CARDIOVIRAL POLY (C) TRACTS AND VIRUS PATHOGENICITY

描述：小核糖核酸病毒属的心脏病毒属和口疮病毒属是 在病毒中以长的多聚嘧啶段的存在而区别 在其基因组的5'非编码区内。 在心脏病毒中， EMCV和Mengo，片段含有几乎纯的胞苷序列 （分别为C115 UCUC 3UC 10和C44 UC 10）或“Poly（C）。“基因 对cDNA的操作清楚地表明，poly（C）的特定长度是一个重要的基因。 Mengo致病性的关键决定因素 野生型心脏病毒是 高毒性并感染许多动物物种，包括灵长类动物， 啮齿动物和猪 当施用时，鼠LD 50在1-100 pfu之间 I.C.相比之下，Mengo poly（C）的工程缺失产生了 LD 50为10[6]-10 9] pfu的病毒株（例如vMC[0]）。 这种衰减 伴随着组织培养中高度的遗传稳定性， 动物 接种受体保护性血清转化， 免疫力 这使得poly（C）现象在基因上 用于有效递送其它异源表位 在门戈cDNA中进行改造。 活的减毒嵌合体， 表达1000个额外的核苷酸作为蛋白质编码序列已被测试。 HIV、SIV和疟疾决定因子是小鼠中极好的、有效的免疫原 和猴子的时候，以这种方式交付。 poly（C）的作用机理 衰减仍然是个谜。 有人提出，野生型病毒依赖于 在它们的长poly（C）上结合并抑制哨兵细胞酶， 例如dsRNA活化的蛋白激酶（PKR）， 腺病毒VAI RNA。 短束病毒不能引诱或诱捕PKR 同样的效率，似乎无法避免随之而来的抗病毒 细胞的反应，本质上，接种宿主而不是杀死 了 支持这一假设的是PKR敲除小鼠的数据， 这种必需基因，因此易受野生型样杀伤， 通常减毒的短聚（C）病毒。 具体目标是 建议是：（1）测试新的“捕鼠器”假设，预测 野生型心脏病毒是致病性的，因为它们的长poly（C） 纤维束能够结合或灭活前哨细胞酶， （2）避免病毒状态;（3）检查遗传 EMCV和Mengo在poly（C）附近的差异，并确定所有局部 可能导致衰减现象的序列;（3） 记录短束门戈的遗传稳定性和病毒持久性， 小鼠使用强制传代技术和回复突变分析;（4）检查 新的门戈构造的异源承载能力， 嵌合体，目的是用作活的减毒疫苗载体 运载系统。

DESCRIPTION: The cardio-and aphthovirus genera of picornaviruses are distinguished among viruses by the presence of long, polypyrimidine tracts within the 5' non-coding regions of their genomes. In cardioviruses, like EMCV and Mengo, the tracts contain virtually pure cytidine sequences (C115UCUC3UC10 and C44UC10, respectively), or "Poly(C)." Genetic manipulation of cDNAs has clearly shown the specific length of poly(C) is a critical determinant of Mengo pathogenicity. Wild-type cardioviruses are highly virulent and infect many species of animals, including primates, rodents and pigs. The murine LD50 is between 1-100 pfu when administered i.c. In contrast, engineered deletion of the Mengo poly(C) has produced viral strains (e.g. vMC[0]) with LD50s of 10[6]-10[9] pfu. This attenuation is accompanied by a high degree of genetic stability in tissue culture and animals. Inoculated recipients protectively seroconvert with long-lived immunity. This has allowed the poly(C) phenomenon to be genetically harnessed for the effective delivery of other heterologous epitopes engineered within the Mengo cDNAs. Live, attenuated chimeras that carry and express 1000 extra nucleotides as protein-coding sequences have been tested. HIV, SIV, and malaria determinants are excellent, potent immunogens in mice and monkeys when delivered in this manner. The mechanism of poly(C) attenuation remains enigmatic. It is proposed that wild-type viruses rely on their long poly(C)s to bind and inactivate sentinel cellular enzymes, such as dsRNA-activated protein kinase (PKR) in a manner analogous to adenovirus VAI RNAs. The short-tract viruses, unable to lure or trap PKR with the same efficiency, seem unable to avoid a consequent antiviral response by the cells, and in essence, vaccinate the host instead of killing it. In support of this hypothesis are data with PKR knockout mice that lack this essential gene, and thus are susceptible to wild-type like killing by the normally attenuated short poly(C) viruses. The specific aims of this proposal are: (1) to test the novel "mousetrap" hypothesis which predicts that wild-type cardioviruses are pathogenic because their long poly(C) tracts enable binding or inactivation of sentinel cellular enzymes and consequent avoidance of an antiviral state; (2) to examine the genetic differences between EMCV and Mengo near the poly(C) and identify all local sequences which may contribute to the attenuation phenomenon; (3) to document the genetic stability and viral persistence of short-tract Mengo in mice using forced-passage techniques and revertant analysis; (4) to examine the heterologous carrying capacity of novel Mengo constructions and chimeras, with the intent of exploitation as live, attenuated vaccine-vector delivery systems.