Essential early events in the flavivirus lifecycle

项目摘要 节肢动物传播的黄病毒，如登革热病毒、西尼罗河病毒、黄热病病毒和寨卡病毒 病毒，是人类疾病的主要原因。我们正在研究早期、融合后和复制前 通过使用黄病毒原型YFV作为我们的模型来研究黄病毒生命周期中的事件。我们最重要的是 假设是黄病毒在脊椎动物和无脊椎动物宿主中交替复制， 进化到使用i)在宿主之间共享的高度保守的因子；以及ii)多个冗余因子 这可能不会在主机之间得到很好的保存。因此，YFV的发现将是 通过与其他黄病毒以及人和蚊子细胞的比较来验证和研究。 目的1研究核衣壳脱壳的后融合过程，剖析核衣壳脱壳的机制。 是哪些细胞解锁了这一决定命运的货物。目标1是我们初步数据的合乎逻辑的扩展，表明 可翻译YFV基因组的传递需要细胞泛素化和VCP/p97，一种细胞 从大分子复合体中提取泛素化蛋白质的ATPase。目标2侧重于 确定裂解病毒NS1-2A多蛋白中间体的细胞蛋白酶(S)。乳沟是 对黄病毒复制是必不可少的(如图所示)，但这种蛋白水解酶的特性仍然难以捉摸 已经二十多年了。我们已经确定了一个相关候选NS1-2A蛋白水解酶的小家族 正在通过严格的组合基因消融来验证它们的活动。这些努力将解决长期的- 黄病毒基因表达和复制的困惑及未来靶点的确定 开发广效抗病毒药物。

Project Summary Arthropod-borne flaviviruses such as dengue virus, West Nile virus, yellow fever virus (YFV), and Zika virus, are a major cause of human disease. We are studying the early, post-fusion and pre-replication events in the flavivirus lifecycle by using the prototype flavivirus, YFV, as our model. Our overarching hypothesis is that flaviviruses, which alternately replicate in vertebrate and invertebrate hosts, have evolved to use i) highly conserved factors shared between hosts; and ii) multiple, redundant factors that may not be well conserved between hosts. Therefore, discoveries made with YFV will be validated and studied by comparison to other flaviviruses and between human and mosquito cells. Aim 1 focuses on the post-fusion process of nucleocapsid uncoating, dissecting the mechanisms by which cells unlock this fateful cargo. Aim 1 is a logical extension of our Preliminary Data showing that the delivery of a translatable YFV genome requires cellular ubiquitylation and VCP/p97, a cellular ATPase that extracts ubiquitylated proteins from large macromolecular complexes. Aim 2 focuses on identifying the cellular protease(s) that cleave the viral NS1-2A polyprotein intermediate. Cleavage is essential for flavivirus replication (shown here), yet the identity of this protease has remained elusive for over two decades. We have identified a small family of related candidate NS1-2A proteases and are validating their activity by rigorous, combinatorial genetic ablation. These efforts will solve long- standing puzzles in flavivirus gene expression and replication and identify targets for future development of broadly acting antivirals.