Packaging of Ebola Virus RNA into Budding VLPs

描述：埃博拉病毒是一种与严重出血热相关的新出现的人类病原体，由于其可能被用作生物恐怖主义制剂，因此仍然是一种威胁。目前，既没有疫苗，也没有抗病毒药物可用于预防或治疗埃博拉病毒感染。本实验室成功利用病毒样颗粒（VLP）研究了埃博拉病毒基质蛋白VP40晚期结构域在VP40 VLP出芽中的作用。我们还描述了其他病毒和宿主成分在VP40 VLP出芽中的作用。基于我们研究VP40 VLP出芽的丰富经验，我们假设我们的VP40 VLP出芽试验可以用来识别埃博拉病毒3E-5E小基因组RNA的包装信号，从而可以作为基于VLP的疫苗开发载体或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小基因组RNA包装成出芽VLPs所需的最小病毒蛋白。我们提出通过产生3E-5E小基因组突变体来鉴定埃博拉病毒RNA包装信号，并在改进的VP40 VLP出芽试验中对其进行检测。我们进一步的目标是确定VP35的包装和与3E-5E小基因组RNA的相互作用以及与VP40的相互作用所需的区域。一旦包装信号被识别，就会产生一个模型RNA，其中包含包装信号以及感兴趣的蛋白质，如红色荧光蛋白。然后，我们将测试我们的VLP在组织培养中转导细胞的能力，并从我们的模型RNA中产生蛋白质。这种系统的开发将增加我们对埃博拉病毒基因组包装分子需求的了解，并可作为抗原递送平台，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.