宿主限制性因子MOV10对HBV复制的影响

Effect of host restriction factor MOV10 on HBV replication

目的研究宿主限制性因子莫罗尼白血病病毒10蛋白(Moloney leukemia virus 10,MOV10)对乙肝病毒(hepatitis B virus,HBV)复制的调控作用。方法首先在肝癌细胞Huh7中转染HBV复制质粒,探讨HBV复制和MOV10表达的相关性;接着在Huh7中通过siRNA干扰或过表达MOV10,提取病毒核心颗粒的DNA,通过荧光定量PCR分析病毒复制水平的变化;进一步构建MOV10保守结构域Ⅱ的酶活突变体(D645A、E646Q和G648A),检测突变后对MOV10抗病毒作用的影响;在人肾上皮细胞293细胞中共转染HBV复制质粒和MOV10表达质粒,通过RNA结合蛋白免疫沉淀(RNA binding protein immunoprecipitation,RIP)研究MOV10与HBVRNA的结合。结果肝癌细胞Huh7转染HBV复制质粒后,MOV10蛋白水平增高;肝癌细胞Huh7中干扰内源性MOV10后,HBV复制水平升高1.5倍,而Huh7细胞中过表达MOV10,显著抑制了HBV的复制;MOV10结构域Ⅱ突变体同样显著抑制HBV的复制;MOV10可以与HBV3.5kbRNA结合。结论在肝癌细胞中,宿主限制性因子MOV10的表达与HBV复制相关,其抑制HBV复制的作用不依赖于其解旋酶活性,可能与HBV3.5kbRNA结合有关。

Objective To investigate the regulatory role of host restriction factor Moloney leukemia virus 10 (MOV10) protein in HBV replication. Methods Firstly, a HBV replication-expression plasmid was transfected into Huh7 cells to investigate the effect of HBV replication on MOV10 expression. Secondly, HBV DNA was extracted and measured by quantitative PCR (qPCR) after knocking down the expression of endogenous MOV10 or enhancing the expression of exogenous MOV10. Furthermore, MOV10 conserved domain Ⅱ enzyme active mutants (D645A, E646Q and G648A) were constructed and analyzed regarding their antiviral activities. The HBV replication plasmid and MOV10 expression plasmid were co-transfected into human renal epithelial cells (HEK293) to investigate whether MOV10 could bind to HBV mRNA using RNA binding protein immunoprecipitation (RIP). Results The expression of MOV10 was increased after transfection of HBV replication plasmid into Huh7 cells. After knocking down the expression of endogenous MOV10 by siRNA in Huh7 cells, HBV replication was increased about 1.5 times compared with control group, while the viral DNA level was significantly decreased in Huh7 cells that overexpressed MOV10. MOV10 domain Ⅱ mutants also significantly inhibited HBV replication. MOV10 could bind to 3.5 kb HBV RNA. Conclusion In liver cancer cells, the expression of the host restriction factor MOV10 was associated with HBV replication. Its inhibitory effect against HBV replication was independent of its helicase activity, but might be associated with its binding activity with 3.5kb HBV RNA.