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

摘要 疱疹病毒的组装严重依赖于病毒蛋白之间的特定相互作用。在核能领域 Exress是由pUL31和pUL34组成的NEC杂二聚体，具有多种功能并相互作用 与多种其他病毒因素有关。然而，在许多情况下，机械的后果和意义 这些相互作用是未知的。类似地，细胞质被膜依赖于一种 少数必需的被膜蛋白(HSV-1中的VP16、pUL36和pUL37)，但它们之间的哪些相互作用(其他 比彼此)更重要，因为这个过程是未知的，尽管有广泛的交互伙伴 通过蛋白质组学和其他方法进行鉴定。在这里，我们建议使用一种新颖的定向进化 关键相互作用的功能识别方法。我们用一个必要的单纯疱疹病毒基因替换它 来自VZV的同源基因，然后对可以使用VZV同源基因用于 集合。在这种定向进化过程中发生的突变的图谱可以识别新的 功能上的相互作用，并确立他人的意义。我们提供的初步数据表明， 用VZV ORF24取代HSV-1 UL34嵌合病毒的方法的实用性。我们有 确定了与HSV-1 ICP4的一种新的功能相互作用，并验证了之前 报告了与ICP22的相互作用。我们建议确定这些关键因素的作用机制 互动，并使用系统来识别其他人。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点是pUL31。

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.