In the present study, high mobility group nucleosome binding domain 1 (HMGN1) was screened out based on our biotin-avidin enrichment system. First, chromatin immunoprecipitation and fluorescent in situ hybridization assays confirmed the binding of HMGN1 with cccDNA in the nucleus. Second, functional experiments in HBV-infected cells showed that the promoting effect of HMGN1 on HBV transcription and replication depended on the functional region of the nucleosomal binding domain, while transfection of the HMGN1 mutant showed no influence on HBV compared with the vector. Third, further mechanistic exploration revealed that the silencing of HMGN1 increased the level of phosphorylase CLK2 and promoted H3 phosphorylation causing the reduced accessibility of cccDNA. Moreover, silenced HMGN1 was mimicked in HBV (r) cccDNA mouse model of HBV infection in vivo. The results showed that silencing HMGN1 inhibited HBV replication in vivo.
In summary, our study identified that a host protein can bind to cccDNA and promote its transcription, providing a candidate strategy for anti-HBV targeting to interfere with the transcriptional activity of cccDNA microchromosomes.
结果:在本研究中,基于我们的生物素-抗生物素蛋白富集系统,筛选出高迁移率基团核小体结合域1(HMGN1)。首先,染色质免疫沉淀和荧光原位杂交测定证实了HMGN1与cccDNA在细胞核中的结合。第二,HBV感染细胞的功能实验表明,HMGN1对HBV转录和复制的促进作用依赖于核小体结合域的功能区,而HMGN1突变体的转染与载体相比对HBV没有影响。第三,进一步的机制探索表明,HMGN1的沉默增加了磷酸化酶CLK2的水平,并促进H3磷酸化,导致cccDNA的可及性降低。此外,在体内HBV感染的HBV(r)cccDNA小鼠模型中模拟沉默的HMGN1。结果表明,沉默HMGN1在体内抑制HBV复制。
结论:总之,我们的研究确定,一个宿主蛋白可以结合到cccDNA,并促进其转录,提供抗HBV靶向干扰cccDNA微染色体转录活性的候选策略。