Voloudakis A E, Malpica C A, Aleman-Verdaguer M-E, Stark D M, Fauquet C M, Beachy R N
Department of Cell Biology, The Scripps Research Institute, La Jolla, California, USA.
Arch Virol. 2004 Apr;149(4):699-712. doi: 10.1007/s00705-003-0247-x. Epub 2003 Dec 8.
The assembly of Tobacco etch potyvirus (TEV) coat protein (CP) and truncated mutants in Escherichia coli was studied. CP from which 28, 63 or 112 amino acids were deleted from the N-terminus polymerized into potyvirus-like particles (PVLPs). These structures were more rigid and progressively smaller in diameter than those produced by full length TEV-CP. CP from which 175 N-terminal amino acids were removed, failed to polymerize. A fragment containing amino acids 131 to 206 of TEV-CP is sufficient for PVLP assembly in E. coli. To determine the function of the highly conserved amino acids Ser152, Arg154, and Asp198 point mutants were generated. The mutant CPDelta63(Asp198Glu) exhibited different spectral properties following circular dichroism analysis showing a lower amount of alpha-helix compared to the wild type molecule. No differences were observed in spectra obtained from fluorescence spectroscopy. The point mutants bind RNA in vitro to the same degree as the wild type protein. However, while the wild type and the Arg154Gln mutant CP were each able to form PVLPs in E. coli, the Asp198Glu and the double mutant Ser152Pro/Arg154Gln mutants did not. These results suggest that the Asp198Glu mutation has an altered secondary structure which affects the capacity of the protein to polymerize but did not affect in vitro protein-RNA interactions.
对烟草蚀纹马铃薯Y病毒（TEV）外壳蛋白（CP）及其截短突变体在大肠杆菌中的组装进行了研究。从N端缺失28、63或112个氨基酸的CP聚合形成了类马铃薯Y病毒颗粒（PVLPs）。这些结构比全长TEV-CP产生的结构更坚硬，直径也逐渐变小。去除175个N端氨基酸的CP未能聚合。包含TEV-CP第131至206位氨基酸的片段足以在大肠杆菌中组装PVLP。为了确定高度保守的氨基酸Ser152、Arg154和Asp198的功能，构建了点突变体。突变体CPDelta63（Asp198Glu）在圆二色性分析后表现出不同的光谱特性，与野生型分子相比，α-螺旋含量较低。荧光光谱获得的光谱未观察到差异。点突变体在体外与RNA的结合程度与野生型蛋白相同。然而，虽然野生型和Arg154Gln突变体CP各自能够在大肠杆菌中形成PVLPs，但Asp198Glu和双突变体Ser152Pro/Arg154Gln突变体则不能。这些结果表明，Asp198Glu突变具有改变的二级结构，影响了蛋白质的聚合能力，但不影响体外蛋白质-RNA相互作用。
