Subcellular Localization and Function of an Epitope-Tagged p7 Viroporin in Hepatitis C Virus-Producing Cells
Subcellular Localization and Function of an Epitope-Tagged p7 Viroporin in Hepatitis C Virus-Producing Cells
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DOI：
10.1128/jvi.02782-12
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发表时间：
2012-11
影响因子：
5.4
通讯作者：
G. Vieyres;C. Brohm;M. Friesland;Juliane Gentzsch;B. Wölk;P. Roingeard;E. Steinmann;T. Pietschmann
中科院分区：
文献类型：
--
作者：
G. Vieyres;C. Brohm;M. Friesland;Juliane Gentzsch;B. Wölk;P. Roingeard;E. Steinmann;T. Pietschmann
摘要丙型肝炎病毒（HCV）病毒孔蛋白p7在感染性病毒子代的产生中起着重要作用。然而，它在病毒复制周期中的作用仍然不完全清楚，部分原因是p7特异性抗体的可用性差。为了克服这一障碍，我们在其N端插入了两个连续的血凝素（HA）表位标签。HA标记的p7降低了约100%的峰值病毒滴度。10-与野生型病毒相比，病毒生产的动力学增加了一倍。然而，HA标记的p7挽救了缺乏p7的突变病毒的病毒生产，从而提供了标签不破坏p7功能的正式证据。在HCV产生细胞中，p7显示出网状染色模式，与HCV包膜糖蛋白2（E2）共定位，但也部分与病毒非结构蛋白2，3和5A。使用免疫共沉淀，我们证实了p7和NS 2之间的特异性相互作用，而我们没有检测到与核心，E2或NS 5A的稳定相互作用。此外，我们没有观察到p7掺入亲和纯化的病毒颗粒。一致地，没有证据支持p7在病毒进入中的作用，因为抗HA抗体不能中和从HA-p7标记的基因组产生的Jc 1病毒。总的来说，这些发现突出了p7和NS 2之间的稳定相互作用，这可能对感染性HCV颗粒的产生至关重要。使用这种功能性表位标记的p7变体应有助于分析HCV复制周期的最后步骤。
ABSTRACT The hepatitis C virus (HCV) viroporin p7 is crucial for production of infectious viral progeny. However, its role in the viral replication cycle remains incompletely understood, in part due to the poor availability of p7-specific antibodies. To circumvent this obstacle, we inserted two consecutive hemagglutinin (HA) epitope tags at its N terminus. HA-tagged p7 reduced peak virus titers ca. 10-fold and decreased kinetics of virus production compared to the wild-type virus. However, HA-tagged p7 rescued virus production of a mutant virus lacking p7, thus providing formal proof that the tag does not disrupt p7 function. In HCV-producing cells, p7 displayed a reticular staining pattern which colocalized with the HCV envelope glycoprotein 2 (E2) but also partially with viral nonstructural proteins 2, 3, and 5A. Using coimmunoprecipitation, we confirmed a specific interaction between p7 and NS2, whereas we did not detect a stable interaction with core, E2, or NS5A. Moreover, we did not observe p7 incorporation into affinity-purified virus particles. Consistently, there was no evidence supporting a role of p7 in viral entry, as an anti-HA antibody was not able to neutralize Jc1 virus produced from an HA-p7-tagged genome. Collectively, these findings highlight a stable interaction between p7 and NS2 which is likely crucial for production of infectious HCV particles. Use of this functional epitope-tagged p7 variant should facilitate the analysis of the final steps of the HCV replication cycle.