摘要
新城疫病毒（Newcastle disease virus，NDV）是一种与养禽业经济息息相关的重要禽类病毒，由其引起的新城疫（Newcastle disease，ND）具有高度的传染性和致死性，严重威胁了世界养禽业的正常发展。新城疫病毒属于副黏病毒科（Paramyxoviridae）、禽腮腺炎病毒属（Avulavirus），基因组为不分节段的单股负链RNA，其基因组按照3′-Leader-NP-P-M-F-HN-L-Trailer5′的顺序编码6种结构蛋白。NDV病毒蛋白的翻译依赖于宿主的翻译系统，其mRNA与大多数真核生物一样都具有5＇帽子结构，一般认为L蛋白具有加帽酶的活性能够在病毒mRNA的5＇端形成缺少2’-O甲基化的“cap0”型帽子结构。新城疫病毒在细胞内具有很强的增殖能力，感染后能够迅速、大量地合成病毒自身RNA和蛋白，完成装配。新城疫病毒RNA的合成是依赖其自身合成的病毒RNA聚合酶，启动病毒的转录机制，但是转录后的翻译机制尚未可知。
大多数真核生物的mRNA都具有3’ poly A尾和5’甲基化的帽子结构，并利用帽子结构依赖性的真核翻译系统进行mRNA的翻译。翻译的起始过程是真核蛋白翻译过程中重要的限速步骤，该过程的核心元件是翻译起始复合体eIF4F（translation initiaton factor4F）。这一复合体由eIF4E、eIF4G和eIF4A三个组分构成，其活性是由一系列的磷酸化级联反应所调节的，包括PI3K/Akt/mTOR和MAPK两个重要的信号通路。病毒作为一种严格的胞内寄生物，完全依赖于宿主的翻译系统合成病毒蛋白质从而实现病毒的复制，因此eIF4F也是病毒感染过程中重要的调控靶标。许多病毒都能够通过影响eIF4F复合体及其调控通路来促进病毒蛋白的合成。为了探究新城疫病毒感染细胞后如何激活和利用宿主的真核翻译系统，我们开展了以下几方面的研究：
1本研究通过构建pET-32a-NP重组表达载体，原核表达NDV NP重组蛋白并免疫小鼠，经过四次亚克隆后，成功制备得到两株针对NP蛋白的单克隆抗体。经过实验验证，这两株抗体均具有ELISA、IFA和Western Blot效价。该单抗的成功研制将为进一步研究NP蛋白功能以及其与宿主细胞的相互作用提供研究工具，并为实现新城疫的临床快速诊断提供理论基础。
2新城疫病毒感染细胞后能够引起eIF4F复合体的磷酸化并促进其装配。为了研究NDV感染能否诱导宿主翻译系统的活化，我们用Herts/33毒株感染人宫颈癌HeLa细胞系，以Western-Blot、和m7GTP pull down的方法鉴定eIF4F复合体的磷酸化水平及装配能力。结果显示，5MOI的Herts/33毒株感染HeLa细胞后6-12h伴随着病毒蛋白的大量合成eIF4E及eIF4G的磷酸化水平明显上升，而紫外灭活的NDV并不能诱导该现象的产生；NDV感染8h后进行m7GTP pull down实验发现，NDV感染后能够引起eIF4F复合体的大量装配。这些结果说明NDV感染细胞后能够通过诱导eIF4F的磷酸化并促进其装配的方式来激活宿主的真核翻译系统。此外，我们还通过间接免疫荧光和RNA干扰实验发现，eIF4E在NDV感染8h后从弥散的散在分布变为点状成簇分布并与NP蛋白存在共定位；当eIF4E或/和eIF4G被RNA干扰时，病毒蛋白的合成和子代病毒的产生均会受到明显抑制，证实了NDV病毒蛋白的合成依赖于帽子结构依赖性的翻译起始过程。
3本研究进一步分析了PI3K/Akt通路在介导NDV感染引起eIF4F活化过程中所起的具体作用。我们利用Herts/33感染HeLa细胞，检测PI3K/Akt通路下游信号分子的磷酸化水平并结合使用抑制剂来确证PI3K/Akt通路是否参与NDV诱导的eIF4F的激活。同时我们还研究了PI3K/Akt通路对病毒诱导的eIF4F的形成以及对病毒复制的影响。结果表明，NDV在感染后6-12h能够激活Akt及下游mTOR，p70S6K和4E-BP1，并且这些下游分子的磷酸化均是由于NDV感染激活PI3K所介导的。研究结果还表明，NDV感染所诱导的eIF4G的磷酸化也是通过激活PI3K/Akt通路下游mTOR激酶实现的。而当使用PI3K的特异性抑制剂LY294002处理细胞时，会导致eIF4F复合体的形成受到抑制从而影响病毒蛋白的合成。此外，我们还发现NDV引起的4E-BP1的过磷酸化能够拮抗Rapamycin的抑制作用，所以Rapamycin既不能抑制抑制NDV诱导的eIF4F复合体的形成，也不能对病毒的复制产生任何影响。以上结果说明，NDV感染后通过激活PI3K/Akt通路诱导eIF4G和4E-BP1发生磷酸化，从而促进eIF4F复合体的形成并保证病毒mRNA的顺利翻译。
