Gershon P D, Shi X, Hodel A E
Department of Biochemistry and Biophysics, Texas A&M University, Houston, USA.
Virology. 1998 Jul 5;246(2):253-65. doi: 10.1006/viro.1998.9209.
Vaccinia protein VP39 has two RNA modifying activities. In monomeric form, it acts as an mRNA cap-specific 2'-O-methyltransferase, specifically modifying the ribose moiety of the first transcribed nucleotide of m7G-capped mRNA. In association with VP55, the catalytic subunit of the vaccinia poly(A) polymerase, VP39 facilitates the rapid elongation of poly(A) tails that are already greater than approximately 35 nt in length. Introducing new assays, we provide evidence that substrates for each of VP39's two activities do not detectably modulate the converse reaction and that VP39's 2'-O-methyltransferase activity is not significantly affected by its association with VP55. In an electrophoretic mobility shift assay, VP39 interacted with a short (5 nucleotide) RNA only when the latter was m7G-capped. Complexes with longer (22 nucleotide) RNAs were more stable (i.e., cap-independent) but were further stabilized by the presence of an m7G cap. An additional complex was observed at elevated RNA:protein molar ratios, indicating the presence of two RNA binding sites per VP39 molecule. Interaction at one of these sites was stabilized by the cap structure. Additional experiments indicated that RNA molecules undergoing poly(A) tail elongation by the VP55-VP39 heterodimer are not favored as cap-methylation substrates.
痘苗病毒蛋白VP39具有两种RNA修饰活性。以单体形式存在时，它作为一种mRNA帽特异性2'-O-甲基转移酶，特异性修饰m7G帽化mRNA第一个转录核苷酸的核糖部分。与痘苗病毒多聚腺苷酸聚合酶的催化亚基VP55结合时，VP39促进长度已大于约35个核苷酸的多聚腺苷酸尾巴的快速延伸。通过引入新的检测方法，我们提供了证据表明VP39的两种活性的底物不会显著调节相反的反应，并且VP39的2'-O-甲基转移酶活性不会因其与VP55的结合而受到显著影响。在电泳迁移率变动分析中，只有当短（5个核苷酸）RNA被m7G帽化时，VP39才会与之相互作用。与更长（22个核苷酸）RNA形成的复合物更稳定（即不依赖帽结构），但m7G帽的存在会进一步使其稳定。在升高的RNA:蛋白质摩尔比下观察到另一种复合物，表明每个VP39分子存在两个RNA结合位点。其中一个位点的相互作用通过帽结构得以稳定。额外的实验表明，由VP55-VP39异二聚体进行多聚腺苷酸尾巴延伸的RNA分子不太适合作为帽甲基化底物。
