Characterization of microRNA binding sites in the eastern equine encephalitis virus 3'NTR

描述（由申请方提供）：北美东部马脑炎病毒（EEEV）是披膜病毒科的一种甲病毒属，被归为NIH优先病原体列表的B类，并且由于其对人类和马具有高度致死性，并且缺乏有效的疫苗和疗法，因此被USDA归为高后果牲畜病原体。在神经毒性疾病的小鼠模型中，EEEV感染仅诱导低水平的IFN-γ，至少部分是由于“失败”而不能有效感染骨髓细胞和淋巴组织;这是一种关键的I型IFN逃避策略。EEEV对骨髓系细胞的感染性的早期和深刻的阻断发生在来自传入病毒基因组的基因表达水平上（并且推测也是子代基因组的翻译），这表明复制中这一必要的第一步完成的效率显著有助于细胞/组织向性。我们最近在Nature上发表了以下内容：i）EEEV基因组的翻译在间充质细胞中的效率比在髓样细胞中的效率高约1,000倍，这是由3'非翻译区（NTR）中的核苷酸序列决定的; ii）髓样细胞特异性microRNA，miR 142 - 3 p，在该区域具有四个潜在的结合位点，并且负责这种翻译抑制;和iii）另一方面，3 ′ NTR的相同区域是在蚊子细胞和蚊子中复制所必需的。具体来说，我们建议确定和确定的EEEV基因组中的突变，恢复在哺乳动物骨髓细胞在体外和体内的翻译的影响;和类似的特点，这些突变对蚊子细胞复制能力的影响。我们预计这些研究将允许EEEV利用的机制的识别和禁用，以避免IFN-γ活性，因此，产生免疫原性，减毒活疫苗候选人，不会感染蚊子。此外，我们将确定一个高度限制性的miRNA结合域，可用于设计抗病毒治疗。最后，我们预计我们关于EEEV 3' NTR的向性决定活性的发现将在其他病毒中重现，因此，这些研究将具有 广泛的适用性。

DESCRIPTION (provided by applicant): North American eastern equine encephalitis virus (EEEV), an Alphavirus in the family Togaviridae, is classified in Category B of the NIH Priority Pathogens List, and as a high consequence livestock pathogen by the USDA because it is highly lethal for humans and equines, and because effective vaccines and therapies are lacking. In a mouse model of neurovirulent disease, EEEV infection induces only low levels of IFN-�, at least in part as a consequence of the "failure" to productively infect myeloid cells and lymphoid tissues; a critical type I IFN evasion strategy. An early and profound block on EEEV infectivity for myeloid-lineage cells occurs at the level of gene expression from the incoming vira genome (and presumably also translation of progeny genomes), suggesting that the efficiency with which this essential first step in replication is completed contributes significantly to cell/tissue tropism. We have recently published in Nature that: i) translation of the EEEV genome occurs with ~1,000-fold greater efficiency in mesenchymal than in myeloid cells dictated by nucleotide sequences in the 3' non-translated region (NTR); ii) myeloid cell-specific microRNA, miR142-3p, has four potential binding sites in this region and is responsible for this translation inhibition; and iii) on the other hand, the same region of the 3' NTR is required for replication in mosquito cells and mosquitoes. Specifically, we propose to identify and determine the effect of mutations in the EEEV genome that restore translation in mammalian myeloid-lineage cells in vitro and in vivo; and similarly characterize the effects of these mutations on mosquito cell replication capability. We anticipate that these studies will allow the identificatio and disablement of mechanisms utilized by EEEV to avoid IFN-� activity and, therefore, generate immunogenic, live-attenuated vaccine candidates that will not infect mosquitoes. Also, we will identify a highly restrictive miRNA binding domain which can be used for design of antiviral therapeutics. Finally, we anticipate that our findings regarding the tropism-determining activity of the EEEV 3' NTR will be recapitulated in other viruses and, thus, the studies will have broad applicability.