Evasion of Host Immune Response by Dengue Virus

为了检验DENV C对诱导i型干扰素的影响，构建了编码未成熟或成熟形式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其抗病毒活性的诱导。DENV C的过表达导致病毒滴度升高，表明抗病毒活性降低。与模拟转染的对照组相比，经poly（I:C）处理后，过表达DENV C的HEK293细胞对干扰素β的诱导降低。与野生型相比，引入DENV C基因氨基末端的突变导致影响干扰素β诱导的能力下降。采用免疫共沉淀法（co-Immunoprecipitation, co-IP）和质谱法（mass spectrometry， MS）鉴定DENV - C的宿主相互作用伙伴。质谱鉴定出许多潜在的相互作用伙伴，包括Nucleolin （NCL）。

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.