PIAS1-mediated SUMOylation of influenza A virus PB2 restricts viral replication and virulence.
PIAS1-mediated SUMOylation of influenza A virus PB2 restricts viral replication and virulence.
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PIAS1介导的甲型流感病毒PB2的SUMO化限制病毒复制和毒力
DOI：
10.1371/journal.ppat.1010446
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发表时间：
2022-04
期刊：
影响因子：
6.7
通讯作者：
中科院分区：
文献类型：
--
作者：
宿主防御系统利用翻译后修饰来抵御入侵的病原体。在此，我们发现信号转导及转录激活因子1的蛋白抑制因子（PIAS1）与甲型流感病毒（IAV）的核蛋白（NP）、聚合酶碱性蛋白1（PB1）和聚合酶碱性蛋白2（PB2）相互作用。慢病毒介导的PIAS1稳定过表达显著抑制了IAV的复制，而通过siRNA敲低或CRISPR/Cas9敲除PIAS1的表达则显著促进了病毒的生长。在细胞培养和小鼠实验中，IAV感染均能显著诱导PIAS1的表达，且PIAS1参与了IAV感染诱导的细胞SUMO化修饰整体水平的升高。我们发现PIAS1抑制了病毒核糖核蛋白（RNP）复合物的活性，而缺乏SUMO E3连接酶活性的PIAS1的C351S或W372A突变体则丧失了抑制病毒RNP复合物活性的能力。值得注意的是，PIAS1的SUMO E3连接酶活性催化了PB2强烈的SUMO化修饰，但对PB1的SUMO化修饰没有作用，对NP的SUMO化修饰作用微弱。此外，PIAS1介导的SUMO化修饰显著降低了IAV PB2的稳定性。在体内实验中，我们发现小鼠体内Pias1表达下调会增强IAV的生长和毒力。综上所述，我们的研究将PIAS1确定为IAV复制和致病过程的一种限制因子。
SUMO化修饰似乎是蛋白质（包括病毒蛋白）一种重要的翻译后修饰机制。在本研究中，我们发现SUMO E3连接酶PIAS1与IAV的RNP复合物中的PB2、PB1和NP蛋白相互作用。研究发现PIAS1的表达抑制了病毒RNP复合物的活性。从机制上来说，PIAS1的SUMO E3连接酶活性导致IAV PB2发生强烈的SUMO化修饰，但分别对PB1和NP的SUMO化修饰没有作用或作用微弱，且PIAS1介导的SUMO化修饰显著降低了PB2的稳定性。在细胞培养和小鼠实验中，IAV感染均能显著诱导PIAS1的表达，这表明PIAS1在抑制IAV复制过程中发挥着积极作用且具有重要生物学意义。值得注意的是，在Pias1+/-小鼠中直接验证了Pias1在限制IAV复制和毒力方面的作用。因此，我们的研究鉴定出一种能与IAV PB2相互作用并使其发生SUMO化修饰的SUMO E3连接酶，从而在体外和体内实验中均降低了病毒的复制和毒力。
Host defense systems employ posttranslational modifications to protect against invading pathogens. Here, we found that protein inhibitor of activated STAT 1 (PIAS1) interacts with the nucleoprotein (NP), polymerase basic protein 1 (PB1), and polymerase basic protein 2 (PB2) of influenza A virus (IAV). Lentiviral-mediated stable overexpression of PIAS1 dramatically suppressed the replication of IAV, whereas siRNA knockdown or CRISPR/Cas9 knockout of PIAS1 expression significantly increased virus growth. The expression of PIAS1 was significantly induced upon IAV infection in both cell culture and mice, and PIAS1 was involved in the overall increase in cellular SUMOylation induced by IAV infection. We found that PIAS1 inhibited the activity of the viral RNP complex, whereas the C351S or W372A mutant of PIAS1, which lacks the SUMO E3 ligase activity, lost the ability to suppress the activity of the viral RNP complex. Notably, the SUMO E3 ligase activity of PIAS1 catalyzed robust SUMOylation of PB2, but had no role in PB1 SUMOylation and a minimal role in NP SUMOylation. Moreover, PIAS1-mediated SUMOylation remarkably reduced the stability of IAV PB2. When tested in vivo, we found that the downregulation of Pias1 expression in mice enhanced the growth and virulence of IAV. Together, our findings define PIAS1 as a restriction factor for the replication and pathogenesis of IAV. SUMOylation appears to be an important posttranslational modification mechanism of proteins, including viral proteins. In the present study, we found that the SUMO E3 ligase PIAS1 interacts with the PB2, PB1, and NP proteins of the RNP complex of IAV. PIAS1 expression was found to suppress the viral RNP complex activity. Mechanistically, the SUMO E3 ligase activity of PIAS1 led to robust SUMOylation of IAV PB2, but had no or a minimal effect on the SUMOylation of PB1 and NP, respectively, and PIAS1-mediated SUMOylation significantly decreased the stability of PB2. The expression of PIAS1 was markedly induced upon IAV infection in cell culture and mice, indicating that PIAS1 is actively involved and biologically important in the inhibition of IAV replication. Of note, the role of Pias1 in restricting the replication and virulence of IAV was directly verified in Pias1+/- mice. Our findings thus identify a SUMO E3 ligase that interacts with and SUMOylates IAV PB2, thereby leading to reduced virus replication and virulence in vitro and in vivo.
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