Identification of a new region in the vesicular stomatitis virus L polymerase protein which is essential for mRNA cap methylation
Identification of a new region in the vesicular stomatitis virus L polymerase protein which is essential for mRNA cap methylation
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DOI：
10.1016/j.virol.2006.02.021
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发表时间：
2006-07-05
期刊：
影响因子：
3.7
通讯作者：
Moyer, Sue A.
中科院分区：
文献类型：
--
作者：
Grdzelishvili, Valery Z.;Smallwood, Sherin;Moyer, Sue A.
水疱性口炎病毒 (VSV) L 聚合酶蛋白具有两种甲基转移酶 (MTase) 活性，可催化病毒 mRNA 帽子结构的鸟嘌呤-N7 和 2'-O-腺苷位置甲基化。为了鉴定 MTase 活性所需的 L 序列，我们分析了 VSV 的宿主范围 (hr) 和温度敏感 (ts) 突变体 hr8，该突变体的 mRNA 帽甲基化有缺陷。 hr8 测序发现了 5 个氨基酸取代，全部位于 L 蛋白中。使用每个已识别的 L 突变生成重组 VSV，并且位于保守结构域 V 和 VI 之间的 L 中单个 G1481R 取代的存在足以使总体 mRNA 甲基化显着降低（约 90%）。 Cap 分析显示残留的鸟嘌呤-N7 甲基化和减少的 2'-O-腺苷甲基化，与原始 hr8 病毒相同。当在允许和不允许 hr8 突变体的条件下测试重组病毒的病毒生长时，相同的单一 L 突变 G1481R 单独负责 hr 和 ts 表型。鉴定出 rG1481R 病毒的自发抑制突变体，该突变体可恢复非允许细胞上的生长和帽甲基化，并将其映射到 L 中的单个变化 L1450I。对结构域 V 和 VI 之间的区域（L 的氨基酸 1419-1672）进行定点诱变，然后拯救重组病毒，鉴定出另外 5 个病毒突变体：K1468A、R1478A/D1479A、 G1481A、G1481N 和 G1672A，均存在 hr 且 mRNA 帽甲基化缺陷。因此，除了先前表征的结构域 VI [Grdzelishvili, VZ.、Smallwood, S.、Tower, D.、Hall, R.L.、Hunt, D.M.、Moyer, S.A., 2005。水泡性口炎病毒 L-聚合酶蛋白中的单个氨基酸变化完全消除了病毒 mRNA 帽甲基化。 J.维罗尔。 79、7327-7337； Li, J., Fontaine-Rodriguez, E.C., Whelan, S.P., 2005。水疱性口炎病毒大聚合酶蛋白保守结构域 VI 内的氨基酸残基对 mRNA 帽甲基转移酶活性至关重要。 J.维罗尔。 [79, 13373-13384]，鉴定出 L 氨基酸 1450-1481 之间的一个新区域，该区域对于 mRNA 帽三乙基化至关重要。 (c) 2006 Elsevier Inc. 保留所有权利。
The vesicular stomatitis virus (VSV) L polymerase protein possesses two methyltransferase (MTase) activities, which catalyze the methylation of viral mRNA cap structures at the guanine-N7 and 2 '-O-adenosine positions. To identify L sequences required for the MTase activities, we analyzed a host range (hr) and temperature-sensitive (ts) mutant of VSV, hr8, which was defective in mRNA cap methylation. Sequencing hr8 identified five amino acid substitutions, all residing in the L protein. Recombinant VSV were generated with each of the identified L mutations, and the presence of a single G1481R substitution in L, located between conserved domains V and VI, was sufficient to produce a dramatic reduction (about 90%) in overall mRNA methylation. Cap analysis showed residual guanine-N7 methylation and reduced 2 '-O-adenosine methylation, identical to that of the original hr8 virus. When recombinant viruses were tested for virus growth under conditions that were permissive and nonpermissive for the hr8 mutant, the same single L mutation, G1481R, was solely responsible for both the hr and ts phenotypes. A spontaneous suppressor mutant of the rG1481R virus that restored both growth on nonpermissive cells and cap methylation was identified and mapped to a single change, L1450I, in L. Site-directed mutagenesis of the region between domains V and VI, amino acids 1419-1672 of L, followed by the rescue of recombinant viruses identified five additional virus mutants, K1468A, R1478A/ D1479A, G1481A, G1481N, and G1672A, that were all hr and defective in mRNA cap methylation. Thus, in addition to the previously characterized domain VI [Grdzelishvili, VZ., Smallwood, S., Tower, D., Hall, R.L., Hunt, D.M., Moyer, S.A., 2005. A single amino acid change in the L-polymerase protein of vesicular stomatitis virus completely abolishes viral mRNA cap methylation. J. Virol. 79, 7327-7337; Li, J., Fontaine-Rodriguez, E.C., Whelan, S.P., 2005. Amino acid residues within conserved domain VI of the vesicular stomatitis virus large polymerase protein essential for mRNA cap methyltransferase activity. J. Virol. 79, 13373-13384], a new region between L amino acids 1450-1481 was identified which is critical for mRNA cap triethylation. (c) 2006 Elsevier Inc. All rights reserved.