■HBV(和由此产生的肝病)的慢性是由HBV共价闭合环状DNA(cccDNA)的肝内持久性决定的,一种附加形式,编码所有病毒转录本。因此,cccDNA是新疗法的关键靶标,最终的治疗目的是完全消除。虽然已建立的cccDNA分子已知在静息肝细胞中是稳定的,我们的目的是了解他们的命运在分裂的细胞使用体外模型。
■我们用HBV感染HepG2-NTCP和HepaRG-NTCP细胞,并通过传代细胞诱导有丝分裂。我们用野生型HBV测量了cccDNA拷贝数(通过精确的PCR测定)和HBV表达细胞(通过免疫荧光)。我们使用表达荧光素酶或RFP的报告病毒来追踪有丝分裂诱导后HBV表达细胞的数量,分别。
■在所有情况下,我们观察到cccDNA水平急剧下降,HBV阳性细胞数,和cccDNA依赖性蛋白表达后,每轮细胞有丝分裂。还原率与子细胞中完全cccDNA损失(与稀释成)的数学模型高度一致。
■我们的结果与以前的HBV感染动物模型一致,并表明HBV持久性可以通过诱导细胞有丝分裂来有效克服。这些结果支持诱导肝脏更新(例如免疫调节剂)的治疗方法,除了直接作用的抗病毒治疗,以实现乙型肝炎治愈。
慢性乙型肝炎影响3亿人(每年导致884,000人死亡),是无法治愈的。为了治愈它,我们需要从肝脏清除HBV基因组。在这项研究中,我们观察了病毒在细胞分裂后的行为。我们发现它完全清除了病毒,制造2个新的未感染细胞。我们的工作为开发治疗慢性乙型肝炎感染的新方法提供了信息。
UNASSIGNED: The chronicity of HBV (and resultant liver disease) is determined by intrahepatic persistence of the HBV covalently closed circular DNA (cccDNA), an episomal form that encodes all viral transcripts. Therefore, cccDNA is a key target for new treatments, with the ultimate therapeutic aim being its complete elimination. Although established cccDNA molecules are known to be stable in resting hepatocytes, we aimed to understand their fate in dividing cells using in vitro models.
UNASSIGNED: We infected HepG2-NTCP and HepaRG-NTCP cells with HBV and induced mitosis by passaging cells. We measured cccDNA copy number (by precise PCR assays) and HBV-expressing cells (by immunofluorescence) with wild-type HBV. We used reporter viruses expressing luciferase or RFP to track number of HBV-expressing cells over time after mitosis induction using luciferase assays and live imaging, respectively.
UNASSIGNED: In all cases, we observed dramatic reductions in cccDNA levels, HBV-positive cell numbers, and cccDNA-dependent protein expression after each round of cell mitosis. The rates of reduction were highly consistent with mathematical models of a complete cccDNA loss in (as opposed to dilution into) daughter cells.
UNASSIGNED: Our results are concordant with previous animal models of HBV infection and show that HBV persistence can be efficiently overcome by inducing cell mitosis. These results support therapeutic approaches that induce liver turnover (e.g. immune modulators) in addition to direct-acting antiviral therapies to achieve hepatitis B cure.
UNASSIGNED: Chronic hepatitis B affects 300 million people (killing 884,000 per year) and is incurable. To cure it, we need to clear the HBV genome from the liver. In this study, we looked at how the virus behaves after a cell divides. We found that it completely clears the virus, making 2 new uninfected cells. Our work informs new approaches to develop cures for chronic hepatitis B infections.