Jiang Linshan, Peng Lanlang, Chen Hong, Liao Fangli, Yang Liping, Ye Yuanyuan, Zhang Gaoli, Sun Hua, Hu Qin, Chen Weixian
Department of Laboratory Medicine, The Second Affiliated Hospital of Chongqing Medical University, No.74 Linjing Road, Yuzhong District, Chongqing, 400010, China.
Virol J. 2025 Jun 11;22(1):189. doi: 10.1186/s12985-025-02820-9.
Chronic hepatitis B virus (HBV) infection remains a serious public health challenge, and primary treatment strategies are unable to cure HBV infection due to the persistence of HBV covalently closed circular DNA (cccDNA) in the nuclei of cells. The HBV X protein (HBx) plays a crucial role in regulating cccDNA transcription. Therefore, targeting HBx to identify host proteins that regulate cccDNA transcription could represent a curative approach.
Here, we used co-immunoprecipitation (co-IP) and mass spectrometry (MS) to identify proteins that interact with HBx. The candidate proteins Lamin A and C (Lamin A/C) were knocked down by RNA interference, and Lamin A/C was overexpressed via lentiviral vectors in HBV-infected cell cultures. Chromatin immunoprecipitation (ChIP) followed by quantitative PCR (ChIP-qPCR) was used to investigate protein-DNA interactions.
A mechanistic study revealed that Lamin A/C bound to HBx, resulting in reduced degradation of HBx by the 26S proteasome. Moreover, Lamin A/C regulated HBV cccDNA transcription in vitro, which was associated with HBx. In addition, Lamin A/C knockdown resulted in transcriptional silencing of the HBV cccDNA but not in HBV-HBx-deficient (∆HBx)-infected cells. Importantly, experimental knockdown of Lamin A/C reduced the level of active histone modifications (H3K4me3 and H3k27Ac) bound to cccDNA and increased the level of inhibited histone modifications (H3K9me3 and H3K27me3) bound to cccDNA, which was suppressed in the HBV-∆HBx system.
Our findings reveal that Lamin A/C acts as a new host factor for HBV cccDNA through an epigenetic regulatory mechanism that can interact with HBx and prevent its degradation.
慢性乙型肝炎病毒（HBV）感染仍然是一项严峻的公共卫生挑战，由于HBV共价闭合环状DNA（cccDNA）在细胞核中持续存在，主要治疗策略无法治愈HBV感染。HBV X蛋白（HBx）在调节cccDNA转录中起关键作用。因此，靶向HBx以鉴定调节cccDNA转录的宿主蛋白可能是一种治愈方法。
在此，我们使用免疫共沉淀（co-IP）和质谱（MS）来鉴定与HBx相互作用的蛋白质。通过RNA干扰敲低候选蛋白核纤层蛋白A和C（Lamin A/C），并通过慢病毒载体在HBV感染的细胞培养物中过表达Lamin A/C。采用染色质免疫沉淀（ChIP）结合定量PCR（ChIP-qPCR）来研究蛋白质-DNA相互作用。
一项机制研究表明，Lamin A/C与HBx结合，导致26S蛋白酶体对HBx的降解减少。此外，Lamin A/C在体外调节HBV cccDNA转录，这与HBx相关。此外，敲低Lamin A/C导致HBV cccDNA转录沉默，但在HBV-HBx缺陷（∆HBx）感染的细胞中未出现这种情况。重要的是，实验性敲低Lamin A/C降低了与cccDNA结合的活性组蛋白修饰（H3K4me3和H3k27Ac）水平，并增加了与cccDNA结合的抑制性组蛋白修饰（H3K9me3和H3K27me3）水平，这在HBV-∆HBx系统中受到抑制。
我们的研究结果表明，Lamin A/C通过一种表观遗传调控机制作为HBV cccDNA的新宿主因子，该机制可与HBx相互作用并防止其降解。
