The Intraviral Protein Interaction Network of Hepatitis C Virus
The Intraviral Protein Interaction Network of Hepatitis C Virus
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DOI：
10.1074/mcp.m113.036301
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发表时间：
2014-07-01
影响因子：
7
通讯作者：
Schindler, Michael
中科院分区：
文献类型：
--
作者：
Hagen, Nicole;Bayer, Karen;Schindler, Michael
丙型肝炎病毒（HCV）是一个全球性的健康问题，也是导致肝硬化、肝细胞癌等慢性肝病的主要原因之一。HCV基因组被翻译成多蛋白，该多蛋白被蛋白水解加工成10种病毒蛋白。HCV蛋白与宿主细胞的相互作用组已经确定;然而，病毒蛋白如何相互作用仍不清楚。我们的目的是使用我们实验室建立的基于流式细胞术的FRET测定法产生这10种HCV蛋白的相互作用网络（Banning，C.，Votteler，J.，霍夫曼，D.，Koppensteiner，H.，温暖，M，Reimer，R.，Kirchhoff，F.，舒伯特，美国，Hauber，J.，和Schindler，M.（2010）基于流式细胞术的FRET测定，以鉴定和分析活细胞中的蛋白质-蛋白质相互作用。将HCV蛋白构建为与发色团CFP和YFP的融合物。所有的HCV融合表达和定位到特定的亚细胞区室，表明它们的功能。FACS-FRET测量确定了总共20种相互作用;其中13种先前已经描述过，现在已经通过我们的方法在活细胞中得到证实。在7种新的蛋白结合对中，HCV p7起着关键作用。它与HCV衣壳蛋白Core和两种糖蛋白E1和E2结合。这些相互作用在表达感染性HCV的Huh7.5肝细胞的相关背景中得到进一步证明。我们的工作证明了通过FACS-FRET快速生成小相互作用网络的可行性，并定义了HCV蛋白质内相互作用的网络。此外，我们的数据支持p7在HCV组装中的重要作用。
Hepatitis C virus (HCV) is a global health problem and one of the main reasons for chronic liver diseases such as cirrhosis and hepatocellular carcinoma. The HCV genome is translated into a polyprotein which is proteolytically processed into 10 viral proteins. The interactome of the HCV proteins with the host cell has been worked out; however, it remains unclear how viral proteins interact with each other. We aimed to generate the interaction network of these 10 HCV proteins using a flow-cytometry-based FRET assay established in our laboratory (Banning, C., Votteler, J., Hoffmann, D., Koppensteiner, H., Warmer, M., Reimer, R., Kirchhoff, F., Schubert, U., Hauber, J., and Schindler, M. (2010) A flow cytometry-based FRET assay to identify and analyse protein-protein interactions in living cells. PLoS One 5, e9344).HCV proteins were constructed as fusions with the chromophores CFP and YFP. All HCV fusions were expressed and localized to specific subcellular compartments, indicating that they were functional. FACS-FRET measurements identified a total of 20 interactions; 13 of these were previously described and have now been confirmed in living cells via our method. Among the seven novel protein binding pairs, HCV p7 plays a pivotal role. It binds to the HCV capsid protein Core and the two glycoproteins E1 and E2. These interplays were further demonstrated in the relevant context of Huh7.5 liver cells expressing infectious HCV.Our work demonstrates the feasibility of rapidly generating small interaction networks via FACS-FRET and defines the network of intra-HCV protein interactions. Furthermore, our data support an important role of p7 in HCV assembly.