Krab Ivo M, Caldwell Christian, Gallie Daniel R, Bol John F
Institute of Biology, Gorlaeus Laboratories, Leiden University, Einsteinweg 55, 2333 CC Leiden, The Netherlands.
Department of Biochemistry, Boyce Hall, University of California, Riverside, CA 92521, USA.
J Gen Virol. 2005 Jun;86(Pt 6):1841-1849. doi: 10.1099/vir.0.80796-0.
The three plus-strand genomic RNAs of Alfalfa mosaic virus (AMV) and the subgenomic messenger for viral coat protein (CP) contain a 5'-cap structure, but no 3'-poly(A) tail. Binding of CP to the 3' end of AMV RNAs is required for efficient translation of the viral RNAs and to initiate infection in plant cells. To study the role of CP in translation, plant protoplasts were transfected with luciferase (Luc) transcripts with 3'-terminal sequences consisting of the 3' untranslated region of AMV RNA 3 (Luc-AMV), a poly(A) tail of 50 residues [Luc-poly(A)] or a short vector-derived sequence (Luc-control). Pre-incubation of the transcripts with CP had no effect on Luc expression from Luc-poly(A) or Luc-control, but strongly stimulated Luc expression from Luc-AMV. From time-course experiments, it was calculated that CP binding increased the half-life of Luc-AMV by 20 % and enhanced its translational efficiency by about 40-fold. In addition to the 3' AMV sequence, the cap structure was required for CP-mediated stimulation of Luc-AMV translation. Glutathione S-transferase pull-down assays revealed an interaction between AMV CP and initiation factor complexes eIF4F and eIFiso4F from wheatgerm. Far-Western blotting revealed that this binding occurred through an interaction of CP with the eIF4G and eIFiso4G subunits of eIF4F and eIFiso4F, respectively. The results support the hypothesis that the role of CP in translation of viral RNAs mimics the role of the poly(A)-binding protein in translation of cellular mRNAs.
苜蓿花叶病毒（AMV）的三条正义链基因组RNA以及病毒外壳蛋白（CP）的亚基因组信使RNA含有5'-帽结构，但没有3'-聚腺苷酸尾巴。CP与AMV RNA的3'末端结合是病毒RNA有效翻译以及在植物细胞中引发感染所必需的。为了研究CP在翻译中的作用，用荧光素酶（Luc）转录本转染植物原生质体，这些转录本的3'-末端序列由AMV RNA 3的3'非翻译区（Luc-AMV）、50个残基的聚腺苷酸尾巴[Luc-聚（A）]或短的载体衍生序列（Luc-对照）组成。转录本与CP的预孵育对Luc-聚（A）或Luc-对照的Luc表达没有影响，但强烈刺激了Luc-AMV的Luc表达。从时间进程实验计算得出，CP结合使Luc-AMV的半衰期增加了20%，并使其翻译效率提高了约40倍。除了3' AMV序列外，帽结构对于CP介导的Luc-AMV翻译刺激也是必需的。谷胱甘肽S-转移酶下拉试验揭示了AMV CP与来自小麦胚芽的起始因子复合物eIF4F和eIFiso4F之间的相互作用。Far-Western印迹显示，这种结合分别通过CP与eIF4F和eIFiso4F的eIF4G和eIFiso4G亚基的相互作用而发生。结果支持了这样的假设，即CP在病毒RNA翻译中的作用类似于聚腺苷酸结合蛋白在细胞mRNA翻译中的作用。