4本研究同样也对MAKP通路在NDV激活eIF4F的过程中所起的调控作用进行了分析。我们首先用Western-Blot检测了NDV感染后MAPK及下游Mnk1的磷酸化状态，发现p38、Erk及Mnk1都在病毒感染后6-12h出现磷酸化。但是通过使用特异性的抑制剂将其分别阻断时，只有p38的抑制剂SB203580既能抑制Mnk1的活化又能影响eIF4E的磷酸化水平，而Erk被阻断时只对病毒诱导的Mnk1的磷酸化有影响。此外我们还通过使用抑制剂结合m7GTP pull down实验，分析了病毒诱导的eIF4E磷酸化对eIF4F复合体装配的影响。当使用p38及Mnk1的抑制剂阻断病毒诱导的eIF4E的磷酸化时，eIF4F复合体的形成并不受影响，因此病毒蛋白的合成及子代病毒的产生也未受到明显的影响。这些结果说明，MAPK信号通路，主要是p38MAPK/Mnk1通路，在新城疫病毒感染后可以通过诱导eIF4E磷酸化来促进eIF4F的翻译起始活性，但是调控作用有限，可能只是影响了翻译起始的效率。
5最后，本研究还发现了NDV的NP蛋白能够与eIF4F复合体相互作用。为了进一步阐明NDV对真核翻译系统的直接调控作用，我们利用m7GTP pull down实验和免疫共沉淀（Co-IP）实验对NDV的病毒蛋白和eIF4F复合体进行互作研究。结果显示NDV的NP蛋白能够与eIF4F复合体相互作用，且该互作不依赖于其他病毒蛋白的介导。我们还对NP蛋白与eIF4F互作的区域进行了分析，发现NP蛋白N端的391个氨基酸对于其与eIF4F的互作是必需的，而C端氨基酸的长度可能起到调节NP蛋白与eIF4F亲和力的作用。
综上所述，本研究主要阐明了新城疫病毒感染后调控宿主真核翻译系统的具体机制，并分析了PI3K/Akt/mTOR和p38MAPK/Mnk1信号通路在该调控过程中发挥的具体作用，此外我们还第一次发现NDV的NP蛋白可以通过与eIF4F的相互作用来促进病毒mRNA的选择性翻译。本研究结果对深入解析NDV与宿主之间的相互作用以及翻译相关信号通路参与调控病毒复制的机制具有重要意义，也为新型抗病毒药物的研发提供了理论基础。
Newcastle disease virus (NDV), the member of the Paramyxoviridae family, is a single-stranded,nonsegmented, negative-sense RNA virus that infects most species of birds, resulting in substantial lossto the poultry industry. The genome contains six major genes that encode the structural proteins in theorder3’-NP-P-M-F-HN-L-5’, as well as two non-structural proteins, W and V via mRNA editing. NDVis dependent on the cellular translational machinery to synthesize viral proteins, and the viral mRNAsare capped and polyadenylated by L protein during synthesis. The structure of the NDV mRNA cap ism7G(5’)ppp(5’)GpPyp, which lacks2’-O-methylation and belongs to the cap0type. All viralreplication events occur within the host cell cytoplasm, and the positive-sense RNA intermediates areformed, which act as mRNA using the host cell translation machinery to translate proteins. However, theregulation of translational machinery in NDV-infected cells and the detailed cellular pathways has notbeen studied.
