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Full-length 5'RACE identifies all major HBV transcripts in HBV-infected hepatocytes and patient serum
Journal of Hepatology ( IF 26.8 ) Pub Date : 2020-02-20 , DOI: 10.1016/j.jhep.2020.01.028
Bernd Stadelmayer 1 , Audrey Diederichs 1 , Fleur Chapus 1 , Michel Rivoire 2 , Gregory Neveu 3 , Antoine Alam 3 , Laurent Fraisse 3 , Kara Carter 3 , Barbara Testoni 1 , Fabien Zoulim 4
Affiliation  

Covalently closed circular DNA (cccDNA) is the episomal form of the HBV genome that stably resides in the nucleus of infected hepatocytes. cccDNA is the template for the transcription of 6 major viral RNAs, preC, pg, preS1/2, S and HBx RNA. All viral transcripts share the same 3' end and are all to various degrees subsets of each other. Especially under infection conditions, it has been difficult to study in depth the transcription of the different viral transcripts. We thus wanted to develop a method with which we could easily detect the full spectrum of viral RNAs in any lab. We set up an HBV full-length 5'RACE (rapid amplification of cDNA ends) method with which we measured and characterized the full spectrum of viral RNAs in cell culture and in chronically infected patients. In addition to canonical HBx transcripts coding for full-length X, we identified shorter HBx transcripts potentially coding for short X proteins. We showed that interferon-β treatment leads to a strong reduction of preC and pgRNAs but has only a moderate effect on the other viral transcripts. We found pgRNA, 1 spliced pgRNA variant and a variety of HBx transcripts associated with viral particles generated by HepAD38 cells. The different HBx RNAs are both capped and uncapped. Lastly, we identified 3 major categories of circulating RNA species in patients with chronic HBV infection: pgRNA, spliced pgRNA variants and HBx. This HBV full-length 5'RACE method should significantly contribute to the understanding of HBV transcription during the course of infection and therapy and may guide the development of novel therapies aimed at targeting cccDNA. Especially under infection conditions, it has been difficult to study the different hepatitis B virus transcripts in depth. This study introduces a new method that can be used in any standard lab to discriminate all hepatitis B viral transcripts in cell culture and in the serum of patients.

中文翻译:


全长 5'RACE 可识别 HBV 感染的肝细胞和患者血清中的所有主要 HBV 转录本



共价闭合环状 DNA (cccDNA) 是 HBV 基因组的游离形式,稳定存在于受感染肝细胞的细胞核中。 cccDNA 是 6 种主要病毒 RNA(preC、pg、preS1/2、S 和 HBx RNA)转录的模板。所有病毒转录本都具有相同的 3' 末端,并且在不同程度上都是彼此的子集。特别是在感染条件下,深入研究不同病毒转录本的转录一直很困难。因此,我们希望开发一种方法,可以在任何实验室轻松检测全谱病毒 RNA。我们建立了一种 HBV 全长 5'RACE(cDNA 末端快速扩增)方法,用于测量和表征细胞培养物和慢性感染患者中的全谱病毒 RNA。除了编码全长 X 的规范 HBx 转录本外,我们还发现了可能编码短 X 蛋白的较短 HBx 转录本。我们发现,干扰素-β 治疗会导致 preC 和 pgRNA 大幅减少,但对其他病毒转录物仅产生中等影响。我们发现了 pgRNA、1 个剪接的 pgRNA 变体和多种与​​ HepAD38 细胞产生的病毒颗粒相关的 HBx 转录本。不同的 HBx RNA 有加帽的和无帽的。最后,我们确定了慢性 HBV 感染患者中 3 个主要的循环 RNA 种类:pgRNA、剪接 pgRNA 变异体和 HBx。这种 HBV 全长 5'RACE 方法应显着有助于理解感染和治疗过程中的 HBV 转录,并可能指导针对 cccDNA 的新疗法的开发。尤其是在感染条件下,深入研究乙型肝炎病毒的不同转录本一直很困难。 这项研究引入了一种新方法,可在任何标准实验室中使用,以区分细胞培养物和患者血清中的所有乙型肝炎病毒转录本。
更新日期:2020-02-20
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