Alanine substitutions within a linker region of the influenza A virus non-structural protein 1 alter its subcellular localization and attenuate virus replication.
Alanine substitutions within a linker region of the influenza A virus non-structural protein 1 alter its subcellular localization and attenuate virus replication.
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甲型流感病毒非结构蛋白 1 连接区内的丙氨酸取代改变其亚细胞定位并减弱病毒复制。
DOI：
10.1099/vir.0.031336-0
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发表时间：
2011
期刊：
影响因子：
--
通讯作者：
Noah,DianaL
中科院分区：
文献类型：
--
作者：
Li,Wei;Noah,JamesW;Noah,DianaL
甲型流感病毒非结构蛋白1（NS 1）是一种多功能蛋白，是重要的毒力因子。它由两个表征良好的结构域组成，这些结构域由一个短的、但在晶体学上没有很好定义的、功能未知的区域连接。为了研究该区域的可能功能，我们引入丙氨酸取代以取代氨基酸位置69和77处的两个高度保守的亮氨酸残基。突变体L 69，77 A NS 1蛋白保留了与野生型（WT）相当的对dsRNA、切割和多聚腺苷酸化特异性因子30以及PI 3 K的p85β亚基的结合能力。使用反向遗传学产生表达L 69，77 A NS 1蛋白的突变型甲型流感病毒。与WT NS 1病毒相比，L 69，77 A NS 1病毒感染在Madin-Darby犬肾（MDCK）细胞中诱导显著更高水平的β干扰素（IFN-β）表达。此外，与WT病毒相比，L 69，77 A NS 1病毒在MDCK细胞中的复制率显著降低，但在Vero细胞中不降低，表明L 69，77 A NS 1蛋白在病毒复制期间不完全拮抗IFN。L 69，77 A NS 1病毒感染不能激活PI 3 K/Akt抗凋亡途径，表明突变型NS 1蛋白可能无法定位，从而在感染期间在体内接近p85β，这得到了转染或病毒感染后突变型NS 1与WT NS 1相比改变的亚细胞定位模式的支持。我们的数据表明，这两个结构域之间的连接区是至关重要的NS 1蛋白在甲型流感病毒感染的功能，可能通过确定蛋白质的正确的亚细胞定位。
The influenza A virus non-structural protein 1 (NS1) is a multifunctional protein and an important virulence factor. It is composed of two well-characterized domains linked by a short, but not well crystallographically defined, region of unknown function. To study the possible function of this region, we introduced alanine substitutions to replace the two highly conserved leucine residues at amino acid positions 69 and 77. The mutant L69,77A NS1 protein retained wild-type (WT)-comparable binding capabilities to dsRNA, cleavage and polyadenylation specificity factor 30 and the p85β subunit of PI3K. A mutant influenza A virus expressing the L69,77A NS1 protein was generated using reverse genetics. L69,77A NS1 virus infection induced significantly higher levels of beta interferon (IFN-β) expression in Madin–Darby canine kidney (MDCK) cells compared with WT NS1 virus. In addition, the replication rate of the L69,77A NS1 virus was substantially lower in MDCK cells but not in Vero cells compared with the WT virus, suggesting that the L69,77A NS1 protein does not fully antagonize IFN during viral replication. L69,77A NS1 virus infection was not able to activate the PI3K/Akt anti-apoptotic pathway, suggesting that the mutant NS1 protein may not be localized such that it has access to p85βin vivoduring infection, which was supported by the altered subcellular localization pattern of the mutant NS1 compared with WT NS1 after transfection or virus infection. Our data demonstrate that this linker region between the two domains is critical for the functions of the NS1 protein during influenza A virus infection, possibly by determining the protein’s correct subcellular localization.
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影响因子：
16
作者：
Nemeroff, ME;Barabino, SML;Krug, RM
通讯作者：
Krug, RM
影响因子：
3.7
作者：
LU, Y;WAMBACH, M;KRUG, RM
通讯作者：
KRUG, RM
影响因子：
5.4
作者：
Donelan, NR;Basler, CF;García-Sastre, A
通讯作者：
García-Sastre, A
影响因子：
3.7
作者：
Noah, DL;Twu, KY;Krug, RM
通讯作者：
Krug, RM
DOI：
10.1073/pnas.0602184103
发表时间：
2006-05-02
影响因子：
11.1
作者：
Min, JY;Krug, RM
通讯作者：
Krug, RM