Signature motifs of GDP polyribonucleotidyltransferase, a non-segmented negative strand RNA viral mRNA capping enzyme, domain in the L protein are required for covalent enzyme-pRNA intermediate formation.
Signature motifs of GDP polyribonucleotidyltransferase, a non-segmented negative strand RNA viral mRNA capping enzyme, domain in the L protein are required for covalent enzyme-pRNA intermediate formation.
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DOI：
10.1093/nar/gkv1286
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发表时间：
2016-01-08
影响因子：
14.9
通讯作者：
Ogino T
中科院分区：
文献类型：
--
作者：
Neubauer J;Ogino M;Green TJ;Ogino T
非常规mRNA加帽酶非节段负链（NNS）RNA病毒RNA聚合酶L蛋白中的（GDP多核糖核苷酸转移酶，PRNTase; Block V）结构域（例如狂犬病、麻疹、埃博拉）含有5个共线序列元件Rx（3）Wx（3-8）ΦxGx x（P/A）（基序A; Φ，疏水性; β，亲水性）、（Y/W）ΦGSxT（基序B）、W（基序C）、HR（基序D）和Gxx Φx（F/Y）QxxΦ（基序E）。我们进行了水泡性口炎病毒（VSV，原型NNS RNA病毒）的L蛋白的定点突变，以检查这些图案在mRNA加帽的参与。与基序D中的催化残基类似，发现基序A中的G1100、基序B中的T1157、基序C中的W1188以及基序E中的F1269和Q1270对于共价L-pRNA中间体形成步骤中的PRNT酶活性是必需的或重要的，但对于产生GDP（pRNA受体）的GT3酶活性不是必需的或重要的。这些残基中的帽缺陷突变诱导mRNA合成在位置+40处终止，随后是异常的停止-开始转录，并消除宿主细胞中的病毒基因表达。这些结果表明，保守的基序构成的PRNTase结构域的活性位点和L-pRNA中间体的形成，随后的帽形成是全长mRNA的成功合成所必需的。
The unconventional mRNA capping enzyme (GDP polyribonucleotidyltransferase, PRNTase; block V) domain in RNA polymerase L proteins of non-segmented negative strand (NNS) RNA viruses (e.g. rabies, measles, Ebola) contains five collinear sequence elements, Rx(3)Wx(3–8)ΦxGxζx(P/A) (motif A; Φ, hydrophobic; ζ, hydrophilic), (Y/W)ΦGSxT (motif B), W (motif C), HR (motif D) and ζxxΦx(F/Y)QxxΦ (motif E). We performed site-directed mutagenesis of the L protein of vesicular stomatitis virus (VSV, a prototypic NNS RNA virus) to examine participation of these motifs in mRNA capping. Similar to the catalytic residues in motif D, G1100 in motif A, T1157 in motif B, W1188 in motif C, and F1269 and Q1270 in motif E were found to be essential or important for the PRNTase activity in the step of the covalent L-pRNA intermediate formation, but not for the GTPase activity that generates GDP (pRNA acceptor). Cap defective mutations in these residues induced termination of mRNA synthesis at position +40 followed by aberrant stop–start transcription, and abolished virus gene expression in host cells. These results suggest that the conserved motifs constitute the active site of the PRNTase domain and the L-pRNA intermediate formation followed by the cap formation is essential for successful synthesis of full-length mRNAs.