人类呼吸道合胞病毒(RSV)是全球婴儿严重急性下呼吸道感染的主要原因。RSV的非结构蛋白NS1通过以多种方式充当I型和III型干扰素(IFN)产生和信号传导的拮抗剂来调节宿主先天免疫应答。很可能,NS1通过与不同的宿主蛋白相互作用来执行此功能。为了获得NS1互动伙伴的全面概述,我们进行了三个互补的蛋白质-蛋白质相互作用筛选,即,BioID,MAPPIT,和吻。为了模仿自然感染,使用重组RSV进行BioID邻近筛选,其中NS1蛋白与生物素连接酶融合。值得注意的是,MED25,中介复合体的一个亚基,在所有三种进行的筛选方法中都被鉴定为潜在的NS1相互作用蛋白。我们通过免疫共沉淀证实了MED25和RSVNS1之间的相互作用,不仅在RSV感染过程中NS1过表达,而且内源性NS1也过表达。我们还证明,RSV的复制可以在MED25敲除A549细胞中增强,提示MED25在RSV感染期间的潜在抗病毒作用。中介子亚基作为转录共激活因子起作用,并参与其靶基因的转录调控。因此,RSVNS1与细胞MED25之间的相互作用可能通过影响宿主转录和宿主对感染的免疫反应而对感染期间的RSV有益。重要性先天免疫反应,包括I型和III型干扰素的生产,在抵御RSV感染的第一道防线中起着至关重要的作用。然而,在RSV感染期间仅观察到I型IFN的弱诱导,这表明RSV已经进化出阻止受感染宿主细胞表达I型IFN的机制。一种独特的RSV蛋白,NS1,在很大程度上是造成这种影响的原因,可能通过与多种宿主蛋白的相互作用。更好地了解RSVNS1与宿主蛋白之间的相互作用可能有助于确定有效抗病毒治疗的靶标。我们通过进行三个互补的蛋白质-蛋白质相互作用筛选来解决这个问题,并将MED25鉴定为RSVNS1相互作用蛋白。我们提出了这种介体复合物亚基在先天抗RSV防御中的作用。
Human respiratory syncytial virus (RSV) is the leading cause of severe acute lower respiratory tract infections in infants worldwide. Nonstructural protein NS1 of RSV modulates the host innate immune response by acting as an antagonist of type I and type III interferon (IFN) production and signaling in multiple ways. Likely, NS1 performs this function by interacting with different host proteins. In order to obtain a comprehensive overview of the NS1 interaction partners, we performed three complementary protein-protein interaction screens, i.e., BioID, MAPPIT, and KISS. To closely mimic a natural infection, the BioID proximity screen was performed using a recombinant RSV in which the NS1 protein is fused to a biotin ligase. Remarkably,
MED25, a subunit of the Mediator complex, was identified in all three performed screening methods as a potential NS1-interacting protein. We confirmed the interaction between MED25 and RSV NS1 by coimmunoprecipitation, not only upon overexpression of NS1 but also with endogenous NS1 during RSV infection. We also demonstrate that the replication of RSV can be enhanced in
MED25 knockout A549 cells, suggesting a potential antiviral role of
MED25 during RSV infection. Mediator subunits function as transcriptional coactivators and are involved in transcriptional regulation of their target genes. Therefore, the interaction between RSV NS1 and cellular
MED25 might be beneficial for RSV during infection by affecting host transcription and the host immune response to infection. IMPORTANCE Innate immune responses, including the production of type I and III interferons, play a crucial role in the first line of defense against RSV infection. However, only a poor induction of type I IFNs is observed during RSV infection, suggesting that RSV has evolved mechanisms to prevent type I IFN expression by the infected host cell. A unique RSV protein, NS1, is largely responsible for this effect, probably through interaction with multiple host proteins. A better understanding of the interactions that occur between RSV NS1 and host proteins may help to identify targets for an effective antiviral therapy. We addressed this question by performing three complementary protein-protein interaction screens and identified
MED25 as an RSV NS1-interacting protein. We propose a role in innate anti-RSV defense for this Mediator complex subunit.