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

HSV/VZV 嵌合病毒用于识别关键病毒疱疹病毒组装相互作用

• 批准号: 10556366
• 负责人: RICHARD J ROLLER
• 金额: $ 50.97万
• 依托单位: UNIVERSITY OF IOWA
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-02-01 至 2027-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10556366
• 关键词: Biological Assay; Capsid Proteins; Cell Culture Techniques; Cells; Complex; Conserved Sequence; Cytoplasm; Data; Defect; Development; Directed Molecular Evolution; Gene Targeting; Genes; Growth; Herpesviridae; Herpesvirus 1; Herpesvirus Type 3; Homologous Gene; Homologous Protein; Human; Immediate-Early Proteins; Maps; Mutation; Nuclear; Nuclear Envelope; Play; Process; Protein Kinase; Protein-Serine-Threonine Kinases; Proteins; Proteomics; Reporting; Role; Simplexvirus; System; Testing; VP 16; Vaccines; Viral; Viral Genes; Viral Genome; Viral Proteins; Virus; Virus Assembly; Virus Replication; mutant; novel; novel strategies; protein complex

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.

概括 疱疹病毒组装在严重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，执行多个功能并相互作用 与其他多个病毒因素。但是，在许多情况下，机械性的后果和意义 这些相互作用是未知的。同样，细胞质包膜取决于A的相互作用 很少有必要的Tegument蛋白（HSV-1中的VP16，PUL36和PUL37），但它们的哪些相互作用（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确定。在这里，我们建议使用新颖的定向进化 功能识别关键相互作用的方法。我们用它的基因代替了基本的HSV基因 VZV的同源物，然后对可以使用VZV同源物进行的病毒进行生长选择 集会。在这种定向演化期间发生的突变的映射可以识别新颖 功能相互作用并确定他人的意义。我们介绍了展示的初步数据 这种方法使用嵌合病毒的效用，其中HSV-1 UL34被VZV ORF24取代。我们有 确定了与HSV-1 ICP4的新功能相互作用，并验证了先前的意义 报告与ICP22的相互作用。我们建议确定这些关键的作用机理 交互并使用系统来识别其他人。此外，我们建议扩大该系统的使用 与核出口相关的另一个基因靶标PUL31。