Desvoyes Bénédicte, Scholthof Herman B
Department of Plant Pathology and Microbiology, Texas A&M University, College Station 77843, USA.
Virology. 2002 Dec 20;304(2):434-42. doi: 10.1006/viro.2002.1714.
This study examined the contribution of the Tomato bushy stunt virus (TBSV) coat protein (CP) and its corresponding RNA to systemic infection of plants. Compared to results obtained with a mutant lacking the 5'-half of the CP gene, the presence of those CP-RNA sequences in another mutant benefited TBSV infection on Nicotiana benthamiana even though wild-type CP expression was eliminated by introduction of a small out-of-frame deletion. RT-PCR of viral RNA associated with rapid infections established by this CP frameshift deletion mutant revealed that in planta recombination had provided the progeny with the ability to express a truncated CP (tCP) with a block of N-proximal 30 residues deleted from the 66 amino acid RNA-binding domain. Subsequent biochemical characterizations revealed the presence of large ribonucleoprotein complexes that were shown to contain viral RNA as well as the approximately 38-kDa tCP. Electron microscopic examination of purified complexes showed particle-like structures that were nonuniform in size and shape compared to wild-type TBSV particles. Inoculation of pepper with the tCP-containing ribonucleoprotein complexes resulted in a rapid systemic infection similar to that caused by wild-type TBSV. In contrast, infections established in pepper by the original CP frameshift deletion mutant transcripts were restricted to inoculated leaves and did not yield recombinants capable of systemically infecting this host. In summary, TBSV possesses the flexibility to form alternative virion-like structures even if a substantial portion of the RNA-binding domain is deleted from the CP; mutants producing the tCP-containing particle-like structures are more effective for virus spread than those devoid of CP expression; and recombination events to produce the alternative tCP-RNA complexes are host-dependent.
本研究检测了番茄丛矮病毒(TBSV)外壳蛋白(CP)及其相应RNA对植物系统感染的作用。与缺失CP基因5′端一半的突变体的结果相比,另一个突变体中那些CP-RNA序列的存在有利于TBSV在本氏烟草上的感染,尽管通过引入一个小的移码缺失消除了野生型CP的表达。对由该CP移码缺失突变体建立的快速感染相关的病毒RNA进行RT-PCR分析表明,在植物体内的重组为子代提供了表达截短CP(tCP)的能力,该截短CP从66个氨基酸的RNA结合结构域中缺失了N端的30个残基。随后的生化特性分析揭示了存在大型核糖核蛋白复合体,该复合体被证明含有病毒RNA以及约38 kDa的tCP。对纯化复合体的电子显微镜检查显示,与野生型TBSV颗粒相比,颗粒状结构的大小和形状不均匀。用含tCP的核糖核蛋白复合体接种辣椒会导致快速的系统感染,类似于野生型TBSV引起的感染。相比之下,最初的CP移码缺失突变体转录本在辣椒中建立的感染仅限于接种叶片,并且没有产生能够系统感染该宿主的重组体。总之,即使CP中大部分RNA结合结构域被缺失,TBSV仍具有形成替代病毒样结构的灵活性;产生含tCP颗粒状结构的突变体比那些缺乏CP表达的突变体在病毒传播方面更有效;并且产生替代tCP-RNA复合体的重组事件是宿主依赖性的。