HSV/VZV chimeric viruses for identifying critical virus herpesvirus assembly interactions

总结 疱疹病毒的装配严重依赖于病毒蛋白之间的特异性相互作用。核 出口，NEC异源二聚体，由pUL 31和pUL 34组成，执行多种功能，并相互作用 与其他多种病毒因子的混合然而，在许多情况下， 这些相互作用是未知的。类似地，细胞质的表达依赖于细胞质中的蛋白质之间的相互作用。 一些必需的被膜蛋白（HSV-1中的VP 16、pUL 36和pUL 37），但它们之间的相互作用（其他 尽管有各种各样的互动伙伴， 通过蛋白质组学和其他方法鉴定。在这里，我们建议使用一种新的定向进化 关键相互作用的功能鉴定方法。我们用它的基因替换了一个基本的HSV基因， 从VZV同源物中筛选病毒，然后对可以使用VZV同源物的病毒进行生长选择， 组装件.在这种定向进化过程中发生的突变的映射可以鉴定新的突变。 功能性互动，并确立他人的重要性。我们提供的初步数据显示， 使用嵌合病毒的这种方法的实用性，其中HSV-1 UL 34被VZV ORF 24取代。我们有 鉴定了与HSV-1 ICP 4的一种新的功能性相互作用，并验证了以前的研究结果的重要性。 报告了与ICP 22的相互作用。我们建议确定这些关键的作用机制， 互动，并使用该系统来识别其他人。此外，我们建议扩大这个系统的使用范围 另一个与核出口相关的基因靶pUL 31。

SUMMARY Herpesvirus assembly is critically dependent on specific interactions between viral proteins. In nuclear egress, the NEC heterodimer, consisting of pUL31 and pUL34, performs multiple functions and interacts with multiple other viral factors. In many cases, however, the mechanistic consequence and significance of those interactions is unknown. Similarly, cytoplasmic envelopment depends upon interactions made by a few essential tegument proteins (VP16, pUL36 and pUL37 in HSV-1), but which of their interactions (other than with each other) are important for this process is unknown despite a wide array of interaction partners identified by proteomic and other approaches. Here we propose to use a novel directed evolution approach for functional identification of crucial interactions. We replace an essential HSV gene with its homolog from VZV and then perform a growth selection for viruses that can use the VZV homolog for assembly. Mapping of the mutations that have occurred during this directed evolution can identify novel functional interactions and establish the significance of others. We present preliminary data showing the utility of this approach using a chimeric virus in which HSV-1 UL34 is replaced by VZV ORF24. We have identified a novel functional interaction with HSV-1 ICP4 and validated the significance of a previously reported interaction with ICP22. We propose to determine the mechanism of action of these critical interactions and use the system to identify others. In addition, we propose to expand the use of this system another gene target relevant to nuclear egress, pUL31.