Evasion of Host Immune Response by Dengue Virus

登革热病毒逃避宿主免疫反应

• 批准号: 8556076
• 负责人: Kathryn Zoon
• 金额: $ 19.16万
• 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/8556076
• 关键词: A549; Affect; Aftercare; Antiviral Response; Apoptosis; Attenuated; Capsid Proteins; Cell Nucleus; Cells; Co-Immunoprecipitations; Confocal Microscopy; Core Protein; Dengue Virus; Gene Proteins; Genes; IRF3 gene; Immune response; Interferon-beta; Interferons; JUN gene; Life Cycle Stages; Mass Spectrum Analysis; Measures; Mediating; Monitor; Mutation; NF-kappa B; Nuclear; Nuclear Localization Signal; Phenotype; Phosphorylation; Play; Poly I-C; Proteins; Research; Role; Site-Directed Mutagenesis; Transfection; Viral; Virion; Virus; Virus Diseases; Virus Replication; Western Blotting; cell type; citrate carrier; expression vector; mutant; overexpression; response; virus core; A549; Affect; Aftercare; Antiviral Response; Apoptosis; Attenuated; Capsid Proteins; Cell Nucleus; Cells; Co-Immunoprecipitations; Confocal Microscopy; Core Protein; Dengue Virus; Gene Proteins; Genes; IRF3 gene; Immune response; Interferon-beta; Interferons; JUN gene; Life Cycle Stages; Mass Spectrum Analysis; Measures; Mediating; Monitor; Mutation; NF-kappa B; Nuclear; Nuclear Localization Signal; Phenotype; Phosphorylation; Play; Poly I-C; Proteins; Research; Role; Site-Directed Mutagenesis; Transfection; Viral; Virion; Virus; Virus Diseases; Virus Replication; Western Blotting; cell type; citrate carrier; expression vector; mutant; overexpression; response; virus core

Dengue virus core protein attenuates the host IFN response. In order to examine the effect that DENV-C protein has on induction of type I IFN, expression vectors were constructed containing the DENV-C gene. After transfection of HEK293 cells with the DENV-C expression vector, cells were treated with poly (I:C) and subsequently monitored for induction of IFN by qRT-PCR. Cells expressing the DENV-C protein showed decreased levels of IFN-beta expression when compared to mock transfected controls. A similar phenotype was observed using other cell types Huh7 and A549. In addition, overexpression of DENV-C decreased AV activity as measured by viral titers after treatment with poly (I:C). Components of the IFN-beta enhanceosome were examined by western blot analysis. No observable differences were detected in either the amount or phosphorylation of IRF3, NF-kappa B or c-Jun, indicating that these proteins were not responsible for the observed phenotype Dengue virus core protein interacts with host proteins. Possible interaction partners for DENV-C were identified by co-immunoprecipitation (Co-IP), followed by mass spectrometry (MS). MS identified a number of possible host interaction partners. Results were then verified by Co-IP and reverse Co-IP followed by western blot analysis. Virus-host protein interactions were also verified by examining co-localization by confocal microscopy. The cellular protein NF45was determined to interact with DENV-C. Mutations in the DENV-C gene affect its ability to abrogate the host IFN response. A number of mutations were introduced into the DENV-C gene using site directed mutagenesis. The DENV-C mutants were then examined for their ability to abrogate induction of type I IFN after treatment with Poly(I:C). Mutations in the N-terminus appear to affect the observed phenotype of DENV-C. In addition, localization of DENV-C and mutants were observed by confocal microscopy. Mutations to the N-terminus of the protein affect the nuclear localization of DENV-C, indicating that the N-terminus is important for the affect that DENV-C has on induction of type I IFN either through mediating interactions with specific host proteins, or by influencing the localization of the DENV-C protein.

登革热病毒核心蛋白减弱了宿主IFN反应。为了检查DENV-C蛋白对I型IFN诱导的影响，构建了包含DENV-C基因的表达载体。 用DENV-C表达载体转染HEK293细胞后，用Poly（I：C）处理细胞，然后对QRT-PCR进行监测以诱导IFN。 与模拟转染的对照相比，表达DENV-C蛋白的细胞显示IFN-β表达水平降低。 使用其他细胞类型HUH7和A549观察到类似的表型。另外，通过使用Poly（I：C）处理后，通过病毒滴度测量的DENV-C的过表达降低了AV活性。 通过Western印迹分析检查了IFN-β增强体的成分。 在IRF3，NF-KAPPA B或C-JUN的量或磷酸化中未检测到可观察到的差异，这表明这些蛋白质对观察到的表型不负责 登革热病毒核心蛋白与宿主蛋白相互作用。通过共免疫沉淀（CO-IP）鉴定出DENV-C的可能相互作用伙伴，然后进行质谱（MS）。 MS确定了许多可能的主机交互合作伙伴。 然后通过co-IP和反向co-IP验证结果，然后进行蛋白质印迹分析。病毒宿主蛋白相互作用还通过共聚焦显微镜检查共定位来验证。 细胞蛋白NF45WAS确定与DENV-C相互作用。 DENV-C基因中的突变会影响其消除宿主IFN反应的能力。使用位点的诱变将许多突变引入DENV-C基因中。 然后检查了DENV-C突变体，以消除用Poly（I：C）治疗后的I型IFN诱导的能力。 N末端中的突变似乎影响了观察到的DENV-C的表型。 另外，通过共聚焦显微镜观察到DENV-C和突变体的定位。 蛋白质N末端的突变会影响DENV-C的核定位置，表明N末端对于DENV-C通过与特定宿主蛋白的相互作用或影响DENV-C-C蛋白的定位而对I型IFN诱导的影响很重要。