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

StephenW

Full-length 5'RACE identifies all major HBV transcripts in HBV-infected hepatocytes and patient serum

    Bernd Stadelmayer
    Audrey Diederichs
    Fleur Chapus
    Kara Carter
    Barbara Testoni
    Fabien Zoulim
    Show all authors

Published: February 19, 2020 DOI: https://doi.org/10.1016/j.jhep.2020.01.028

Highlights

    •
    HBV full-length 5'RACE discriminates all viral transcripts including HBx.
    •
    Viral particles contain pgRNA, pgRNA splice variants and different HBx RNAs.
    •
    HBx RNAs in viral particles are both capped and uncapped.
    •
    The composition of circulating viral RNA species can vary among patients.

Background & Aims
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, ie 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.
Methods
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.
Results
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.
Conclusions
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.
Lay summary
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 .

StephenW

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

    伯恩·斯塔德迈尔
    奥黛丽·狄德瑞希斯（Audrey Diederichs）
    芙蓉沙普斯
    卡拉·卡特
    芭芭拉·泰斯托尼
    法比恩·祖利姆（Fabien Zoulim）
    显示所有作者

发布时间：2020年2月19日DOI：https：//doi.org/10.1016/j.jhep.2020.01.028

强调

    •
    HBV全长5'RACE区分包括HBx在内的所有病毒转录物。
    •
    病毒颗粒包含pgRNA，pgRNA剪接变体和不同的HBx RNA。
    •
    病毒颗粒中的HBx RNA均已加盖和未加盖。
    •
    患者之间循环病毒RNA种类的组成可能有所不同。

背景与目标
共价封闭的环状DNA（cccDNA）是HBV基因组的游离形式，稳定存在于被感染的肝细胞核中。 cccDNA是转录6种主要病毒RNA（即preC，pg，preS1 / 2，S和HBx RNA）的模板。所有病毒转录物共享相同的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，很难深入研究不同病毒转录本的转录。因此，我们希望开发一种可以在任何实验室中轻松检测出病毒RNA完整光谱的方法。
方法
我们建立了HBV全长5'RACE（cDNA末端的快速扩增）方法，通过该方法我们可以测量和表征细胞培养物中和慢性感染患者中病毒RNA的全谱。
结果
除了编码全长X的规范HBx转录本以外，我们还鉴定了可能编码短X蛋白的较短HBx转录本。我们表明，干扰素-β处理可导致preC和pgRNA的强烈减少，但对其他病毒转录本仅具有中等作用。我们发现了pgRNA，1个剪接的pgRNA变体以及与HepAD38细胞产生的病毒颗粒相关的各种HBx转录本。不同的HBx RNA均已封端和未封端。最后，我们确定了慢性HBV感染患者的3种主要循环RNA种类：pgRNA，剪接的pgRNA变异体和HBx。
结论
在感染和治疗过程中，这种HBV全长5'RACE方法应有助于理解HBV转录，并可能指导针对cccDNA的新疗法的开发。
放置摘要
特别是在感染条件下，很难深入研究不同的乙肝病毒转录本。这项研究引入了一种新方法，可以在任何标准实验室中用来区分细胞培养和患者血清中的所有乙型肝炎病毒转录物。

StephenW

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