Packaging of Ebola Virus RNA into Budding VLPs

描述：埃博拉病毒是一种新出现的人类病原体，与严重出血热有关，由于它可能被用作生物恐怖主义制剂，因此仍是一种威胁。目前，既没有疫苗，也没有抗病毒药物来预防或治疗埃博拉病毒感染。本实验室已成功地利用病毒样颗粒(VLP)研究了埃博拉病毒基质蛋白VP40晚期结构域在VP40 VLP萌发中的作用。我们还描述了其他病毒和宿主成分在VP40 VLP萌发中的作用。根据我们研究VP40 VLP萌发的丰富经验，我们假设我们的VP40 VLP萌发试验可以用于识别埃博拉病毒3E-5E微型基因组RNA的包装信号，从而可以作为基于VLP的载体用于疫苗开发或作为基因治疗载体的平台。我们的初步数据表明，VP40和VP35是将3E-5E微型基因组RNA包装成萌芽VLP所需的最低病毒蛋白。我们建议通过产生3E-5E微型基因组的突变体来识别埃博拉病毒RNA包装信号，并在我们改进的VP40 VLP萌发试验中对它们进行测试。我们进一步的目标是确定VP35的包装所需的区域，以及与3E-5E微型基因组RNA的相互作用，以及与VP40的相互作用。一旦识别了包装信号，就会产生一个模型RNA，其中包含包装信号和感兴趣的蛋白质，如红色荧光蛋白。然后，我们将测试我们的VLP在组织培养中转换细胞和从我们的模型RNA中生产蛋白质的能力。这种系统的开发将增加我们对埃博拉病毒基因组包装的分子要求的了解，并可用作抗原递送平台，以产生有效的体液和细胞免疫反应。

DESCRIPTION: Ebola virus is an emerging human pathogen that is associated with a severe hemorrhagic fever and remains a threat due to its possible use as a bioterrorism agent. Currently, there are neither vaccines, nor antivirals available to prevent or treat Ebola virus infections. Our laboratory has successfully used virus like particles (VLPs) to study the role of Ebola virus matrix protein VP40 late domains in VP40 VLP budding. We have also described a role for other viral and host components in VP40 VLP budding. Based on our considerable experience investigating VP40 VLP budding we hypothesize that our VP40 VLP budding assay can be utilized to identify the packaging signal of the Ebola virus 3E-5E minigenome RNA, and thus could serve as a platform for a VLP based vector for vaccine development or as a gene therapy vector. Our preliminary data indicate that VP40 and VP35 are the minimal viral proteins required to package the 3E-5E minigenome RNA into budding VLPs. We propose to identify the Ebola virus RNA packaging signal by generating mutants of the 3E-5E minigenome and test them in our modified VP40 VLP budding assay. We further aim to identify regions of VP35 required for packaging and interactions with the 3E-5E minigenome RNA as well as interactions with VP40. Once the packaging signal is identified, a model RNA will be generated containing the packaging signal as well as a protein of interest such as red fluorescent protein. We will then test our VLP for its ability to transduce cells in tissue culture and produce protein from our model RNA. Development of such a system would increase our understanding of the molecular requirements of Ebola virus genome packaging and could be used as an antigen delivery platform to produce an efficacious humoral and cellular immune response.