武汉野田村病毒B2蛋白抑制RNAi介导的抗病毒免疫机制的研究

RNA interference (RNAi) is well known as an antiviral innate immunity conserved in plants, invertebrate and mammals. In the RNAi antiviral immunity, host Dicer functions as the pattern recognition receptor (PRR) to recognize viral dsRNA as a pathogen-associated molecular pattern (PAMP), leading to process viral dsRNA into siRNAs that are subsequently transferred to an Argonaute (AGO) protein. To combat the host RNAi immunity, viruses encode viral suppressors of RNA silencing (VSRs) that target one or more components in the RNAi machinery. VSRs can function at either RNA level through sequestering viral dsRNA and/or siRNA, or protein level such as targeting AGO protein. In recent years, the mechanisms of RNAi immunity and VSRs are intensively studied for their significant impacts in innate immunity and viral pathogenesis. In our previous studies, we have uncovered that the Wuhan Nodavirus (WhNV) B2 protein is a VSR and suppresses RNAi by sequestering both viral dsRNA and siRNA. Moreover, our following studies indicate that WhNV B2 contains another mechanism(s) to suppress RNAi immunity and this unidentified mechanism functions through B2 interaction with some protein(s) of the RNAi pathway. In this project, we aim to construct a WhNV B2-mediated RNAi suppression model in Drosophila cells, and use this model to identify the host factors that interact with WhNV B2 and function in the RNAi immunity. For this purpose, a group of candidate proteins, including Dicer-2, AGO2, R2D2, FMRP, VIG, Tudor-SN, Hsp90 and Hsp70 will be examined for their binding abilities to WhNV B2. Besides, we will conduct the mass spectrometry analysis of the immunocomplex containing WhNV B2 and yeast two-hybrid screening of the Drosophila cDNA library for potential partner proteins of B2 protein. After that, we aim to uncover the detailed molecular mechanisms of how WhNV B2 suppresses the RNAi antiviral immunity through interacting with the protein(s) in the RNAi pathway, study the interrelationship among the diverse activities of WhNV B2, and build up an experimental system to study the VSRs from other viruses and the components of the RNAi pathway. The accomplishment of this project will significantly deepen our understanding to the RNAi suppressing mechanisms of VSRs and the RNAi-mediated antiviral immunity, and also provide the molecular bases for designing VSR-targeting antiviral therapies.

RNA干扰(RNAi)介导的抗病毒免疫是一种广泛存在于植物、昆虫和哺乳动物的先天免疫机制；而许多病毒通过编码RNAi抑制因子来达到逃逸宿主RNAi免疫的目的。尽管对病毒逃逸宿主RNAi免疫机制的研究在病毒学、免疫学及抗病毒疗法上都具有重要意义，但目前对其认识还存在着很多空白。本项目通过对模式病毒- - 武汉野田村病毒(WhNV)RNAi抑制因子B2蛋白的研究，致力于建立一个WhNV B2抑制果蝇RNAi免疫的细胞模型，在此基础上筛选鉴定与B2相互作用、并在RNAi通路发挥作用的宿主蛋白，深入探索B2在蛋白水平抑制RNAi的分子机制、及其与已报道的B2蛋白RNA水平RNAi抑制机制间的相互关系，并建立研究其他病毒RNAi抑制因子与RNAi通路的细胞模型。本研究在RNAi抗病毒免疫及其病毒逃逸机制上具有十分重要的理论价值，同时也将为抗病毒疗法的研究提供必要的理论基础，从而推动病毒学、免疫学的发展

RNA干扰（RNAi）介导的抗病毒免疫是一种广泛存在于真菌、植物与无脊椎动物中的抗病毒天然免疫机制；而许多病毒通过编码病毒RNAi抑制子（VSR）来达到拮抗并逃逸RNAi抗病毒免疫的目的。尽管目前对病毒逃逸宿主RNAi 免疫机制的研究在病毒学、免疫学及抗病毒疗法上都具有重要意义，但目前对其认识还存在着很多空白。在本课题中，我们通过对模式病毒- 武汉野田村病毒(Wuhan Nodavirus, WhNV)所编码的VSR B2蛋白的研究，建立了一个WhNV B2 抑制果蝇RNAi 免疫的细胞模型，并在此基础上成果的筛选与鉴定到RNAi通路中最重要的蛋白Dicer-2能与WhNV B2相互作用, B2通过直接抑制Dicer-2的RNase III结构域的双链RNA切割活性，以及Dicer-2 PAZ结构域的siRNA转运或许，成功抑制RNAi 通路。该工作也是首次发现宿主Dicer蛋白能为病毒蛋白所直接抑制，揭示了一种逃逸RNAi抗病毒免疫的全新机制。同时，我们的研究还发现，Dicer-2蛋白能以一种RNAi非依赖的方式参与调控果蝇的Toll天然免疫通路，为研究RNAi与Toll这两个重要的昆虫天然免疫通路的相互作用打下来基础。此外，通过我们的研究，还建立了筛选其他病毒VSR的细胞模型，该筛选模型在后续的多项研究中起到了重要作用。本项目所资助的研究对RNAi抗病毒免疫及其病毒逃逸机制上具有十分重要的理论价值，推动了相关领域的发展。