Tamm T, Truve E
Gene Technology Center, Institute of Chemical Physics and Biophysics, Tallinn Technical University, Akadeemia tee 23, EE12618, Tallinn, Estonia.
Virus Res. 2000 Feb;66(2):197-207. doi: 10.1016/s0168-1702(00)00125-8.
Cocksfoot mottle virus (CfMV) has a positive-sense ssRNA genome containing four open reading frames (ORFs). ORF1 encoded protein (P1) is the putative movement protein; the product of ORF2a (P2a) contains VPg and the motifs characteristic of serine proteases. P2b, encoded by ORF2b, is the putative RNA-dependent RNA polymerase. P3, the coat protein, is encoded by ORF3. CfMV P1, P2a, P2b, and P3, containing a six histidine tag at the amino terminus, were expressed in Escherichia coli, purified and their RNA-binding activities were analysed. The northwestern blot assay showed that His-tagged P1, P2a, P2b, and P3 were able to interact with ssRNA transcripts in a sequence-nonspecific manner. The filter-binding assay confirmed the ssRNA-binding capacity of recombinant P1, P2a, and P3. The RNA-binding activities of His-tagged P3 and native coat protein were similar. P1 and P2a binding to ssRNA decreased markedly by increasing NaCl concentrations. In contrast, P3 had the RNA-binding optimum at 100-200 mM NaCl. We discuss the possible amino acid motifs involved in the RNA-binding of CfMV proteins.
鸭茅斑驳病毒（CfMV）具有一个正义单链RNA基因组，包含四个开放阅读框（ORF）。ORF1编码的蛋白（P1）是假定的运动蛋白；ORF2a（P2a）的产物包含VPg和丝氨酸蛋白酶的特征基序。由ORF2b编码的P2b是假定的RNA依赖RNA聚合酶。外壳蛋白P3由ORF3编码。在氨基末端含有六个组氨酸标签的CfMV P1、P2a、P2b和P3在大肠杆菌中表达、纯化，并分析它们的RNA结合活性。蛋白质印迹分析表明，带有His标签的P1、P2a、P2b和P3能够以序列非特异性方式与单链RNA转录本相互作用。滤膜结合试验证实了重组P1、P2a和P3的单链RNA结合能力。带有His标签的P3和天然外壳蛋白的RNA结合活性相似。随着NaCl浓度的增加，P1和P2a与单链RNA的结合显著降低。相反，P3在100 - 200 mM NaCl浓度下具有最佳RNA结合活性。我们讨论了CfMV蛋白RNA结合中可能涉及的氨基酸基序。
