Molecular Biology Of Varicella Zoster Virus Infection

水痘带状疱疹病毒感染的分子生物学

• 批准号: 8946264
• 负责人: Jeffrey Cohen
• 金额: $ 20.24万
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8946264
• 关键词: 1-Phosphatidylinositol 3-Kinase; Antibodies; Antigens; Apoptosis; Biological Assay; Caspase; Cell Cycle; Cell Cycle Progression; Cell Death; Cell Proliferation; Cell division; Cells; Chickenpox; Chickenpox Vaccine; DNA Damage; DNA Double Strand Break; DNA Repair; Diagnostic; Fluorescent Antibody Technique; Genes; Goals; Herpes zoster disease; Herpesvirus Type 3; Mammalian Cell; Mediator of activation protein; Membrane; Molecular Biology; Neurons; Pathogenesis; Pathway interactions; Persons; Phosphorylation; Prevention program; Proteins; Recruitment Activity; Reporter; Sensitivity and Specificity; Serum; Signal Pathway; Signal Transduction; Signaling Protein; Site; System; Vaccines; Viral Proteins; Virus; Virus Diseases; Virus Replication; cell type; genome-wide; improved; keratinocyte; knock-down; overexpression; receptor; response; tissue/cell culture

Varicella-zoster virus (VZV) causes chickenpox and shingles. Virus infection of cells is known to trigger several signaling pathways that are important for cell proliferation and prevention of programmed cell death (also known as apoptosis). We have used a virus genome wide approach to identify functions of VZV proteins. Nearly all of the 71 VZV genes were individually expressed in tissue culture cells along with a reporter system that responds to specific signaling proteins. Using this approach, last year we showed that VZV ORF12 protein activates the phosphatidylinositol 3-kinase (PI3K)/Akt pathway (a cell signaling pathway) to regulate cell cycle progression (important for cell division). Since VZV replicates in both dividing (keratinocytes) and non-dividing (neurons) cells, the ability of the VZV ORF12 protein to regulate the cell cycle is likely important for VZV replication in various cell types in the body. This year we found that VZV triggers the phosphorylation of two proteins, Bim and BAD, that are important for inducing programmed cell death (also known as apoptosis). Phosphorylation of these proteins reduces their ability to cause cell death. VZV-infected cells that were knocked-down for expression of Bim survived longer and produced higher titers of virus compared with wild-type cells infected with the virus. In contrast, VZV-infected cells that over-expressed Bim showed reduced survival and reduced virus replication. Inhibition of caspase activity in cells overexpressing Bim restored levels of virus replication to those seen in wild-type cells. Mammalian cells activate DNA damage response pathways in response to virus infections. H2AX and ATM are activated and recruited to the site of double-stranded DNA breaks and are important for repairing the DNA. We found that VZV-infected cells had elevated levels of phosphorylated H2AX and ATM compared with uninfected cells. Cells infected with VZV deleted for ORF61 or ORF63, but not ORF67, had higher levels of phosphorylated H2AX and ATM compared with cells infected with wild-type virus. We have also been developing a more sensitive assay to detect antibody to VZV in recipients of the varicella vaccine. We are comparing our assay using sera from persons who received the vaccine, with results obtained from the fluorescent antibody to membrane antigen (FAMA) assay, to determine the sensitivity and specificity of our assay.

水痘带状疱疹病毒（VZV）引起水痘和带状疱疹。已知细胞的病毒感染会触发几种对细胞增殖和预防程序性细胞死亡（也称为凋亡）至关重要的信号通路。我们已经使用了病毒基因组广泛的方法来识别VZV蛋白的功能。几乎所有71个VZV基因都在组织培养细胞中单独表达，以及对特定信号蛋白响应的报告基因。使用这种方法，去年我们表明，VZV ORF12蛋白激活磷脂酰肌醇3-激酶（PI3K）/AKT途径（细胞信号通路）以调节细胞周期进程（对细胞分裂很重要）。 由于VZV在分裂（角质形成细胞）和非分裂（神经元）细胞中都复制，因此VZV ORF12蛋白调节细胞周期的能力对于体内各种细胞类型的VZV复制可能很重要。 今年，我们发现VZV触发了两种BIM和坏蛋白的磷酸化，这对于诱导程序性细胞死亡（也称为凋亡）很重要。 这些蛋白质的磷酸化降低了它们引起细胞死亡的能力。 与感染了病毒感染的野生型细胞相比，被撞倒以表达BIM的VZV感染细胞存活更长，并且产生了更高的病毒滴度。 相比之下，过表达BIM的VZV感染细胞显示存活率降低和病毒复制降低。 抑制过表达BIM的细胞中caspase活性的抑制，使病毒复制水平恢复了野生型细胞中的病毒复制水平。 哺乳动物细胞激活DNA损伤反应途径，以响应病毒感染。 H2AX和ATM被激活并招募到双链DNA断裂部位，对于修复DNA很重要。 我们发现，与未感染的细胞相比，VZV感染的细胞的磷酸化H2AX和ATM水平升高。 与感染了野生型病毒的细胞相比，被VZV感染的细胞被删除的ORF61或ORF63而不是ORF67的细胞具有较高水平的磷酸化H2AX和ATM。 我们还一直在开发一种更敏感的测定法，以检测水痘疫苗接受者中对VZV的抗体。我们正在使用接收疫苗的人的血清进行比较，并从荧光抗体到膜抗原（FAMA）测定法获得结果，以确定我们的测定的敏感性和特异性。