干扰素在肝癌细胞Huh7.0中诱导SAMHD1抑制HBV复制 <sup>*</sup>

doi:10.13523/j.cb.20190301

中国生物工程杂志

2019, Vol. 39

Issue (3): 1-6 DOI: 10.13523/j.cb.20190301

研究报告

干扰素在肝癌细胞Huh7.0中诱导SAMHD1抑制HBV复制 ^*

乔淼,胡杰,毛彬力,皮思蝶,胡源

重庆医科大学感染性疾病分子生物学教育部重点实验室重庆 400016

The Inhibition of IFN Induce SAMHD1 to the Replication of HBV in Huh7.0 Cells

Miao QIAO,Jie HU,Bin-li MAO,Si-die PI,Yuan HU

Key Laboratory of Molecular Biology on Infectious Disease, Ministry of Education,Chongqing Medical University, Chongqing 400016, China

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摘要：

目的: 研究干扰素调节宿主限制性因子SAMHD1的表达而抑制HBV复制的分子机制。方法: 首先不同剂量的干扰素α、β和γ处理Huh7.0细胞,通过荧光定量PCR和Western blot检测SAMHD1在转录和翻译后的表达水平;进一步通过siRNA干扰内源性的SAMHD1,再评价干扰素α、β对病毒的抑制效应;最后通过免疫荧光检测了干扰素α诱导内源性SAMHD1 的细胞定位及Southern blot检测了SAMHD1细胞定位对病毒复制的影响。结果: 在Huh7.0细胞中,SAMHD1的RNA水平和蛋白表达明显受干扰素α和β的诱导升高;干扰SAMHD1后干扰素α和β对HBV复制的抑制作用消失;SAMHD1定位在细胞核内,干扰素α诱导SAMHD1同样定位在细胞核内,缺失核定位信号后SAMHD1丧失了其抑制病毒复制的作用。结论: 在Huh7细胞中,干扰素α、β能诱导 SAMHD1表达上调来抑制HBV的复制,SAMDH1的抗病毒作用依赖于其细胞定位。

关键词： 乙型肝炎病毒; 干扰素; SAMHD1; 抑制

Abstract:

Objective: To analyze the mechanism of the inhibition of IFNs induce SAMHD1 to hepatitis B virus(HBV)in liver cancer cells Huh7.0. Methods: (1) Recombiant expression plasmids of SAMHD1 (sterile alpha motif and histidine/ aspartic acid domain-containing protein 1) mutants that were defective in dNTPase (deoxynucleoside triphosphate triphosphohydrolase) activity and its nuclear location mutation were constructed. (2) The mRNA and protein levels of SAMHD1 in Huh7.0 cells by IFNs treatment were measured by real time and Western blot. (3) HBV core-associated DNA levels in transfected Huh7.0 cells were measured by Southern blot. (4) The location of SAMHD1 and its nuclear mutation in the Huh7.0 cells was measured by immunofluorescence. Results: (1) Both the RNA and protein levels of SAMHD1 were upregulated by interferons. (2) The antiviral function of SAMHD1 was lost when knocking down the expression of SAMHD1 by siRNA. (3) SAMHD1 lost its restriction towards HBV after mutating its NLS signal. Conclusion: IFNs can upregulate the expressing of SAMHD1 in Huh7 cells. SAMHD1 is mainly located in the nucleus and its restriction towards HBV dependent on its location.

Key words: Hepatitis B virus Interferons SAMHD1 Inhibition

收稿日期: 2018-09-12 出版日期: 2019-04-12

ZTFLH:

Q789

基金资助: * 国家自然科学基金(81471945);重庆市科委自然科学基金(cstc2018jcyjAX0166);重庆市教委重庆市青少年创新人才培养雏鹰计划(第七期)资助项目

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引用本文:

乔淼,胡杰,毛彬力,皮思蝶,胡源. 干扰素在肝癌细胞Huh7.0中诱导SAMHD1抑制HBV复制 ^*[J]. 中国生物工程杂志, 2019, 39(3): 1-6.

Miao QIAO,Jie HU,Bin-li MAO,Si-die PI,Yuan HU. The Inhibition of IFN Induce SAMHD1 to the Replication of HBV in Huh7.0 Cells. China Biotechnology, 2019, 39(3): 1-6.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20190301 或 https://manu60.magtech.com.cn/biotech/CN/Y2019/V39/I3/1

表1 SAMHD1的定点突变引物序列和特异性siRNA干扰序列

图1 肝癌细胞Huh7.0中干扰素α、β、γ的SAMHD1的诱导效应

图2 干扰SAMHD1后IFNα、β对HBV的抑制作用

图3 免疫荧光试验检测SAMHD1及核定位缺失的定位

图4 SAMHD1抑制HBV复制依赖于细胞定位

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