Biophysical studies on the RNA cores of satellite tobacco mosaic virus
Biophysical studies on the RNA cores of satellite tobacco mosaic virus
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DOI：
10.1016/s0006-3495(01)76206-6
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发表时间：
2001-05-01
影响因子：
3.4
通讯作者：
McPherson, A
中科院分区：
文献类型：
--
作者：
Day, J;Kuznetsov, YG;McPherson, A
卫星烟草花叶病毒（STMV）的探测使用各种蛋白酶。使用凝胶电泳、准弹性光散射（QELS）和原子力显微镜（AFM）分析降解的后果。研究了蛋白质衣壳完全降解的30分钟至多小时的蛋白质水解速率。对于每种蛋白酶，通过QELS显示直径为17 nm的病毒体的降解产生直径为10 nm的颗粒，这是通过X射线衍射分析在病毒体中观察到的RNA核心的直径。这是通过原子力显微镜直接可视化验证。使用QELS，进一步表明新鲜制备的RNA核心保持为单独的、稳定的、10 nm凝聚颗粒12至24 h。然后形成颗粒簇，随后形成直径为500至1000 nm的非常大的聚集体。原子力显微镜显示，聚集体是由凝聚的RNA核心的组，而不是由于核酸的解折叠。AFM没有观察到核心颗粒展开成伸展构象，直到样品被加热远超过90 ℃。RNA核心颗粒的质谱分析显示存在一种主要多肽，其氨基酸序列对应于外壳蛋白的残基2至25。先前观察到氨基酸13至25与RNA直接接触，并且推测其被保护免于蛋白酶消化。低分辨率差分傅立叶分析表明完整病毒体中氨基末端链（氨基酸2-12）的延伸过程。任何单独的链似乎都有几种路径选择，这解释了在高分辨率下观察到的无序。这些带正电荷的链，作为虚拟的多胺，接合RNA的螺旋片段。氨基酸残基2至25与RNA的紧密结合可能有助于核酸核心的凝聚构象的稳定性。
Satellite tobacco mosaic virus (STMV) was probed using a variety of proteases. Consequences of the degradation were analyzed using gel electrophoresis, quasi-elastic light scattering (QELS), and atomic force microscopy (AFM). Proteolysis rates of 30 minutes for complete degradation of the protein capsid, up to many hours, were investigated. With each protease, degradation of virions 17 nm in diameter was shown by QELS to result in particles of 10 nm diameter, which is that of the RNA core observed in the virion by x-ray diffraction analysis. This was verified by direct visualization with atomic force microscopy. Using QELS, it was further shown that freshly prepared RNA cores remain as individual, stable, 10-nm condensed particles for 12 to 24 h. Clusters of particles then formed, followed by very large aggregates of 500 to 1000 nm diameter. AFM showed that the aggregates were composed of groups of the condensed RNA cores and were not due to unfolding of the nucleic acid. No unfolding of the core particles into extended conformation was seen by AFM until the samples were heated well beyond 90 degreesC. Mass spectrometry of RNA core particles revealed the presence of a major polypeptide whose amino acid sequence corresponded to residues 2 through 25 of the coat protein. Amino acids 13 through 25 were previously observed to be in direct contact with the RNA and are presumably protected from protease digestion. Low resolution difference Fourier analyses indicated the courses of the remainders of the amino terminal strands (amino acids 2-12) in intact virions. Any individual strand appears to have several choices of path, which accounts for the observed disorder at high resolution. These positively charged strands, serving as virtual polyamines, engage the helical segments of RNA. The intimate association of amino acid residues 2 through 25 with RNA likely contributes to the stability of the condensed conformation of the nucleic acid cores.