乙型肝炎病毒的天然preS1缺失突变体丢失的小包膜蛋白表达常

StephenW

Lost small envelope protein expression from naturally occurring preS1 deletion mutants of hepatitis B virus is often accompanied by increased HBx and core protein expression as well as genome replication
Shuwen Fu  1 , Jing Zhang  1 , Quan Yuan  2 , Qianru Wang  1 , Qiang Deng  1 , Jisu Li  3 , Ningshao Xia  2 , Yongxiang Wang  4 , Yumei Wen  1 , Shuping Tong  4   3
Affiliations
Affiliations

    1
    Department of Pathobiology, Key Laboratory of Medical Molecular Virology, School of Basic Medical Sciences, Fudan University, Shanghai, China.
    2
    State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, National Institute of Diagnostics and Vaccine Development in Infectious Diseases, School of Public Health, Xiamen University, Xiamen, China.
    3
    Liver Research Center, Rhode Island Hospital and Warren Alpert Medical School of Brown University, Providence, Rhode Island, USA.
    4
    Department of Pathobiology, Key Laboratory of Medical Molecular Virology, School of Basic Medical Sciences, Fudan University, Shanghai, China [email protected] [email protected].

    PMID: 33910956 DOI: 10.1128/JVI.00660-21

Abstract

Hepatitis B virus (HBV) transcribes co-terminal mRNAs of 0.7-3.5kb from the 3.2-kb covalently closed circular (ccc) DNA, with the 2.1-kb RNA being most abundant. The 0.7-kb RNA produces HBx protein, a transcriptional transactivator, while the 3.5-kb pgRNA drives core and P protein translation as well as genome replication. The large (L) and small (S) envelope proteins are translated from the 2.4-kb and 2.1-kb RNAs, respectively, with majority of the S protein secreted as noninfectious subviral particles and detected as hepatitis B surface antigen (HBsAg). pgRNA transcription could inhibit transcription of subgenomic RNAs. The present study characterized naturally occurring in-frame deletions in the 3' preS1 region, which not only codes for L protein but also serves as the promoter for 2.1-kb RNA. Human hepatoma cell line Huh7 was transiently transfected with subgenomic expression constructs for envelope (and HBx) proteins, dimeric construct, or one mimicking cccDNA. The results confirmed lost 2.1-kb RNA transcription and HBsAg production from many deletion mutants, which was accompanied by increased other (especially 2.4-kb) RNAs, intracellular HBx and core proteins, replicative DNA, but impaired virion and L protein secretion. Highest intracellular L protein was rather achieved by mutants with residual S protein expression or retaining matrix domain in L protein. Site-directed mutagenesis of a high replicating deletion mutant suggested that increased HBx protein expression and blocked virion secretion both contributed to high replication phenotype. Our findings could help explain why such deletions are selected at late stage of chronic HBV infection and how they contribute to viral pathogenesis.IMPORTANCEExpression of hepatitis B e antigen (HBeAg) and overproduction of hepatitis B surface antigen (HBsAg) by wild-type hepatitis B virus (HBV) are implicated in the induction of immune tolerance to achieve chronic infection. How HBV survives the subsequent immune clearance phase remains incompletely understood. Our previous characterization of core promoter mutations to reduce HBeAg production revealed ability of the 3.5-kb pgRNA to diminish transcription of co-terminal RNAs of 2.4kb, 2.1kb, and 0.7kb. The later stage of chronic HBV infection often selects for in-frame deletions in the preS region. Here we found that many 3' preS1 deletions prevented transcription of the 2.1-kb RNA for HBsAg production, which was often accompanied by increased intracellular 3.5-, 0.7-, and especially 2.4-kb RNAs, HBx and core proteins, replicative DNA, but lost virion secretion. These findings established biological consequences of preS1 deletions, thus shedding light on why they are selected and how they contribute to hepatocarcinogenesis.

Copyright © 2021 Fu et al.

StephenW

乙型肝炎病毒的天然preS1缺失突变体丢失的小包膜蛋白表达常常伴随着HBx和核心蛋白表达的增加以及基因组复制
傅树文1，张静1，全院2，王谦如1，邓强1，李继苏3，宁少霞2，王永祥4，文玉梅1，树屏堂4 3
隶属关系
隶属关系

    1个
    复旦大学基础医学院，病理生物学教研室，医学分子病毒学重点实验室，上海
    2个
    厦门大学公共卫生学院，国家传染病诊断和疫苗开发研究所，分子疫苗学和分子诊断学国家重点实验室
    3
    美国罗得岛普罗维登斯布朗大学罗德岛医院肝脏研究中心和沃伦·阿尔珀特医学院。
    4
    复旦大学基础医学院，病理生物学教研室，医学分子病毒学重点实验室，上海[email protected] [email protected]。

    PMID：33910956 DOI：10.1128 / JVI.00660-21

抽象的

乙型肝炎病毒（HBV）从3.2-kb共价闭合的环状（ccc）DNA转录0.7-3.5kb的共末端mRNA，其中2.1-kb RNA最为丰富。 0.7-kb RNA产生HBx蛋白，一种转录反式激活因子，而3.5-kb pgRNA驱动核心和P蛋白翻译以及基因组复制。大（L）和小（S）包膜蛋白分别从2.4 KB和2.1 KB RNA进行翻译，其中大多数S蛋白以非感染性亚病毒颗粒的形式分泌，并被检测为乙型肝炎表面抗原（HBsAg）。 pgRNA转录可以抑制亚基因组RNA的转录。本研究的特征是在3'preS1区域中自然发生的框内缺失，该缺失不仅编码L蛋白，而且还充当2.1 KB RNA的启动子。用包膜（和HBx）蛋白的亚基因组表达构建体，二聚体构建体或一种模拟cccDNA瞬时转染人肝癌细胞系Huh7。结果证实，许多缺失突变体丧失了2.1-kb RNA转录和HBsAg的产生，并伴随着其他（尤其是2.4-kb）RNA，细胞内HBx和核心蛋白，复制性DNA的增加，但病毒体和L蛋白的分泌受损。最高的细胞内L蛋白是通过具有残留S蛋白表达或在L蛋白中保留基质结构域的突变体实现的。高复制缺失突变体的定点诱变表明，HBx蛋白表达增加和病毒体分泌受阻均促成高复制表型。我们的发现可以帮助解释为什么在慢性HBV感染的晚期选择此类缺失以及它们如何导致病毒发病机理。乙型肝炎病毒（HBV）参与诱导免疫耐受以实现慢性感染。 HBV如何在随后的免疫清除阶段中生存仍然不完全清楚。我们先前对降低HBeAg产生的核心启动子突变的表征揭示了3.5-kb pgRNA减少2.4kb，2.1kb和0.7kb共端RNA转录的能力。慢性HBV感染的后期阶段通常选择preS区域内的框内缺失。在这里，我们发现许多3'preS1缺失阻止了HBsAg产生的2.1-kb RNA的转录，这通常伴随着细胞内3.5-，0.7-尤其是2.4-kb RNA，HBx和核心蛋白，复制性DNA的增加，但是病毒体分泌丢失。这些发现确定了preS1缺失的生物学结果，从而阐明了为什么选择它们以及它们如何促进肝癌的发生。

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