Ng James C K, Josefsson Caroline, Clark Anthony J, Franz Alexander W E, Perry Keith L
Department of Botany and Plant Pathology, Purdue University, West Lafayette, IN 47907, USA.
Virology. 2005 Feb 5;332(1):397-405. doi: 10.1016/j.virol.2004.11.021.
The physical stability of virions of Cucumber mosaic virus (CMV) mutants was investigated to determine if relative stability correlated with efficiency of aphid transmission. Virion stability was evaluated by a urea disruption assay and by testing the infectivity of virus following purifications. All viruses were infectious when purified using a low salt buffer without organic solvent, whereas two of seven viruses were less stable and inactivated following purification with a high salt buffer and chloroform. These two viruses were both reassortants derived from the spontaneous transmission-defective mutant CMV-M (F1F2M3 and F1F2M3-L129P). F1F2M3 was relatively unstable, being disrupted between 0 and 1 M urea versus the wild-type CMV-Fny (F1F2M3) that was destabilized at 3-4 M urea. Modifications of F1F2M3 at three amino acid positions (129, 162, 168), singly or in combination, increased the relative stability of virions. A second class of transmission-defective CMVs with engineered mutations in the betaH-betaI surface loop of the CMV-Fny capsid protein (CP) exhibited near wild-type levels of stability. Lastly, a single Pro to Leu substitution at CP position 129 of CMV-Fny (F1F2M3-P129L) conferred the induction of necrosis in tobacco plants and reduced aphid transmissibility, but did not markedly alter the physical stability of virions. Thus, only among CMV-M derivatives harboring the CP mutation of Thr to Ala at position 162 were increases in stability correlated with restoration of transmissibility by the aphid Aphis gossypii.
研究了黄瓜花叶病毒(CMV)突变体病毒粒子的物理稳定性,以确定相对稳定性是否与蚜虫传播效率相关。通过尿素破坏试验和测试纯化后病毒的感染性来评估病毒粒子的稳定性。当使用不含有机溶剂的低盐缓冲液纯化时,所有病毒均具有感染性,而七种病毒中的两种在使用高盐缓冲液和氯仿纯化后稳定性较差并失活。这两种病毒均为源自自发传播缺陷型突变体CMV-M(F1F2M3和F1F2M3-L129P)的重配体。F1F2M3相对不稳定,在0至1 M尿素之间被破坏,而野生型CMV-Fny(F1F2M3)在3-4 M尿素时才不稳定。在三个氨基酸位置(129、162、168)对F1F2M3进行单一位点或组合修饰,可提高病毒粒子的相对稳定性。第二类在CMV-Fny衣壳蛋白(CP)的βH-βI表面环中具有工程突变的传播缺陷型CMV表现出接近野生型水平的稳定性。最后,CMV-Fny(F1F2M3-P129L)的CP位置129处的单个脯氨酸到亮氨酸的取代导致烟草植株坏死并降低了蚜虫的传播能力,但并未明显改变病毒粒子的物理稳定性。因此,只有在CMV-M衍生物中,衣壳蛋白第162位苏氨酸突变为丙氨酸的那些突变体,其稳定性的增加才与棉蚜传播能力的恢复相关。