Roles of Experimentally Evolved Vaccinia Mutations in Antagonizing Cell Defenses

描述（由申请人提供）：除了作为病原体、疫苗载体和溶瘤剂的医学重要性外，痘病毒还提供了易于处理的模型系统，用于发现病毒与其寄生的宿主细胞之间“军备竞赛”的基本分子和进化机制。这些冲突推动宿主和病毒的快速进化 蛋白质，并可以创建物种屏障病毒传播。其中一种情况是dsRNA激活的蛋白激酶R（PKR）途径，它具有广泛的抗病毒活性，但可以被多种病毒因子以物种特异性方式阻断。实验进化与深度测序技术相结合，为揭示PKR及其拮抗剂之间军备竞赛的新因素和机制提供了一种强有力的策略。例如，编码在一种细胞类型中仅弱阻断PKR的拮抗剂的牛痘病毒（VV）突变体的连续传代产生了不仅能够在用于连续传代的细胞中而且能够在来自其他物种的细胞中更有效地复制的子代病毒。进一步的研究表明，编码弱拮抗剂的基因的扩增或其他两个VV基因A24 R和A35 R中的任一个中的突变，这两个基因先前都没有被牵连作为PKR途径的调节剂，显着改善了来自多个物种的细胞中的VV复制。这些研究旨在阐明A24 R和A35 R突变使VV能够克服PKR和相关宿主防御系统的机制。因此，该项目将揭示这些保守的痘病毒基因的功能和PKR途径及其VV拮抗剂的作用和调节的新见解。更一般地说，这些结果将有助于了解大型DNA病毒如何进化以克服宿主防御，并将对了解病毒跨物种传播的风险和障碍产生影响。

DESCRIPTION (provided by applicant): In addition to their medical importance as pathogens, vaccine vectors and oncolytic agents, poxviruses offer tractable model systems for discovering fundamental molecular and evolutionary mechanisms underlying the "arms races" between viruses and the host cells they parasitize. These conflicts drive rapid evolution of host and viral proteins and can create species barriers to viral transmission. One such case is the dsRNA-activated protein kinase R (PKR) pathway, which has broad antiviral activity but can be blocked, in a species-specific manner, by a variety of viral factors. Experimental evolution coupled with deep sequencing technology offers a powerful strategy for uncovering new factors and mechanisms underlying the arms race between PKR and its antagonists. For example, serial passage of a vaccinia virus (VV) mutant that encodes an antagonist that only weakly blocks PKR in one cell type yielded progeny viruses that were able to replicate much more efficiently not only in the cells used for the serial passage but also in cells from other species. Further studies revealed that amplification of the gene encoding the weak antagonist or mutations in either of two other VV genes, A24R and A35R, neither of which has previously been implicated as a modulator of the PKR pathway, dramatically improves VV replication in cells from multiple species. The proposed studies aim to elucidate the mechanisms by which mutations in A24R and A35R enable VV overcome the PKR and related host defense systems. Thus, this project will reveal new insights into the functions of these conserved poxvirus genes and into the roles and regulation of the PKR pathway and its VV antagonists. More generally, the results will aid in understanding how large DNA viruses can evolve to overcome host defenses and will have implications for understanding the risks and barriers to cross species transmission of viruses.