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

摘要 疱疹病毒的组装很大程度上取决于病毒蛋白之间的特定相互作用。在核 egress，由 pUL31 和 pUL34 组成的 NEC 异二聚体，执行多种功能并相互作用 与多种其他病毒因素。然而，在许多情况下，机械后果和意义 这些相互作用是未知的。同样，细胞质包封取决于由 少数必需的被膜蛋白（HSV-1 中的 VP16、pUL36 和 pUL37），但它们的相互作用（其他 尽管有广泛的互动伙伴，但对于这一过程的重要性尚不清楚 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颖的定向进化 关键相互作用的功能识别方法。我们用其替换了必需的 HSV 基因 来自 VZV 同源物，然后对可以使用 VZV 同源物的病毒进行生长选择 装配。绘制定向进化过程中发生的突变图谱可以识别新的突变 功能性互动并确立他人的重要性。我们提供的初步数据表明 该方法使用嵌合病毒，其中 HSV-1 UL34 被 VZV ORF24 取代。我们有 确定了与 HSV-1 ICP4 的新型功能相互作用，并验证了先前研究的重要性 报告了与 ICP22 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，我们建议扩大该系统的使用范围 另一个与核出口相关的基因靶标，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.