烟草花叶病毒RNA发生poly(A)-rich型多聚腺苷酸化的研究

It has been well known that a tRNA-like structure but not a poly(A) tail is at the 3′-termini of the Tobacco mosaic virus (TMV) RNA. However,we recently identified a group of TMV RNA species containing heteropolymeric polyadenylation tails [hereafter referred to as 'TMV-poly(A)-rich'] from the TMV infected host plants including Chenopodium amaranticolor and Nicotiana benthamiana. So far, the RNA species containing the similar type of 3′-end tails have been isolated from the prokaryotic and eukaryotic cells, and were all linked to the poly(A)-assisted RNA degradation pathway, an ancient RNA surveillance mechanism. Such kind of RNAs, however, has never been found in RNA viruses, indicating an unknown mechanism by which host plants recognize and degrade the virus RNA. On the basis of this novel finding, a series of studies will be carried out as follows: 1) characterization of the specific nature of TMV-poly(A)-rich RNA molecules, 2) dissection of the synthesis/degradation pathway for TMV-poly(A)-rich, 3) investigation of whether the heteropolymeric adenylation occurs widely in multiple virus-host interactions, 4) elucidation of the possible roles of TMV-poly(A)-rich during the viral life cycle. All these data not only will increase the basic knowledge of TMV, a model system for virus, but also are expected to disclose one novel mechanism of host-virus interaction, providing new light for study of the other viruses.

众所周知，烟草花叶病毒(TMV)RNA 3′末端为典型的类tRNA结构。但最近，申请者从感染TMV的植物中分离了一类具有poly(A)-rich尾的TMV RNA--TMV-poly(A)-rich。迄今，具有类似3′特殊序列的RNA分子在原核和真核生物中均已发现，且证实都与poly(A)辅助的RNA降解途径相关。但此类RNA分子是首次发现于RNA病毒，在整个病毒界也鲜有报道。因而，TMV-poly(A)-rich可能代表了寄主识别、降解病毒RNA的一种未知机制。本项目围绕此新发现展开，力图阐明TMV-poly(A)-rich的分子特征，解析其合成/降解的分子机制，求证poly(A)-rich多聚腺苷酸化在不同寄主-病毒互作体系中发生的普遍性，探讨其在病毒生命活动中的生物意义。预期结果将进一步拓展TMV这一重要模式病毒的认知水平，同时揭示寄主-病毒互作的新机制,为其他病毒的研究提供新思路。

烟草花叶病毒(Tobacco mosaic virus, TMV)是一种典型的正链RNA病毒，其基因组3′-末端序列折叠构成类tRNA结构，而非poly(A)尾巴。与此传统认知不同，本研究揭示至少有一少部分TMV基因组RNA(gRNA)具有3′多聚腺苷酸尾巴。数据分析表明，大部分3′多聚腺苷酸序列并非直接添加于完整TMV gRNA的3′-末端，而是与3′-端核苷酸缺失的TMV gRNA分子相连接。同时，根据组成核苷酸，这类3′多聚腺苷酸序列可分为poly(A)和poly(A)-rich两大类，其中后者比例占优，且其分子特征与目前在生物界中已鉴定的具有促进RNA降解功能的3′-多聚腺苷酸序列基本一致。分子检测表明在叶绿体RNA中没有或者均有微量的具有poly(A)和poly(A)-rich尾巴的TMV RNA分子；利用VIGS沉默叶绿体多聚核苷酸磷酸化酶(polynucleotide phosphorylase, PNPase)，一种特异生成3′poly(A)-rich序列的重要聚合酶，也没有影响TMV RNA的多聚腺苷酸化。由此推断，植物叶绿体不是TMV RNA发生多聚腺苷酸化的亚细胞位点。此外，基于多聚腺苷酸化的TMV RNA分子的发现，本研究进一步分析了其它不同科属、且普遍认定的缺乏3′ poly(A)的正链RNA病毒，结果表明所有6种检测的病毒均具有连接poly(A)和poly(A)-rich尾巴的RNA分子。因此，这类此前未知的多聚腺苷酸化病毒RNA分子可能反映了一种普遍存在的病毒-寄主互作机制，值得继续深入研究。