Translation initiation is considered to be a rate limiting process for overall protein synthesis ineukaryotes. Most eukaryotic mRNAs present a5’7-methyl GTP terminal cap structure as well as apoly(A) tail at3’ end, and translated by cap-dependent translation. Cap-dependent translation beginswith7-methyl-GTP cap recognition by eukaryotic translation initiaton factor4F (eIF4F) whichcomposed of eIF4E, eIF4G and eIF4A. eIF4F functions are regulated by cellular signal pathways ineukaryotic cells by means of phosphorylation cascade reactions. A major pathway that regulates eIF4Fcomplex assembly is the PI3K/Akt/mTOR pathway. Another pathway that regulates the translationinitiation is MAP kinase-interacting serine/threonine-protein kinase1(Mnk1). A number of viruses canalso stimulate various host cell signalling pathways to phosphorylate translational control proteins,enhancing the activity of eIF4F components and inactivating translational repressor proteins. In thepresent work, we tried to illustrate the regulation of host translational machinery by Newcastle diseasevirus in host protein translation initiation.
To determine whether eIF4F is modified by NDV infection, the phosphorylation of eIF4G and eIF4Ewas analyzed by Western blotting. The results showed that both phosphorylation was significantlyincreased at6–12hpi, correlating with robust synthesis of viral proteins. Treating HeLa cells withUV-inactivated NDV Herts/33did not alter eIF4F phosphorylation or NDV NP protein expression. Thebinding between eIF4G and eIF4E was investigated, significantly increased eIF4G and eIF4E wasdetected in the eIF4F complex from NDV infected cells. In addition, eIF4E is redistributed post NDVinfection and depleting of eIF4E or/and eIF4G reduces viral protein synthesis. These data indicate thatNDV infection stimulated the phosphorylation of eIF4F and also promoted the assembly of eIF4Fcomplexes. Both eIF4E and eIF4G are important host factors for NDV protein synthesis and growth.
To determine the host intracellular pathways involved in NDV infection, we first examined thePI3K/Akt pathway by determining the level of phosphorylated downstream proteins. We also analysised the effect of PI3K/Akt pathway on the formation of eIF4F complexes and viral protein synthesis inNDV-infected cells. The results showed that Akt and mTOR phosphorylation was enhanced at6to12hpi and phosphorylation of p70S6K and4E-BP1, two downsteam effectors of Akt/mTOR signaling,were also elevated. In addition, the role of PI3K in Akt and downstream proteins phosphorylation afterNDV infection was confirmed by LY294002. We also found that NDV-induced eIF4G phosphorylationrequires mTOR activity. Inhibition of upstream PI3K signaling with LY294002resulted in a reduction inthe assembly of eIF4F. In addition,4E-BP1phosphorylation induced by NDV infection is resistant torapamycin treatment. In that case, rapamycin treatment did not affect the assembly of eIF4F andsynthesis of viral proteins. These data suggest that NDV infection induced phosphorylation of eIF4Gand4E-BP1as well as enhaced assembly of eIF4F through PI3K/Akt pathway.
We also examined the MAPK pathway involved in NDV infection and eIF4F activation. We show herethat Mnk1is strongly phosphorylated for NDV infection, correlating with the phosphorylation of eIF4Eat Ser209. Both Erk and p38are activated in NDV-infected cells, however, only p38activation isdirectly and correlates with eIF4E activation. The role of eIF4E phosphorylation in eIF4F assembly inNDV-infected cells, was examined using specific inhibitors. Phosphorylation of eIF4E contributedneither to NDV mediated eIF4F complex assembly, nor to NDV infection. While p38MAPK/Mnk1pathway is not essential for translation, they might impart a level of control that governs the efficiencywith which translation initiates on viral mRNAs.
To further identify the direct interaction between NDV and host protein translation machinery, weperformed m7GTP pull down and co-immunoprecipitation to confirm the direct interaction betweenNDV NP protein and eIF4F complex. Mapping of the eIF4F-binding domain on the NP protein usingNDV NP truncation mutants showed that the N-terminal391amino acids are necessary for NP tointeract with eIF4F, and the length of C-terminal portion of NP may play a role in regulating thecontaction capacity of NP protein and eIF4F complex.
Overall, our study provides insight into how NDV manipulates host signaling pathways to regulateprotein translation initiation. NDV infection activates PI3K/Akt/mTOR and p38MAPK/Mnk1pathwaysto upregulate phosphorylation of eIF4E and eIF4G, as well as assembly of eIF4F complex to stimulatecap-dependent translation. To favor viral gene translation and replication, the translation initiationmachinery is redistributed and interacts with NP. Further understanding of the mechanisms employed byNDV to recruit and interact with eIF4F complexs will provide additional insight into how NDVinterferes with host translation and optimizes viral gene expression. Our studies also provide insightsinto NDV-host interaction and may provide host cell targets for therapeutic intervention against NDVinfection.
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