Evasion of Host Immune Response by Dengue Virus

登革病毒核心蛋白减弱宿主IFN应答。为了检测DENV-C蛋白对I型IFN诱导的影响，构建了含有DENV-C基因的表达载体。 在用DENV-C表达载体转染HEK 293细胞后，用poly（I：C）处理细胞，随后通过qRT-PCR监测IFN的诱导。 与模拟转染对照相比，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Huh 7和A549观察到类似的表型。此外，DENV-C的过表达降低了AV活性，如用poly（I：C）处理后通过病毒滴度测量的。 通过蛋白质印迹分析检查IFN-β增强体的组分。 在IRF 3、NF-κ B或c-Jun的量或磷酸化方面均未检测到可观察到的差异，表明这些蛋白质与观察到的表型无关 登革病毒核心蛋白与宿主蛋白相互作用。DENV-C的可能相互作用伴侣通过免疫共沉淀（Co-IP），然后通过质谱（MS）进行鉴定。 MS确定了一些可能的主机交互伙伴。 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。通过共聚焦显微镜检查共定位也验证了病毒-宿主蛋白质相互作用。 细胞蛋白NF 45被确定为与DENV-C相互作用。 DENV-C基因中的突变影响其消除宿主IFN应答的能力。使用定点诱变将许多突变引入DENV-C基因。 然后检查DENV-C突变体在用Poly（I：C）处理后消除I型IFN诱导的能力。 N-末端的突变似乎影响所观察到的DENV-C的表型。 此外，通过共聚焦显微镜观察DENV-C和突变体的定位。 蛋白质的N-末端的突变影响DENV-C的核定位，表明N-末端对于DENV-C通过介导与特定宿主蛋白质的相互作用或通过影响DENV-C蛋白质的定位而对I型IFN的诱导的影响是重要的。

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.