Arguello-Astorga Gerardo, Ascencio-Ibáñez J Trinidad, Dallas Mary Beth, Orozco Beverly M, Hanley-Bowdoin Linda
Department of Molecular and Structural Biochemistry, North Carolina State University, Raleigh, NC 27695-7622, USA.
J Virol. 2007 Oct;81(20):11005-15. doi: 10.1128/JVI.00925-07. Epub 2007 Aug 1.
The geminivirus replication protein AL1 interacts with retinoblastoma-related protein (RBR), a key regulator of the plant division cell cycle, to induce conditions permissive for viral DNA replication. Previous studies of tomato golden mosaic virus (TGMV) AL1 showed that amino acid L148 in the conserved helix 4 motif is critical for RBR binding. In this work, we examined the effect of an L148V mutation on TGMV replication in tobacco cells and during infection of Nicotiana benthamiana plants. The L148V mutant replicated 100 times less efficiently than wild-type TGMV in protoplasts but produced severe symptoms that were delayed compared to those of wild-type infection in plants. Analysis of progeny viruses revealed that the L148V mutation reverted at 100% frequency in planta to methionine, leucine, isoleucine, or a second-site mutation depending on the valine codon in the initial DNA sequence. Similar results were seen with another geminivirus, cabbage leaf curl virus (CaLCuV), carrying an L145A mutation in the equivalent residue. Valine was the predominant amino acid recovered from N. benthamiana plants inoculated with the CaLCuV L145A mutant, while threonine was the major residue in Arabidopsis thaliana plants. Together, these data demonstrated that there is strong selection for reversion of the TGMV L148V and CaLCuV L145A mutations but that the nature of the selected revertants is influenced by both the viral background and host components. These data also suggested that high mutation rates contribute to the rapid evolution of geminivirus genomes in plants.
双生病毒复制蛋白AL1与视网膜母细胞瘤相关蛋白（RBR）相互作用，RBR是植物分裂细胞周期的关键调节因子，可诱导有利于病毒DNA复制的条件。先前对番茄金色花叶病毒（TGMV）AL1的研究表明，保守螺旋4基序中的氨基酸L148对RBR结合至关重要。在这项工作中，我们研究了L148V突变对烟草细胞中TGMV复制以及本氏烟草植株感染过程的影响。L148V突变体在原生质体中的复制效率比野生型TGMV低100倍，但在植株中产生了严重症状，不过与野生型感染相比症状出现延迟。对子代病毒的分析表明，L148V突变在植物体内以100%的频率回复突变为甲硫氨酸、亮氨酸、异亮氨酸，或根据初始DNA序列中的缬氨酸密码子发生第二位点突变。在另一种双生病毒卷心菜叶卷曲病毒（CaLCuV）中，其等效残基发生L145A突变，也观察到了类似结果。从接种CaLCuV L145A突变体的本氏烟草植株中回收的主要氨基酸是缬氨酸，而在拟南芥植株中主要是苏氨酸。总之，这些数据表明，TGMV L148V和CaLCuV L145A突变的回复突变存在强烈选择，但所选回复突变体的性质受病毒背景和宿主成分两者影响。这些数据还表明，高突变率有助于双生病毒基因组在植物中的快速进化。
