Grdzelishvili Valery Z, Smallwood Sherin, Tower Dallas, Hall Richard L, Hunt D Margaret, Moyer Sue A
Department of Molecular Genetics and Microbiology, University of Florida College of Medicine, Gainesville, FL 32610, USA.
Virology. 2006 Jul 5;350(2):394-405. doi: 10.1016/j.virol.2006.02.021. Epub 2006 Mar 13.
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, V.Z., 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 methylation.
水泡性口炎病毒（VSV）的L聚合酶蛋白具有两种甲基转移酶（MTase）活性，可催化病毒mRNA帽结构在鸟嘌呤-N7和2'-O-腺苷位置的甲基化。为了鉴定MTase活性所需的L序列，我们分析了VSV的一个宿主范围（hr）和温度敏感（ts）突变体hr8，它在mRNA帽甲基化方面存在缺陷。对hr8进行测序鉴定出五个氨基酸替换，均位于L蛋白中。用每个鉴定出的L突变产生重组VSV，位于保守结构域V和VI之间的L中单个G1481R替换足以使总体mRNA甲基化显著降低（约90%）。帽分析显示残留的鸟嘌呤-N7甲基化和降低的2'-O-腺苷甲基化，与原始hr8病毒相同。当在允许和不允许hr8突变体生长的条件下测试重组病毒的生长时，相同的单个L突变G1481R是hr和ts表型的唯一原因。鉴定出rG1481R病毒的一个自发抑制突变体，其恢复了在非允许细胞上的生长和帽甲基化，并定位到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，随后拯救重组病毒，鉴定出另外五个病毒突变体K1468A、R1478A/D1479A、G1481A、G1481N和G1672A，它们均为hr且在mRNA帽甲基化方面存在缺陷。因此，除了先前已表征的结构域VI [Grdzelishvili, V.Z., 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病毒mRNA帽甲基化。《病毒学杂志》79, 7327 - 7337；Li, J., Fontaine-Rodriguez, E.C., Whelan, S.P., 2005. 水泡性口炎病毒大聚合酶蛋白保守结构域VI内对mRNA帽甲基转移酶活性至关重要的氨基酸残基。《病毒学杂志》79, 13373 - 13384] 外，还鉴定出L氨基酸1450 - 1481之间的一个新区域，它对mRNA帽甲基化至关重要。
