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Identification of cellular interaction partners of the influenza virus ribonucleoprotein complex and polymerase complex using proteomic-based approaches

机译:使用基于蛋白质组学的方法鉴定流感病毒核糖核蛋白复合物和聚合酶复合物的细胞相互作用伴侣

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摘要

Cellular factors that associate with the influenza A viral ribonucleoprotein (vRNP) are presumed to play important roles in the viral life cycle. To date, interaction screens using individual vRNP components, such as the nucleoprotein or viral polymerase subunits, have revealed few cellular interaction partners. To improve this situation, we performed comprehensive, proteomics-based screens to identify cellular factors associated with the native vRNP and viral polymerase complexes. Reconstituted vRNPs were purified from human cells using Strep-tagged viral nucleoprotein (NP-Strep) as bait, and co-purified cellular factors were identified by mass spectrometry (MS). In parallel, reconstituted native influenza A polymerase complexes were isolated using tandem affinity purification (TAP)-tagged polymerase subunits as bait, and co-purified cellular factors were again identified by MS. Using these techniques, we identified 41 proteins that co-purified with NP-Strep-enriched vRNPs and four cellular proteins that co-purified with the viral polymerase complex. Two of the polymerase-associated factors, importin-beta 3 and PARP-1, represent novel interaction partners. Most cellular proteins previously shown to interact with either viral NP and/or vRNP were also identified using our method, demonstrating its sensitivity. Co-immunoprecipitation studies in virus-infected cells using selected novel interaction partners, including nucleophosmin (NPM), confirmed their association with vRNP. Immunofluorescence analysis further revealed that NPM is recruited to sites of viral transcription and replication in infected cells. Additionally, overexpression of NPM resulted in increased viral polymerase activity, indicating its role in viral RNA synthesis. In summary, the proteomics-based approaches used in this study represent powerful tools to identify novel vRNP-associated cellular factors for further characterization.
机译:推测与甲型流感病毒核糖核蛋白(vRNP)相关的细胞因子在病毒生命周期中起重要作用。迄今为止,使用单个vRNP组分(如核蛋白或病毒聚合酶亚基)的相互作用筛选显示出很少的细胞相互作用伴侣。为了改善这种情况,我们进行了基于蛋白质组学的全面筛选,以鉴定与天然vRNP和病毒聚合酶复合物相关的细胞因子。使用带有Strep标签的病毒核蛋白(NP-Strep)作为诱饵从人细胞中纯化重组的vRNP,并通过质谱(MS)鉴定共纯化的细胞因子。平行地,使用串联亲和纯化(TAP)标记的聚合酶亚基作为诱饵,分离了重组的天然甲型流感病毒聚合酶复合物,并再次通过质谱鉴定了共纯化的细胞因子。使用这些技术,我们鉴定了41种与富含NP-Strep的vRNPs共纯化的蛋白和4种与病毒聚合酶复合物共纯化的细胞蛋白。聚合酶相关因子中的两个,importin-beta 3和PARP-1,代表了新型的相互作用伴侣。使用我们的方法还可以鉴定出大多数先前显示与病毒NP和/或vRNP相互作用的细胞蛋白,从而证明了其敏感性。使用选定的新型相互作用伴侣(包括核磷素(NPM))在病毒感染的细胞中进行免疫共沉淀研究,证实了它们与vRNP的关联。免疫荧光分析进一步表明,NPM被募集到病毒在感染细胞中转录和复制的位点。此外,NPM的过表达导致病毒聚合酶活性增加,表明其在病毒RNA合成中的作用。总而言之,本研究中使用的基于蛋白质组学的方法代表了强大的工具,可用于鉴定新型vRNP相关的细胞因子,以进一步表征。

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