Pseudotyped virus to study SARS virus entry

严重急性呼吸综合征（SARS）的病原体已被确定为冠状病毒（SARS-CoV）。虽然完整的基因组序列已经在很短的时间内被确定，但在开发抗病毒策略所必需的病毒蛋白质功能的理解方面仍然存在很大的差距。表面糖蛋白S对于冠状病毒的发病机制至关重要。它是冠状病毒感染引起的保护性免疫反应的主要目标。此外，它还参与复制周期的早期和晚期阶段。在感染开始时，S蛋白介导病毒体与细胞表面上的受体的结合以及随后的病毒和细胞膜之间的融合事件。我们计划利用我们的专业知识产生水泡性口炎病毒（VSV）假型，其中包含SARS-CoV的S蛋白，而不是VSV G蛋白。代替G基因，VSV基因组包含GFP基因，其表达允许通过荧光显微镜容易地检测感染的细胞。由于S蛋白是通过共转染相应的基因反式提供的，因此假型只能进行单个复制循环。新形成的病毒粒子不含表面糖蛋白，因此不具有感染性。这种方法允许独立于其他病毒蛋白分析S蛋白，并且可以在比SARS-CoV实验所需的更低的生物安全水平条件下应用。假型将用于分析S蛋白在与靶细胞相互作用中的功能。此外，将在小鼠和猴子中分析这些病毒样颗粒作为SARS-CoV感染疫苗的潜力。假型也应该是一个有趣的工具，以筛选针对SARS冠状病毒的S蛋白的抗病毒药物。

The etiologic agent involved in severe acute respiratory syndrome (SARS) has been identified as a coronavirus (SARS-CoV). Though the complete genomic sequence has been determined in a short time, there are still large gaps in the understanding of functions of the viral proteins which is necessary to develop antiviral strategies. The surface glycoprotein S is of major importance for the pathogenesis of coronaviruses. It is the main target of the protective immune response elicited by a coronavirus infection. Furthermore it is involved in the early and late stages of the replication cycle. In the initiation of an infection, the S protein mediates the binding of the virion to receptors on the cell surface and the subsequent fusion event between the viral and the cellular membrane. We plan to use our expertise to generate vesicular stomatitis virus (VSV) pseudotypes that contain the S protein of SARS-CoV instead of the VSV G protein. In place of the G gene, the VSV genome contains the GFP gene the expression of which allows easy detection of infected cells by fluorescence microscopy. As the S protein is provided in trans by cotransfection of the corresponding gene, the pseudotypes are only able to perform a single replication cycle. Newly formed virions do not contain a surface glycoprotein and therefore are not infectious. This approach allows to analyze the S protein independent of other viral proteins and it can be applied at lower biosafety level conditions than are required for experiments performed with SARS-CoV. The pseudotypes will used to analyze the function of the S protein in the interaction with target cells. Furthermore, the potential of these virus-like particles as a vaccine against SARS-CoV infection will be analyzed in mice and monkeys. Pseudotypes should also be an interesting tool to screen for antivirals directed against the S protein of SARS-CoV.