核心蛋白突变在隐匿性HBV感染发生中的作用

StephenW

Role of core protein mutations in the development of occult HBV infection

    Jingna Chen a
    Bochao Liu a
    Xi Tang
    Chengyao Li
    Linhai Li
    Tingting Li
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Published:January 13, 2021DOI:https://doi.org/10.1016/j.jhep.2020.12.023

   
Highlights

    ●
    Amino acid (aa) substitutions in the core protein (Cp) of occult HBV infection (OBI) strains were identified.
    ●
    An OBI mutant strain SZA carrying nine aa substitutions in Cp was characterized in vitro (cells) and in vivo (mice).
    ●
    The functional impact of individual aa substitutions in pHBV1.3-SZA Cp replicons was assessed in vitro and in vivo.
    ●
    Cp W62R mutation significantly reduced HBV protein production.
    ●
    Cp W62R and its combination mutations might contribute to the occurrence of OBI.

Abstract
Background & Aims
Occult hepatitis B virus (HBV) infection (OBI) is associated with transfusion-transmitted HBV infection and hepatocellular carcinoma. Studies on OBI genesis concentrated on mutations in the S region and the regulatory elements. The role of mutations in the core region of OBIs remains unclear.
Methods
Here, an OBI strain (SZA) carrying 9 amino acid (aa) substitutions in the core protein/capsid (Cp) was selected by sequence alignment and Western blot analysis from 26 genotype B OBI samples to extensively explore the impact of Cp mutations on viral antigen production in vitro and in vivo.
Results
A large panel of 30 Cp replicons were generated by a replication-competent pHBV1.3 carrying SZA or wild-type (wt) Cp in a 1.3-fold over-length of HBV genome, in which the various Cp mutants were individually introduced by repairing site mutations of SZA-Cp or creating site mutations of wt-Cp by site-directed mutagenesis. The expression of HBcAg, HBeAg and HBsAg and viral RNA was quantified from individual SZA and wt Cp mutant replicons in transfected Huh7 cells or infected mice, respectively. An overall comparison between intracellular or extracellular viral protein production related to individual Cp mutants was measured with Western blot, chemiluminescent microparticle immunoassay, immunofluorescent staining or immunohistochemical staining indicated that W62R mutation in Cp had a critical impact on reduction of HBcAg and HBeAg production during HBV replication, while its combined mutations with P50H or/and S74G played a limited role in influencing viral protein production in vivo.
Conclusions
W62R and its combination mutations in HBV Cp might massively affect HBcAg and HBeAg production during viral replication, which, in turn, might contribute to the occurrence of OBI.

StephenW

核心蛋白突变在隐匿性HBV感染发生中的作用

    陈静娜
    刘伯超
    西塘
    李成耀
    李林海
    李婷婷
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发布时间：2021年1月13日DOI：https：//doi.org/10.1016/j.jhep.2020.12.023

   
强调

    ●
    确定了隐性HBV感染（OBI）菌株核心蛋白（Cp）中的氨基酸（aa）替代。
    ●
    在体外（细胞）和体内（小鼠）表征了在Cp中带有9个氨基酸取代的OBI突变株SZA。
    ●
    在体外和体内评估了pHBV1.3-SZA Cp复制子中单个氨基酸取代的功能影响。
    ●
    Cp W62R突变显着降低了HBV蛋白的产生。
    ●
    Cp W62R及其组合突变可能有助于OBI的发生。

抽象
背景与目标
隐匿性乙型肝炎病毒（HBV）感染（OBI）与输血传播的HBV感染和肝细胞癌有关。 OBI起源的研究集中于S区的突变和调控元件。突变在OBIs核心区域的作用尚不清楚。
方法
在这里，通过序列比对和蛋白质印迹分析，从26个基因型B OBI样本中选择了在核心蛋白/衣壳（Cp）中带有9个氨基酸（aa）取代的OBI菌株（SZA），以广泛研究Cp突变对病毒的影响体内外抗原产生。
结果
由具有复制能力的pHBV1.3在HBV基因组的1.3倍超长长度中携带SZA或野生型（wt）Cp产生了一大批30 Cp复制子，其中通过修复分别引入了各种Cp突变体SZA-Cp的位点突变或通过定点诱变创建wt-Cp的位点突变。分别从转染的Huh7细胞或感染的小鼠中的单个SZA和wt Cp突变体复制子中定量了HBcAg，HBeAg和HBsAg以及病毒RNA的表达。通过蛋白质印迹，化学发光微粒免疫测定，免疫荧光染色或免疫组化染色测量了与单个Cp突变体相关的细胞内或细胞外病毒蛋白产量的总体比较，表明Cp中的W62R突变对HBV复制过程中HBcAg和HBeAg产量的降低具有关键影响，而其与P50H或/和S74G的组合突变在影响体内病毒蛋白产生中的作用有限。
结论
在病毒复制期间，W62R及其在HBV Cp中的组合突变可能会严重影响HBcAg和HBeAg的产生，进而可能导致OBI的发生。