Evasion of Host Immune Response by Dengue Virus

为了研究DENV C在诱生I型干扰素中的作用，构建了编码未成熟或成熟DENV C蛋白的表达载体。DENV C过表达后，用Poly(I：C)处理HEK293细胞，通过检测VSV感染后的病毒滴度来确定诱导抗病毒活性。DENV C的过度表达导致病毒滴度升高，表明抗病毒活性降低。与对照组相比，高表达DENV C的HEK293细胞在PolyI：C处理后表现出对干扰素β的诱导减少。与野生型相比，DENV C基因氨基末端的突变导致影响β干扰素诱导的能力降低。通过共免疫沉淀(co-IP)和质谱仪(MS)鉴定DENV C的宿主相互作用伙伴。MS确定了一些潜在的相互作用伙伴，包括核仁素(NCL)。 DENV C与NCL的相互作用也得到了表征。DENV C通过FRET和使用纯化的重组蛋白直接与NCL相互作用。用siRNA敲除NCL，或用NCL结合适体(AS1411)处理细胞，阻断C蛋白与NCL之间的相互作用，可显著降低DENV感染后的病毒滴度。Western印迹和qRT-PCR分析表明，在感染后的早期时间点，病毒的RNA或蛋白质水平没有差异，表明NCL在病毒形态发生中发挥了作用。我们支持这一假设，表明AS1411治疗改变了天然电泳法显示的病毒衣壳的迁移特征，以及通过RT-PCR扩增病毒RNA的可及性。我们的数据表明DENV C蛋白与NCL之间存在关键的相互作用，这是抗病毒治疗药物开发的一个潜在的新靶点。 我们目前的工作包括进一步确定宿主DENV C相互作用伙伴的特征，以及识别参与DENV介导的颠覆天然免疫反应的新途径。

In order to examine the effect of DENV C on induction of type-I interferon, expression vectors were constructed encoding either immature or mature forms of the DENV C protein. After overexpression of DENV C, HEK293 cells were treated with poly(I:C) and induction of antiviral activity was determined by measuring virus titers after infection with VSV. Overexpression of DENV C leads to an elevation of virus titers, indicating a reduction in antiviral activity. HEK293 cells overexpressing DENV C displayed decreased induction of interferon beta after treatment with poly(I:C) when compared to mock transfected controls. Mutations introduced to the amino-terminus of the DENV C gene resulted in a decreased ability to influence induction of interferon beta compared to wild-type counterparts. Host Interaction partners for DENV C were identified by co-Immunoprecipitation (co-IP) followed by mass spectrometry (MS). MS identified a number of potential interaction partners, including Nucleolin (NCL). DENV C interactions with NCL were also characterized. DENV C was shown to directly interact with NCL via FRET and through the use of purified recombinant proteins. Knockdown of NCL with siRNA, or blocking the interaction between the C protein and NCL by treating cells with a NCL binding aptamer (AS1411), resulted in a significant reduction of viral titers after DENV infection. Western blot and qRT-PCR analysis revealed no differences in viral RNA or protein levels at early time points post-infection, suggesting a role for NCL in viral morphogenesis. We have supported this hypothesis by showing that treatment with AS1411 alters the migration characteristics of the viral capsid as visualized by native electrophoresis, as well as the accessibility of viral RNA to amplification by RT-PCR. Our data suggests a critical interaction between DENV C protein and NCL, which represents a potential new target for the development of antiviral therapeutics. Our current work involves the further characterization of host DENV C interaction partners as well as the identification of novel pathways involved in DENV mediated subversion of the innate immune response.