Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, New Delhi, India.
Department of Biosciences, Jamia Millia Islamia, New Delhi, India.
J Cell Biochem. 2019 Oct;120(10):17858-17871. doi: 10.1002/jcb.29054. Epub 2019 Jul 16.
Hepatitis B virus (HBV) genome consists of circular partially double stranded DNA of 3.2 kb size which gets converted into covalently closed circular DNA (cccDNA) during its life cycle. It then acts as a template for formation of pregenomicRNA (pgRNA) of 3.5 kb. Absence of appropriate animal models prompted a need to establish a better in vitro culture system to uncover the propagation and survival mechanisms of the virus. There is scarcity of data to represent the significance of varying length of replication competent viral genome on the secretion of viral secretory proteins/antigens and in turn on the overall effects on the accomplishment of the viral life cycle. The present study was undertaken to ascertain a suitable replication competent construct in which the viral life cycle of HBV with varying clinical relevance can be studied efficiently. Two constructs (pHBV 1.3 and pHBV 1X) of different sizes were used to transfect hepatoma cells and consequently the secretory antigens were monitored. In vector free approach (pHBV 1X), 3.2 kb viral DNA is directly transfected in the culture system whereas in vector mediated approach more than full length of viral genome is cloned in a vector (pHBV 1.3X) and transfected to obtain a 3.5 kb pgRNA intermediate. HBV secretes two important antigens; HBsAg and HBeAg. HBsAg is a hallmark of infection and is the first to be secreted in the blood stream whereas HBeAg is a secretory protein and remains associated with the viral replication. The construct pHBV 1.3X referring to as more than full length, by virtue of being capable of undergoing transcription without the synthesis of cccDNA intermediate (unlike the clinical situation where an intermediate step of cccDNA synthesis is an essential component to initiate the viral life cycle) appears to be better system for studying viral life cycle in in vitro culture system. The reasons could be assigned to the fact that as low as 100 ng of viral DNA was shown to quantify the replicative phenotypes with this construct. The better efficiency of this construct at prima facie, appears to be mediated through the significantly higher levels of pgRNA transcript during the viral life cycle.
乙型肝炎病毒 (HBV) 基因组由大小为 3.2kb 的环状部分双链 DNA 组成，在其生命周期中转化为共价闭合环状 DNA (cccDNA)。然后，它作为形成 3.5kb 前基因组 RNA (pgRNA)的模板。由于缺乏适当的动物模型，需要建立更好的体外培养系统来揭示病毒的繁殖和生存机制。缺乏数据来代表具有不同长度的复制能力病毒基因组对病毒分泌蛋白/抗原的分泌的重要性，以及对病毒生命周期完成的整体影响。本研究旨在确定一种合适的复制能力构建体，以便有效地研究具有不同临床相关性的 HBV 病毒生命周期。使用两种不同大小的构建体 (pHBV 1.3 和 pHBV 1X) 转染肝癌细胞，随后监测分泌抗原。在无载体方法 (pHBV 1X) 中，3.2kb 病毒 DNA 直接转染到培养系统中，而在载体介导方法中，将超过全长的病毒基因组克隆到载体 (pHBV 1.3X) 中并转染以获得 3.5kb pgRNA 中间体。HBV 分泌两种重要的抗原；HBsAg 和 HBeAg。HBsAg 是感染的标志，是第一个在血液中分泌的，而 HBeAg 是一种分泌蛋白，与病毒复制相关。构建体 pHBV 1.3X 被称为全长，由于能够在不合成 cccDNA 中间体的情况下进行转录（与临床情况不同，cccDNA 合成的中间步骤是启动病毒生命周期的必要组成部分），因此似乎是体外培养系统中研究病毒生命周期的更好系统。原因可能归因于这样一个事实，即仅用 100ng 病毒 DNA 即可定量该构建体的复制表型。这个构建体的效率更高，这似乎是通过在病毒生命周期中 pgRNA 转录的水平显著提高来介导的。
