热休克蛋白刺激乙型肝炎病毒中APOBEC-3介导的胞苷脱氨

StephenW

Heat shock proteins stimulate APOBEC-3-mediated cytidine deamination in the hepatitis B virus

    Zhigang Chen1, Thomas L. Eggerman2, Alexander V. Bocharov1, Irina N. Baranova1, Tatyana G. Vishnyakova1, Roger Kurlander1 and Amy P. Patterson3*

    1 Clinical Center, National Institutes of Health, United States;
    2 National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, United States;
    3 National Heart, Lung and Blood Institute, National Institutes of Health, United States

    ↵* Corresponding author; email: amy.patterson{at}nih.gov

    Author contributions: ZC, TLE, and APP designed the study and wrote the paper. AVB, INB, TGV, and RK provided technical assistance, data analyses and consulting. All authors reviewed the results and approved the final version of the manuscript.

Abstract

Apolipoprotein B mRNA-editing enzyme catalytic subunit 3 (APOBEC-3) enzymes are cytidine deaminases that are broadly and constitutively expressed. They are often upregulated during carcinogenesis and candidate genes for causing the major single-base substitution in cancer-associated DNA mutations. Moreover, APOBEC-3s are involved in host innate immunity against many viruses. However, how APOBEC-3 mutational activity is regulated in normal and pathological conditions remains largely unknown. Heat shock protein levels are often elevated in both carcinogenesis and viral infection and are associated with DNA mutations. Here, using mutational analyses of hepatitis B virus (HBV), we found that Hsp90 stimulates deamination activity of APOBEC3G (A3G), A3B, and A3C during co-expression in human liver HepG2 cells. Hsp90 directly stimulated A3G deamination activity when the purified proteins were used in in vitro reactions. Hsp40, 60, and 70 also had variable stimulatory effects in the cellular assay, but not in vitro. Sequencing analyses further demonstrated that Hsp90 increased both A3G cytosine mutation efficiency on HBV DNA and total HBV mutation frequency. In addition, Hsp90 shifted A3G's cytosine region selection in HBV DNA and increased A3G's 5-end nucleoside preference for deoxycytidine (5'CC). Furthermore, the Hsp90 inhibitor 17-AAG dose dependently inhibited A3G and A3B mutational activity on HBV viral DNA. Hsp90 knockdown by siRNA or by Hsp90 active-site mutation also decreased A3G activity. These results indicate that heat shock proteins, in particular Hsp90, stimulate APOBEC3-mediated DNA deamination activity, suggesting a potential physiological role in carcinogenesis and viral innate immunity.

    cancer cytidine deaminase heat shock protein 90 (Hsp90) hepatitis B virus (HBV, Hep B) mutagenesis APOBEC3G cofactor

    Received September 26, 2016.
    Accepted June 21, 2017.

    Copyright © 2017, The American Society for Biochemistry and Molecular Biology

StephenW

热休克蛋白刺激乙型肝炎病毒中APOBEC-3介导的胞苷脱氨

    Zhigang Chen1，Thomas L. Eggerman2，Alexander V. Bocharov1，Irina N. Baranova1，Tatyana G. Vishnyakova1，Roger Kurlander1和Amy P. Patterson3 *

    美国国立卫生研究院1临床中心;
    2美国国家卫生研究院国家糖尿病与消化和肾脏病研究所;
    3国立国立心脏血管研究所，美国国立卫生研究院

    ↵*通讯作者;电子邮件：amy.patterson {at} nih.gov

    作者贡献：ZC，TLE和APP设计研究并撰写论文。 AVB，INB，TGV和RK提供技术支持，数据分析和咨询。所有作者审查结果并批准了手稿的最终版本。

抽象

载脂蛋白B mRNA编辑酶催化亚基3（APOBEC-3）酶是广泛和组成型表达的胞苷脱氨酶。它们通常在致癌过程中被上调，并且在癌症相关DNA突变中引起主要单碱基置换的候选基因。此外，APOBEC-3s涉及许多病毒的宿主先天免疫。然而，APOBEC-3突变活动在正常和病理状态下的调控仍然很大程度上是未知的。热休克蛋白水平通常在癌变和病毒感染中均升高，并与DNA突变有关。在这里，使用乙型肝炎病毒（HBV）的突变分析，我们发现Hsp90在人肝HepG2细胞共表达期间刺激APOBEC3G（A3G），A3B和A3C的脱氨活性。当纯化的蛋白质用于体外反应时，Hsp90直接刺激A3G脱氨活性。 Hsp40,60和70在细胞测定中也具有不同的刺激作用，但不是在体外。测序分析进一步证实，Hsp90增加了HBV DNA和总HBV突变频率的A3G胞嘧啶突变效率。此外，Hsp90转移了A3G在HBV DNA中的胞嘧啶区选择，增加了A3G对脱氧胞苷（5'CC）的5末端核苷偏好。此外，Hsp90抑制剂17-AAG剂量依赖性地抑制HBV病毒DNA上的A3G和A3B突变活性。通过siRNA或Hsp90活性位点突变的Hsp90敲低也降低了A3G活性。这些结果表明，热休克蛋白，特别是Hsp90，刺激APOBEC3介导的DNA脱氨活性，表明在癌发生和病毒先天免疫中潜在的生理作用。

    癌细胞脱氨酶热休克蛋白90（Hsp90）乙型肝炎病毒（HBV，Hep B）诱变APOBEC3G辅因子

    收到2016年9月26日。
    于2017年6月21日接受。

    版权所有©2017，美国生物化学和分子生物学学会