CARDIOVIRAL POLY (C) TRACTS AND VIRUS PATHOGENICITY

描述：微小核糖核酸病毒的心脏和噬菌体病毒属是 病毒的不同之处在于存在长的多嘧啶束 在它们基因组的5‘非编码区内。在心脏病毒中，比如 EMCV和Mengo，这些区域含有几乎纯的胞苷序列 (分别为C115UCUC3UC10和C44UC10)，或“Poly(C)”。遗传 对cDNA的操作清楚地表明聚(C)的特定长度是一个 芒果致病性的关键决定因素。野生型心脏病毒有 高毒力并感染包括灵长类在内的许多动物物种， 啮齿动物和猪。小鼠给药的半数致死量在1-100pfu之间 I.C.相比之下，Mengo Poly(C)的工程缺失已经产生了 LD50为10[6]-10[9]pfu的病毒株(例如VMC[0])。这种衰减 在组织培养中伴随着高度的遗传稳定性 动物。保护性血清转换为长寿疫苗的受者 豁免权。这使得聚(C)现象可以从基因上 用于有效传递其他异源表位 在Mengo cDNA中进行了改造。活的，弱化的嵌合体，携带和 表达1000个额外的核苷酸作为蛋白质编码序列已经被测试。 HIV、SIV和疟疾决定因素在小鼠中是极好的、有效的免疫原 和猴子，当以这种方式交付时。聚(C)的作用机理 衰减仍然是个谜。据认为，野生型病毒依赖于 在他们的长聚(C)S结合和失活哨兵细胞酶， 例如dsRNA激活蛋白激酶(PKR)，其方式类似于 腺病毒VAI RNA。短程病毒，不能引诱或捕获PKR 以同样的效率，似乎无法避免随之而来的抗病毒 细胞的反应，本质上是给宿主接种疫苗而不是杀死 它。支持这一假设的是缺乏PKR基因敲除小鼠的数据 这种必要的基因，因此很容易被野生型杀死 通常减毒的短多聚(C)病毒。这样做的具体目的是 建议有：(1)检验新的“捕鼠器”假说 野生型心脏病毒是致病的，因为它们的长聚(C) 束使前哨细胞酶结合或失活，并 因此避免了抗病毒状态；(2)检查基因 EMCV和Mengo在Poly(C)附近的差异和识别所有局部 可能导致衰减现象的序列；(3) 记录短程Mengo的遗传稳定性和病毒持久性 使用强迫传代技术和突变分析的小鼠；(4)检查 新型Mengo结构的异种承载能力和 嵌合体，意图用作活的减毒疫苗载体 传输系统。

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.