A型流感病毒NS1蛋白羧基端PL结构域影响NS1在细胞核内的定位

PL Domain at the Carboxyl Terminus of Influenza A Virus NS1 Protein Influences The Nuclear Localization of NS1

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.

The C-terminal four residues of influenza A virus non-structural protein 1 （NS1） comprise the binding site for PDZ （the domain of PSD95, Dig and ZO-1） and is named PL domain （PDZ ligand domain）. Previous study showed that NS1 proteins from different subtypes/strains of influenza A virus varied in their amino acids sequence of PL domain, which may affect the interaction between NS1 and cellular proteins and is closely associated with the pathogenicity of influenza A virus. To further explore the role of PL domain in the biological properties of NS1 protein, four wild type NS1-expressing plasmids harboring the NS1 encoding sequence from different influenza A virus subtypes （H1N1, H3N2, H5N1 and H9N2） were constructed firstly, based on pEGFP-c1 vector. The mutant NS1 （H3N2）-expressing plasmids were subsequently generated by either deletion or replacement of PL domain from other influenza A virus subtypes. Comparative analysis of the localization of these NS1 proteins in HeLa cells showed that wild type NS1 protein from H3N2 virus mainly localized in the nucleoli, whereas wild type NS1 proteins from H1N1, H5N1 and H9N2 viruses or mutant NS1 proteins from H3N2 virus mainly localized in the nuclei （but not nucleoli）. In MDCK cells, none of the above NS1 proteins located in the nucleoli. The results indicated that PL domain can significantly influence the nuclear localizing pattern of NS1 protein in HeLa cells, which may be responsible for the variation of NS1 protein in its biological function; the distributed pattern of NS1 protein is closely associated with the origin of host cells.