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

描述（由申请人提供）：北美东部马脑炎病毒（EEEV）是托加病毒科的一种阿尔法病毒，被美国国立卫生研究院列为B类优先病原体清单，并被美国农业部列为高后果家畜病原体，因为它对人类和马具有高度致命性，并且由于缺乏有效的疫苗和治疗方法。在神经毒性疾病的小鼠模型中，EEEV感染仅诱导低水平的IFN-，至少部分是由于无法有效感染髓细胞和淋巴组织；一个关键的I型IFN规避策略。EEEV对髓系细胞的早期和深刻的感染障碍发生在来自传入病毒基因组的基因表达水平上（可能也包括后代基因组的翻译），这表明完成复制的这一重要第一步的效率对细胞/组织趋向性有重要贡献。我们最近在《自然》杂志上发表：i) EEEV基因组的翻译在间质细胞中比在髓细胞中效率高约1000倍，这是由3'非翻译区（NTR）的核苷酸序列决定的；ii)髓细胞特异性microRNA miR142-3p在该区域有四个潜在的结合位点，并负责翻译抑制；iii)另一方面，蚊子细胞和蚊子的复制需要相同的3' NTR区域。具体来说，我们建议在体外和体内鉴定并确定EEEV基因组突变对哺乳动物髓系细胞恢复翻译的影响；同样地描述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鉴定和禁用EEEV利用的机制来避免IFN-活性，从而产生不会感染蚊子的免疫原性减毒活疫苗候选物。此外，我们将确定一个高度限制性的miRNA结合域，可用于抗病毒治疗的设计。最后，我们预计我们关于EEEV 3' NTR的亲性决定活性的发现将在其他病毒中得到概括，因此，研究将具有里程碑式的意义

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.