目的:探讨Zw10结合因子-1(zeste white 10 interactor-1,Zwint-1)及其变异体(Zwint-1v)在HeLa细胞不同细胞周期中的亚定位。方法:采用胸腺嘧啶核苷双阻断法将HeLa细胞同步化,通过瞬时转染及间接免疫荧光检测,观察Zwint-1及Zwint-1v在HeLa细胞不同细胞周期中的亚定位。结果:在HeLa细胞间期,Zwint-1野生型定位于细胞质,而Zwint-1v定位于细胞核;在分裂期,Zwint-1野生型及Zwint-1v定位于纺锤体与着丝粒上。Zwint-1v定化较野生型更趋近于细胞核。结论:Zwint-1及Zwint-1v在HeLa细胞不同细胞周期的亚定位存在差异。
Non-structural protein 1(NS1) of the influenza virus plays a crucial role in modulating the host immune response and facilitating virus replication.The formation of a homodimer or an oligomer is necessary for NS1 to exert its function efficiently.In the present study,the NS1 protein from the A/Shantou/602/06(H3N2) virus(herein abbreviated as NS32) was found to interact with NS1 from A/Shantou/169/06(H1N1),A/Chicken/Guangdong/1/05(H5N1) and A/Quail/Hong Kong/G1/97(H9N2)(abbreviated as NS11,NS51 and NS92,respectively) viruses,although NS32 shares 17.4% 20.9% sequence diversity with NS11,NS51 and NS92.This indicates that the heterologous interactions between NS1 proteins from different influenza A virus subtypes/strains may be a common event during co-infection.
LI WeiZhongZHANG HengWANG GeFeiZHANG ChiZENG XiangXingLIU HuiCHEN XiaoXuanXU YanXuanLI KangSheng
A型流感病毒NS1蛋白羧基端4个氨基酸可以与PDZ结构域(the domain of PSD95,Dig and ZO-1)相结合,称为PL结构域(PDZ ligand domain).对不同亚型或毒株的流感病毒而言,其NS1蛋白PL结构域的组成存在比较大的差异.有研究发现这种差异能够影响NS1与宿主细胞蛋白的相互作用进而影响病毒的致病力.为进一步探讨PL结构域对NS1蛋白生物学特性的影响,首先构建出4种不同亚型流感病毒(H1N1、H3N2、H5N1、H9N2)来源的NS1绿色荧光蛋白表达质粒.在此基础上,对野生型H3N2病毒NS1表达质粒进行人工改造,将其PL结构域缺失或者替换为其他亚型流感病毒的PL结构域,制备出4种重组NS1蛋白表达质粒.通过比较上述不同NS1蛋白在HeLa细胞中的定位情况发现,只有野生型H3N2病毒的NS1蛋白可以定位于核仁当中,而野生型H1N1、H5N1、H9N2病毒的NS1蛋白以及PL结构域缺失或替代的H3N2病毒NS1蛋白都不能定位于核仁.而通过比较上述NS1蛋白在流感病毒易感的MDCK细胞中的定位,进一步发现所有这些蛋白均不定位于核仁.上述结果表明:PL结构域的不同可以明显影响NS1蛋白在HeLa细胞核内的定位和分布,这有可能造成其生物学功能的差异.同时,NS1蛋白在细胞核内的定位还与宿主细胞的来源有着密切关系.
Non-structural protein 1(NS1)is an important virulence factor of the highly pathogenic H5N1 avian influenza virus.A five-amino-acid(5 aa)deletion at position 80–84 and an aspartic acid to glutamic acid substitution at position 92(D92E)are two major NS1 mutations that are highly correlated with enhanced virulence.To investigate the effect of these mutations in H5N1 virulence,three H5N1-NS1 variants were constructed:NS51(lacking 5 aa at position 80–84),NS51(I)(carrying a 5-aa insertion at position 80–84)and NS51(IM)(carrying both the 5-aa insertion and the D92E mutation).We examined the effects of these mutations on interferon(IFN)induction,tumor-necrosis factor(TNF)a response,p53 activity and apoptosis.We found that the D92E mutation eliminated NS1’s repressive effect on IFN induction,while the 5-aa deletion resulted in enhanced resistance to TNFa responses.We also observed that all three variants exhibited a similar suppressive effect on p53 transcriptional activity,although none of them significantly influenced apoptosis of host cells.Our findings shed new light on the role of NS1 in the pathogenicity of H5N1 virus.
