Mechanism of alphavirus packaging: designing of pseudoinfectious viruses

性状（由申请方提供）：披膜病毒科甲病毒属包含多种广泛分布的重要人类和动物病原体。委内瑞拉（VEEV）、东部（EEEV）和西部（WEEV）马脑炎病毒是美国严重的公共卫生威胁，可被生物恐怖分子应用。然而，迄今为止，既没有有效的抗病毒药物，也没有安全有效的疫苗来预防这些感染。现有的实验性疫苗要么效力差，要么在人体中表现出非常强烈的不良反应。我们最近对甲病毒RNA包装的分子机制的研究导致了对甲病毒RNA特异性包装信号的结构和功能的理解，该信号存在于其他致脑炎甲病毒的基因组中，并被同源蛋白和衣壳蛋白识别。这些数据将应用于拟议的研究中，以开发设计具有不可逆的高度减毒表型但产生病毒特异性抗原的活甲病毒变体的全新策略，所述病毒特异性抗原是与野生型病毒一样有效地诱导灭菌免疫所需的。通过将多个合理设计的突变引入衣壳蛋白或新鉴定的通用RNA包装信号，我们将开发出保留高水平RNA复制的甲病毒，并产生主要释放（策略1）或 仅以非感染性、无基因组的病毒样颗粒的形式（策略2），因此， 在体内不产生病毒血症。这些病毒将能够诱导细胞免疫应答和高水平中和抗体的平衡组合。

DESCRIPTION (provided by applicant): The Alphavirus genus in the Togaviridae family contains a variety of widely distributed important human and animal pathogens. Venezuelan (VEEV), eastern (EEEV) and western (WEEV) equine encephalitis viruses represent a serious public health threat in the US and can be applied by bioterrorists. However, to date, neither effective antiviral nor safe and efficient vaccines have been developed for preventing any of these infections. The existing experimental vaccines are either of poor efficacies or demonstrate very strong adverse reactions in humans. Our recent studies of the molecular mechanism of alphavirus RNA packaging led to understanding of structure and function of the alphavirus RNA-specific packaging signal, which is present in the genomes of other encephalitogenic alphaviruses and recognized by both homologous and capsid proteins. These data will be applied in the proposed research to develop fundamentally new strategies of designing live alphavirus variants possessing irreversible, highly attenuated phenotypes, but producing virus-specific antigens, required for induction of sterilizing immunity as efficiently as a wt virus. By introducing multiple, rationally designed mutations into capsid protein or newly identified universal RNA packaging signal, we will develop alphaviruses that retain high levels of RNA replication, and production of structural proteins which are released either mostly (strategy 1) or exclusively (strategy 2) in the form of non-infectious, genome-free virus-like particles and, thus, develop no viremia in vivo. These viruses will be capable of inducing a balanced combination of cellular immune response and high levels of neutralizing antibodies.