Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA; Cancer Virology Program, UPMC Hillman Cancer Center, Pittsburgh, PA, USA; Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, IN, USA.
Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, IN, USA.
Antiviral Res. 2023 Mar;211:105552. doi: 10.1016/j.antiviral.2023.105552. Epub 2023 Feb 1.
HBV cccDNA is the persistent form of viral genome, which exists in host cell nucleus as an episomal minichromosome decorated with histone and non-histone proteins. cccDNA is the authentic viral transcription template and resistant to current antivirals. Growing evidence shows that the transcriptional activity of cccDNA minichromosome undergoes epigenetic regulations, suggesting a new perspective for anti-cccDNA drug development through targeting histone modifications. In this study, we screened an epigenetic compound library in the cccDNA reporter cell line HepBHAe82, which produces the HA-tagged HBeAg in a cccDNA-dependent manner. Among the obtained hits, a bromodomain-containing protein 4 (BRD4) inhibitor MS436 exhibited marked inhibition of cccDNA transcription in both HBV stable cell line HepAD38 and HepG2-NTCP or primary human hepatocyte infection system under noncytotoxic concentrations. Chromatin immunoprecipitation (ChIP) assay demonstrated that MS436 dramatically reduced the enrichment of H3K27ac, an activating histone modification pattern, on cccDNA minichromosome. RNAseq differential analysis showed that MS436 does not drastically change host transcriptome or induce any known anti-HBV factors/pathways, indicating a direct antiviral effect of MS436 on cccDNA minichromosome. Interestingly, the MS436-mediated inhibition of cccDNA transcription is accompanied by cccDNA destabilization in HBV infection and a recombinant cccDNA system, indicating that BRD4 activity may also play a role in cccDNA maintenance. Furthermore, depletion of BRD4 by siRNA knockdown or PROTAC degrader resulted in cccDNA inhibition in HBV-infected HepG2-NTCP cells, further validating BRD4 as an antiviral target. Taken together, our study has demonstrated the practicability of HepBHAe82-based anti-HBV drug screening system and provided a proof-of-concept for targeting HBV cccDNA with epigenetic compounds.
HBV cccDNA 是病毒基因组的持续性形式，它以附着组蛋白和非组蛋白的小染色体形式存在于宿主细胞的细胞核内。cccDNA 是病毒真实的转录模板，并且对当前的抗病毒药物具有抗性。越来越多的证据表明，cccDNA 小染色体的转录活性受到表观遗传调控，这表明通过靶向组蛋白修饰来开发抗 cccDNA 药物具有新的前景。在这项研究中，我们在 HepBHAe82 cccDNA 报告细胞系中筛选了一个表观遗传化合物文库，该细胞系以 cccDNA 依赖的方式产生 HA 标记的 HBeAg。在所获得的命中物中，一种含有溴结构域蛋白 4（BRD4）的抑制剂 MS436 在非细胞毒性浓度下，在 HBV 稳定细胞系 HepAD38 和 HepG2-NTCP 或原代人肝细胞感染系统中，对 cccDNA 转录表现出明显的抑制作用。染色质免疫沉淀（ChIP）实验表明，MS436 显著降低了 cccDNA 小染色体上 H3K27ac 的富集，H3K27ac 是一种激活组蛋白修饰模式。RNAseq 差异分析表明，MS436 不会剧烈改变宿主转录组或诱导任何已知的抗 HBV 因子/途径，表明 MS436 对 cccDNA 小染色体具有直接的抗病毒作用。有趣的是，在 HBV 感染和重组 cccDNA 系统中，MS436 介导的 cccDNA 转录抑制伴随着 cccDNA 的不稳定性，这表明 BRD4 活性也可能在 cccDNA 的维持中发挥作用。此外，siRNA 敲低或 PROTAC 降解剂耗尽 BRD4 会导致 HBV 感染的 HepG2-NTCP 细胞中的 cccDNA 抑制，进一步验证了 BRD4 作为抗病毒靶点的作用。总之，我们的研究证明了基于 HepBHAe82 的抗 HBV 药物筛选系统的实用性，并为使用表观遗传化合物靶向 HBV cccDNA 提供了概念验证。
