EXPRESSION OF SEMLIKI FOREST VIRUS NSP1-SPECIFIC METHYLTRANSFERASE IN INSECT CELLS AND IN ESCHERICHIA-COLI
EXPRESSION OF SEMLIKI FOREST VIRUS NSP1-SPECIFIC METHYLTRANSFERASE IN INSECT CELLS AND IN ESCHERICHIA-COLI
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DOI：
10.1128/jvi.68.11.7418-7425.1994
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发表时间：
1994-11-01
影响因子：
5.4
通讯作者：
KAARIAINEN, L
中科院分区：
文献类型：
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作者：
LAAKKONEN, P;HYVONEN, M;KAARIAINEN, L
我们已经表达了塞姆利基森林病毒（SFV）的特异性非结构蛋白nsP1在昆虫细胞和大肠杆菌中的其他病毒蛋白的情况下。在用重组苜蓿银纹夜蛾核型多角体病毒（Autographa californica nuclear polyhedrosis virus，AcNPV）-nsP1感染的Sf9细胞中合成了大量的nsP1。与野生型AcNPV感染的细胞相比，这些细胞具有高水平的鸟嘌呤-7-甲基转移酶活性。甲基转移酶活性和nsP1主要存在于线粒体颗粒部分（P15）。与SFV感染的BHK细胞的情况一样，用脱氧胆酸盐（DOC）处理可增加酶活性。通过凝胶过滤和蔗糖梯度离心分析DOC处理从AcNPV-nsP1感染的细胞的P15释放的物质。甲基转移酶活性和nsP1都是聚集体。nsP1在E.在37 ℃的大肠杆菌中，在15，000 × g下沉淀，而在15 ℃表达后，nsP1和甲基转移酶活性都在上清液级分中。巧合的是，E.大肠杆菌的生长被Triton X-100和DOC完全抑制。蔗糖梯度分析表明，即使是“可溶性”nsP1-甲基转移酶的聚集体。结果表明，SFV感染的BHK细胞和AcNPV-nsP1感染的Sf 9细胞的P15组分中甲基转移酶活性以及E.大肠杆菌催化的[H-3]甲基从S-腺苷甲硫氨酸线性掺入GTP 60分钟的时间。三种来源的酶对S-腺苷甲硫氨酸具有相似的底物特异性和Km值。除了GTP，它们都甲基化dGTP和GpppG，但不甲基化m（7）GTP或GpppA，或体外转录的具有GpppA和GpppG帽的RNA。SFV特异性nsP1甲基转移酶的独特性质进行了讨论。
We have expressed the Semliki Forest virus (SFV)-specific nonstructural protein nsP1 both in insect cells and in Escherichia coli in the absence of other viral proteins. A substantial amount of nsP1 was synthesized in Sf9 cells infected with the recombinant Autographa californica nuclear polyhedrosis virus (AcNPV) AcNPV-nsP1. These cells had a high level of guanine-7-methyltransferase activity compared with that of wild-type AcNPV-infected cells. The methyltransferase activity and nsP1 were mostly in the mitochondrial pellet fraction (P15). The enzymatic activity was increased by treatment with deoxycholate (DOC), as in the case of SFV-infected BHK cells. The material released by DOC treatment from P15 of the AcNPV-nsP1-infected cells was analyzed by gel filtration and sucrose gradient centrifugation. Both the methyltransferase activity and nsP1 were in aggregates. nsP1 expressed in E. coli at 37 degrees C sedimented at 15,000 x g, whereas after expression at 15 degrees C, both nsP1 and methyltransferase activity were in the supernatant fraction. Paradoxically, the activity from E. coli was completely inhibited by Triton X-100 and DOC. Sucrose gradient analysis showed that even the ''soluble'' nsP1-methyltransferase was in aggregates. The methyltransferase activities in the P15 fractions of SFV-infected BHK cells and AcNPV-nsP1-infected Sf9 cells and in E. coli catalyzed linear incorporation of the [H-3]methyl group from S-adenosylmethionine to GTP for a 60 min period. The enzymes from the three sources had similar substrate specificities and K-m values for S-adenosylmethionine. In addition to GTP, they all methylated dGTP and GpppG, but not m(7)GTP or GpppA, or in vitro-transcribed RNAs with GpppA and GpppG caps. The unique properties of SFV-specific nsP1 methyltransferase are discussed.