Induction of Interferon to Block HIV-1 Replication

描述(由申请人提供)：即使在HIV-1感染者成功进行抗逆转录病毒治疗后，解除对宿主保护性免疫的管制仍是一个主要问题。该提案旨在验证这样一种假设，即诱导抗病毒细胞因子的产生将使细胞能够阻止HIV-1复制，并可能被证明是一种重要的新方法，以补充抗逆转录病毒治疗和减轻艾滋病相关病理。作为原理的证据，我们证明了(Krasnoselskaya-Riz等人，2002)，NF90的一个独特的C末端变体NF90ctv的表达刺激干扰素反应级联反应以阻止HIV-1复制。我们现在建议通过定义NF90的“活性”结构域和与其相关的蛋白质来研究抗病毒反应的机制，以更好地了解细胞免疫防御的调节。其基本原理是，NFg0的功能域介导病毒耐药基因的表达，通过与调节内源性病毒抑制途径的特定蛋白相关联来实现。实验方法将利用：(I)设计为表达标志表位标记的NF90的人CD4+/CXCR4+细胞，以评估其对HIV-1复制的影响。(Ii)将通过研究NF90的特定突变体及其抑制HIV-1复制的能力来分析NF90的功能结构域。(Iii)NF90相关蛋白将在抗FLAG抗体柱上进行亲和分级，并用质谱仪进行分析。(Iv)NF90结合蛋白的生物相关性将通过体内NF90与宿主因子相互作用的竞争来评估，利用与果蝇触角虫同源域多肽、AP-NF90多肽融合的NFg0结构域的嵌合结构，这些多肽可有效地运输到核室。拟议的研究将确定在人类细胞中调节先天抗病毒反应的细胞途径，并将验证增强HIV-1耐药性免疫反应的治疗方法。

DESCRIPTION (provided by applicant): Deregulation of the protective immunity of the host is a major concern even after successful anti-retroviral therapy of HIV-1 infected individuals. The proposal is designed to test the hypothesis that induction of antiviral cytokines production would enable the cells to block HIV-1 replication, and may prove to be a significant novel approach to complement anti-retroviral therapy and alleviate AIDS related pathology. As proof of principle we showed (Krasnoselskaya-Riz et al., 2002), that expression of a unique C-terminal variant of NF90, NF90ctv, stimulates interferon response cascade to block HIV-1 replication. We now propose to examine the mechanism of the antiviral response by defining "active" domains of NF90, and the proteins that associate with it to gain a better understanding of the regulation of cellular immune defense. The rationale being, that functional domains of NFg0 that mediate expression of viral resistance genes, do so by association with specific proteins that regulate the endogenous viral inhibitory pathways. The experimental approach will be to utilize: (i) Human CD4+/CXCR4+ cells designed to express FLAG-epitope tagged NF90 to evaluate its effects on HIV-1 replication. (ii) Functional domains of NF90 will be analyzed by studying specific mutants of NF90 and their ability to inhibit HIV-1 replication. (iii) NF90-associated proteins will be affinity fractionated on anti-FLAG antibody columns and analyzed by mass spectrometer. (iv) Biological relevance of NF90-associted proteins will be assessed by competition of NF90-host factor interactions in vivo, utilizing chimeric constructs of the NFg0-domains fused to Drosophila Antennapedia homeodomain peptide, AP-NF90 peptides that are efficiently transported to the nuclear compartment. Proposed studies will identify the cellular pathways that regulate innate antiviral response in human cells and will validate therapeutic approach to augment immune response for HIV-1 resistance.