DHX36) to pull-down HBV cccDNA, and compared HBV infection in HepG2 cells transfected with wild-type and mutant HBV plasmids. This study provides insights into the presence of a G-quadruplex in the HBV pre-core promoter region essential for HBV replication. The evaluation of this critical host-protein interaction site in the HBV cccDNA may ultimately facilitate the development of novel anti-HBV therapeutics against the resilient cccDNA template.

中文翻译：

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乙型肝炎病毒前核心启动子区G-四链体结构的鉴定和表征

在全球范围内，约有2.5亿人长期感染乙型肝炎病毒（HBV），肝硬化和肝细胞癌的风险增加。HBV以共价闭合的环状DNA（cccDNA）的形式持续存在，它充当所有HBV mRNA转录本的模板。核苷酸类似物不能直接靶向HBV cccDNA，也不能根除HBV。我们发现了一个独特的结构基序，即HBV前核心启动子区域的G-四链体，几乎在所有基因型中都保守，并且是病毒生命周期关键步骤（包括产生前基因组RNA，核心基因和聚合酶蛋白和衣壳化。因此，对HBV前核的深入了解可能会导致开发新的抗HBV cccDNA靶标。我们利用生物物理方法来表征G-四链体的存在，使用已知的四链体结合蛋白（DHX36）提取HBV cccDNA的检测方法，并比较了用野生型和突变HBV质粒转染的HepG2细胞中的HBV感染情况。这项研究提供了有关HBV复制前必不可少的HBV前核心启动子区域中G四联体的见解。对HBV cccDNA中这个关键的宿主-蛋白质相互作用位点的评估可能最终有助于开发针对弹性cccDNA模板的新型抗HBV治疗药物。