4型登革病毒NS2A蛋白通过UXT-V1逃避宿主免疫的机制研究

Study on the mechanism of evading host immunity by UXT-V1 by type 4 dengue virus NS2A protein

目的探讨4型登革病毒NS2A蛋白通过泛转录表达因子剪接变异体1(UXT-V1)逃避宿主免疫的机制。方法在HEK-293T细胞中,4型登革病毒感染后过表达NS2A蛋白,通过实时荧光定量PCR(qPCR)检测干扰素-β(IFN-β)、白细胞介素-8(IL-8)、干扰素诱导蛋白54(ISG54)的表达量;通过荧光素酶报告实验检测NS2A蛋白对IFN-β与核因子κB(NF-κB)通路的影响;通过免疫共沉淀检测NS2A蛋白与UXT-V1的相互作用;通过核蛋白抽取检测IRF3和p65的核定位。结果4型登革病毒NS2A蛋白与宿主UXT-V1存在相互作用。此外,4型登革病毒NS2A蛋白抑制宿主IFN-β、IL-8和ISG54的mRNA水平,阻断IRF3和p65的核转移,抑制宿主IFN-β与NF-κB通路。结论4型登革病毒NS2A蛋白通过与UXT-V1的相关作用阻断宿主固有免疫关键因子IRF3和p65的核定位,抑制宿主IFN-β与NF-κB通路,最终到达其免疫逃避的目的。

Objective To investigate the mechanism by which the ND2A protein of type 4 dengue virus escapes host immunity through ubiquitously expressed transcript splice variant 1(UXT-V1).Methods In HEK-293T cells,NS2A protein was overexpressed after infection with type 4 dengue virus,and the expression levels of IFN-β,IL-8 and ISG54 were detected by qPCR.NS2A protein was detected by luciferase reporter assay for IFN-β.Interaction with NF-κB pathway;interaction of NS2A protein with UXT-V1 was detected by co-immunoprecipitation;nuclear localization of IRF3 and p65 was detected by nuclear protein extraction.Results The type 4 dengue virus NS2A protein interacted with host UXT-V1.In addition,the type 4 dengue virus NS2A protein inhibits mRNA levels of host IFN-β,IL-8 and ISG54,blocks nuclear transfer of IRF3 and p65,and inhibits host IFN-βand NF-κB pathways.Conclusion The type 4 dengue virus NS2A protein blocks the nuclear localization of the host innate immune key factors IRF3 and p65 through the correlation with UXT-V1,inhibits the host IFN-βand NF-κB pathway,and finally reaches the goal of immune evasion.