烟草花叶病毒入侵寄主引发PABPN细胞质重定位的研究

Eukaryotic poly(A) binding protein (PABP), which includes cytoplasmic PABP (PABPC) and nuclear PABP (PABPN), is a kind of important target proteins often manipulated by different viruses. Recently, we identified that when Tobacco mosaic virus (TMV), a well-known plus strand RNA virus, infects the host plant, the host PABPN can be evicted completely out of nuclear and redistributed to cytoplasm. To date, multiple mechanisms of virus manipulation of the host PABPC or PABPN have been uncovered, but there is no any report that the virus infection causes the PABPN cytoplasm redistribution, thus indicating that the TMV infection-mediated PABPN cytoplasm redistribution is a novel finding, and may represent a previously unknown mechanism of virus manipulation of the host PABPN. Accordingly, in this study, a series of experiments will be performed in order to fully dissect the molecular basis of the PABPN cytoplasm redistribution caused by the TMV infection, to investigate the effect of the TMV infection-mediated PABPN cytoplasm redistribution on the life cycle of virus itself as well as the host, and to elucidate the biological significance of the TMV infection-mediated PABPN cytoplasm finally. We believe that the expected data will not just unravel a novel mechanism of viral manipulation of the host PABPN, but also disclose a new type of virus-host interaction.

真核PABP[Poly(A) binding protein]包括细胞质型PABP(PABPC)与细胞核型PABP(PABPN)，是病毒侵染寄主的重要靶标蛋白。在前期研究中，申请人发现烟草花叶病毒(TMV)入侵寄主，能够导致其PABPN无法正常进入细胞核，进而重定位于细胞质。迄今，科学家已揭示了病毒调控寄主PABP的多种分子模式，但尚没有任何关于病毒入侵引发寄主PABPN细胞质重定位的报道。因而，TMV入侵引发寄主PABPN细胞质重定位是一项全新发现，代表了病毒调控寄主PABPN的一种新模式。基于此，本项目拟进一步解析TMV引发PABPN细胞质重定位的分子基础，探寻TMV调控PABPN细胞质重定位对病毒自身的作用，以及对寄主重要生命活动的影响，阐明TMV入侵引发PABPN细胞质重定位的生物学意义。预期结果将阐释病毒调控寄主PABPN的一种新模式，并由此揭示病毒与寄主互作的新机制。

本项目针对烟草花叶病毒(Tobacco mosaic virus, TMV)入侵寄主引发PABPN细胞质重定位开展研究，结果显示瞬时表达TMV编码的126kD蛋白、54kD蛋白、MP、CP这四种蛋白均不能影响PABPN的细胞核定位，而利用EMSA检测证实TMV基因组RNA (genomic RNA)与PABPN也没有发生互作，由此推测TMV不是通过病毒编码蛋白或者RNA直接与PABPN作用，而是通过间接方式引发其重定位，其可能的作用机制正在进一步研究。在上述工作进行中，项目组发现TMV侵染烟草，诱导一种核定位的RNA结合蛋白(RNA binding protein, RBP) NtRBP45的上调表达；利用本氏烟16c株系分析发现，全长NtRBP45促进siRNA积累，具有增强转录后基因沉默的功能。利用农杆菌介导法转化本氏烟，获得稳定表达NtRBP45的转基因植株，分别用TMV-GFP、Pto DC3000 HopQ 1进行攻毒，但结果显示转基因植株没有表现出明显的抗病毒特征，但对DC3000抗性显著。这些试验证据揭示了NtRBP45是植物基因沉默途径的一个正调控因子，其在寄主-病原互作过程中的作用值得继续探讨。