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

描述(申请人提供)：北美东部马脑炎病毒(EEEV)是Togaviridae科的一种甲型病毒，被NIH优先病原体名单归类为B类病毒，并被美国农业部列为高后果牲畜病原体，因为它对人和马具有高度致命性，而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感染只能诱导低水平的干扰素-�，至少部分原因是无法有效地感染髓系细胞和淋巴组织；这是一种关键的I型干扰素逃避策略。EEEV对髓系细胞的感染性早期和深刻的阻断发生在传入病毒基因组的基因表达水平(可能也包括后代基因组的翻译)，这表明完成这一必要的第一步复制的效率对细胞/组织趋向性有重要贡献。我们最近在《自然》杂志上发表了以下文章：i)EEEV基因组的翻译效率在间质细胞中比在髓系细胞中高出约1000倍，这是由3‘非翻译区(NTR)的核苷酸序列决定的；ii)髓系细胞特异的microRNA miR142-3p在该区域有四个潜在的结合位点，并负责这种翻译抑制；iii)另一方面，在蚊子细胞和蚊子中复制需要3’NTR的相同区域。具体地说，我们建议在体外和体内鉴定和确定EEEV基因组中恢复哺乳动物髓系细胞翻译的突变的影响；并类似地表征这些突变对蚊子细胞复制能力的影响。我们预计，这些研究将允许识别和禁用EEEV用来避免干扰素-�活性的机制，从而产生不会感染蚊子的免疫原性、减毒活疫苗候选。此外，我们还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趋向性决定活性的发现将在其他病毒中重现，因此，研究将 适用范围广。

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.