目的:转录终止微调基因表达并有助于确定RNA在真核细胞中的功能。乙型肝炎病毒(HBV)的转录终止受到所有病毒转录本共有的经典聚腺苷酸化信号(cPAS)的识别。目前尚不清楚这种cPAS的使用及其对病毒基因表达和复制的影响。
结果:要解开HBV转录本终止的调节,我们在体外感染的肝细胞和慢性感染的患者中实施了3'RACE-PCR分析与单分子测序相结合。先前未鉴定的转录连读的检测表明cPAS在体外和体内HBV复制期间未被系统地识别。基因表达下调实验证明了RNA解旋酶DDX5和DDX17在促进病毒转录连读中的作用,那是,反过来,与HBVRNA失稳和HBx蛋白表达降低相关。RNA和染色质免疫沉淀,连同cPAS序列的突变,表明DDX5和DDX17在将cPAS识别与转录连读功能连接中的直接作用,HBVRNA稳定性和复制。
结论:我们的发现将DDX5和DDX17确定为HBV转录保真度的关键决定因素和HBV复制的宿主限制因子。
■乙型肝炎病毒(HBV)共价闭合环状(ccc)DNA降解或功能失活仍然是实现HBV治愈的圣杯。转录保真度是基因表达调控的基石。这里,我们证明了两个解旋酶,DDX5和DDX17,抑制HBV多腺苷酸化信号的识别和转录终止,从而降低HBVRNA的稳定性,并作为有效的cccDNA转录和病毒复制的限制因子。DDX5和DDX17在HBV慢性感染患者中下调的观察表明解旋酶在体内HBV持久性中的作用。这些结果为旨在确定中和cccDNA转录的新靶标的研究人员开辟了新的视角。
OBJECTIVE: Transcription termination fine-tunes gene expression and contributes to the specification of RNA function in eukaryotic cells. Transcription termination of HBV is subject to the recognition of the canonical polyadenylation signal (cPAS) common to all viral transcripts. However, the regulation of this cPAS and its impact on viral gene expression and replication is currently unknown.
METHODS: To unravel the regulation of HBV transcript termination, we implemented a 3\' RACE (rapid amplification of cDNA ends)-PCR assay coupled to single molecule sequencing both in in vitro-infected hepatocytes and in chronically infected patients.
RESULTS: The detection of a previously unidentified transcriptional readthrough indicated that the cPAS was not systematically recognized during HBV replication in vitro and in vivo. Gene expression downregulation experiments demonstrated a role for the RNA helicases DDX5 and DDX17 in promoting viral transcriptional readthrough, which was, in turn, associated with HBV RNA destabilization and decreased HBx protein expression. RNA and chromatin immunoprecipitation, together with mutation of the cPAS sequence, suggested a direct role of DDX5 and DDX17 in functionally linking cPAS recognition to transcriptional readthrough, HBV RNA stability and replication.
CONCLUSIONS: Our findings identify DDX5 and DDX17 as crucial determinants of HBV transcriptional fidelity and as host restriction factors for HBV replication.
UNASSIGNED: HBV covalently closed circular (ccc)DNA degradation or functional inactivation remains the holy grail for the achievement of HBV cure. Transcriptional fidelity is a cornerstone in the regulation of gene expression. Here, we demonstrate that two helicases, DDX5 and DDX17, inhibit recognition of the HBV polyadenylation signal and thereby transcriptional termination, thus decreasing HBV RNA stability and acting as restriction factors for efficient cccDNA transcription and viral replication. The observation that DDX5 and DDX17 are downregulated in patients chronically infected with HBV suggests a role for these helicases in HBV persistence in vivo. These results open new perspectives for researchers aiming at identifying new targets to neutralise cccDNA transcription.