干扰素在肝癌细胞Huh7.0中诱导SAMHD1抑制HBV复制

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

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。

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.