CARDIOVIRAL POLY(C) TRACTS AND VIRUS PATHOGENICITY

一系列含有孟戈病毒全长拷贝的cDNA质粒， 已经开发了MengoEMC（脑心肌炎）病毒嵌合基因组 和表征了 分离株彼此不同， 野生型Mengo和EMC病毒序列仅在其5 '非- 编码poly（C）片段。 从这些结构衍生的RNA转录物 对HeLa细胞完全具有感染性， 工程化后代病毒。 然而，在接种小鼠后， 心脏病毒的宿主，携带缩短版本的工程病毒 聚（C）链（例如：C8，C12或C13UC10）的显著性 相对于原始野生型病毒减弱的致病性，或 相对于具有野生型-Mengo长度的新工程化子代病毒， 多聚腺苷酸束（C50UC 10）。 衰减表现为106 - 1010倍 病毒LD50的增加。 此外，接受亚致死剂量的动物 从任何短聚（C）菌株的特征性发展高水平 中和抗体和获得性终身保护性免疫 对抗野生型病毒的（致死）攻击。 这就是我们的目标 建议探索和表征聚（C）的分子基础- 介导的衰减，最终目的是利用这种 现象和从中吸取的原则，为发展 新型有效的小核糖核酸病毒活疫苗。 具体目标是：（1） 用基因工程制造一系列全长的心脏病毒质粒 含有改变的poly（C）片段、不连续性、病毒嵌合 5 '非编码区内的序列、重排和其他变异 （2）测试这些结构对 转录物感染性和子代病毒活力在几个相关的 组织培养细胞系;（3）评估 感染性基因工程子代病毒在小鼠中，为了 评估宿主的保护性反应、病毒病原学、复制， 清除和序列逆转潜力;（4）制定和测试 在分子水平上解释小核糖核酸病毒5 '的作用的假说 非编码序列，特别是poly（C）片段，在限制性 确定病毒-宿主相互作用。 细胞因子的潜在作用 诱导作为增强野生型病毒的贡献因素 将检查感染。

A series of cDNA plasmids containing full-length copies of Mengovirus and MengoEMC (encephalomyocarditis) virus chimeric genomes have been developed and characterized. The isolates differ from each other and from the wildtype Mengo and EMC virus sequences only in the lengths of their 5' non- coding poly(C) tracts. RNA transcripts derived from these constructions are completely infectious to HeLa cells and the resulting genetically engineered progeny viruses. However, upon inoculation of mice, the natural host for cardioviruses, the engineered viruses carrying shortened versions of the poly(C) tract (eg: C8, C12 or C13UC10) exhibit dramatically attenuated pathogenicity relative to the original wildtype viruses or relative to a newly engineered progeny virus with a wildtype-Mengo length poly(C) tract (C50UC10). The attenuation is manifest in a 106-1010 fold increase in the virus LD50. Moreover, animals receiving sublethal doses from any of the short poly(C) strains characteristically develop high level of neutralizing antibodies and acquired lifelong protective immunity against (lethal) challenge with wildtype virus. It is the goal of this proposal to explore and characterize the molecular basis for poly(C)- mediated attenuation with the eventual objective of exploiting this phenomena and the principles to be learned from it, for the development of new and effective, live picornavirus vaccines. The specific aims are: (1) To genetically engineer a spectrum of full-length cardiovirus plasmids containing altered poly(c) tracts, discontinuities, viral chimeric sequences, rearrangements and other variations within the 5' non-coding regions; (2) To test the relative effects of such constructions on transcript infectivity and progeny virus viability in several relevant tissue culture cell lines; (3) To assess the pathogenic effects of infectious genetically engineered progeny viruses in mice, in order to evaluate the host protective responses, viral etiology, replication, clearance and sequence reversion potential; (4) To formulate and test hypotheses to explain at the molecular level, the role of picornaviral 5' non-coding sequences, and in particular poly(C) tracts, in the restrictive determination of virus-host interactions. The potential role of cytokine induction as a contributing element to the enhancement of wildtype viral infections will be examined.