STRUCTURE AND NUCLEOLAR FUNCTION OF SARS N PROTEIN

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。严重急性呼吸综合征冠状病毒（SARS CoV）是一种重要的新发致病因子，但对SARS CoV的致病机制知之甚少。我们提出了一个模型，其中SARS冠状病毒核衣壳(N)蛋白在病毒感染期间定位到宿主细胞的核仁并改变核仁功能：这是一种重要的病毒策略，旨在损害宿主细胞，同时使复制病毒受益。对SARS病毒N蛋白的分析显示了几个被预测参与N蛋白定位到核仁的区域。该项目的第一部分是确定SARS冠状病毒N蛋白是否确实定位于核仁，并表征参与将N转运到细胞核和核仁中的蛋白质结构域。第二部分是与COBRE Core C Protein Purification Group合作，构建N蛋白的详细结构模型，该模型将用于未来的研究，以更好地了解SARS病毒如何引起疾病的机制并确定潜在抗病毒药物的靶点。为了研究SARS冠状病毒感染细胞中的N蛋白，我们研制了几种单克隆抗体（mAb）试剂。令我们惊讶的是，用N特异性单抗46-4染色的感染细胞共聚焦显微镜清楚地显示，N蛋白不能像最初预测的那样定位在细胞核或核仁中。单独表达或用增强型绿色荧光蛋白（EGFP）标记的N也得到了类似的结果。核输运活动的缺乏是一个出乎意料的发现，但在几个实验中得到了证实。尽管SARS冠状病毒的N蛋白被预测为核蛋白，但它并不定位于核仁，而主要保留在细胞质中。我们目前正在研究N的结构，以确定为什么它不能定位到细胞核。这项工作的一个重要的附带好处是创造了几种抗体试剂，这将进一步增强对SARS冠状病毒N蛋白结构和功能的理解，并证明对开发用于诊断SARS冠状病毒感染的N蛋白特异性测试有用。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Severe acute respiratory syndrome coronavirus (SARS CoV) is an important emerging disease-causing agent, but very little is known about how SARS CoV causes disease. We propose a model in which the SARS CoV nucleocapsid (N) protein is proposed to localize to the nucleolus of the host cell during virus infection and alter nucleolar function: an important viral strategy designed to harm the host cell while benefiting the replicating virus. The analysis of the SARS virus N protein shows several regions that are predicted to be involved in the localization of the N protein to the nucleolus. The first part of this project is to determine if SARS CoV N protein does in fact localize to the nucleolus, and to characterize the protein domains involved in transporting N into the nucleus and nucleolus. The second part, in collaboration with the COBRE Core C Protein Purification Group, is to construct a detailed structural model of the N protein, which will be used in future studies to better understand the mechanism of how the SARS virus causes disease and identify targets for potential antiviral drugs. To study the N protein in SARS CoV-infected cells, we developed several monoclonal antibody (mAb) reagents. To our surprise, confocal microscopy of infected cells stained with the N-specific mAb, 46-4, showed clearly that N protein fails to localize in the nucleus or nucleolus as originally predicted. Similar results were obtained for N expressed alone or tagged with enhanced green fluorescent protein (EGFP). Lack of nuclear transport activity was an unexpected finding, but was confirmed in several experiments. Even though the N protein of SARS CoV is predicted to be a nuclear protein, it does not localize to the nucleolus and is primarily retained in the cytoplasm. We are currently investigating the structure of N as a means to determine why it fails to localize to the nucleus. An important side benefit of this work is the creation of several antibody reagents, which will further enhance the understanding of SARS CoV N protein structure and function and prove useful for the development of N protein-specific tests for the diagnosis of SARS CoV infection.