Enzymatic defects of the nsP2 proteins of Semliki Forest virus temperature-sensitive mutants
Enzymatic defects of the nsP2 proteins of Semliki Forest virus temperature-sensitive mutants
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DOI：
10.1128/jvi.02078-06
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发表时间：
2007-03-01
影响因子：
5.4
通讯作者：
Ahola, Tero
中科院分区：
文献类型：
--
作者：
Balistreri, Giuseppe;Caldentey, Javier;Ahola, Tero
我们已经分析了影响Semliki森林病毒温度敏感（TS）突变体中病毒RNA合成的突变的生化后果。在多功能复制酶蛋白nsP 2中映射的六个突变中，三个位于N-末端解旋酶区域，三个位于C-末端蛋白酶结构域。表达、纯化野生型和突变型nsP 2，并在24 ℃和35 ℃下体外测定核苷酸三磷酸酶（NTH）、RNA三磷酸酶（RTH）和蛋白酶活性。蛋白酶结构域突变体（ts 4、ts 6和ts 11）在35 ℃下具有降低的蛋白酶活性，但显示正常的NTR和RTR。解旋酶结构域突变ts 1没有酶的后果，而ts 13 a和ts 9降低NTR和蛋白酶的活性，但在不同的和突变体特异性的方式。这些解旋酶结构域突变体对蛋白酶功能的影响表明nsP 2内的结构域间相互作用。蛋白酶活性不直接需要NTR活性。NTT和RTT结果的相似性以及竞争实验表明，这两种反应利用相同的活性位点。还在重组病毒中研究了突变，首先在允许温度下培养，然后转移到限制温度。在感染了携带ts 4、ts 6、ts 11和ts 13 a突变的病毒的细胞中，非结构性多蛋白的加工通常被延迟，并且在ts 9中出现了特定的缺陷。除ts 13 a外的所有突变均与亚基因组26 S mRNA的产生大幅减少相关，表明蛋白酶和解旋酶结构域均影响病毒复制过程中亚基因组启动子的识别。
We have analyzed the biochemical consequences of mutations that affect viral RNA synthesis in Semliki Forest virus temperature-sensitive (ts) mutants. Of the six mutations mapping in the multifunctional replicase protein nsP2, three were located in the N-terminal helicase region and three were in the C-terminal protease domain. Wild-type and mutant nsP2s were expressed, purified, and assayed for nucleotide triphosphatase (NTPase), RNA triphosphatase (RTPase), and protease activities in vitro at 24 degrees C and 35 degrees C. The protease domain mutants (ts4, ts6, and ts 11) had reduced protease activity at 35'C but displayed normal NTPase and RTPase. The helicase domain mutation ts1 did not have enzymatic consequences, whereas ts13a and ts9 reduced both NTPase and protease activities but in different and mutant-specific ways. The effects of these helicase domain mutants on protease function suggest interdomain interactions within nsP2. NTPase activity was not directly required for protease activity. The similarities of the NTPase and RTPase results, as well as competition experiments, suggest that these two reactions utilize the same active site. The mutations were also studied in recombinant viruses first cultivated at the permissive temperature and then shifted up to the restrictive temperature. Processing of the nonstructural polyprotein was generally retarded in cells infected with viruses carrying the ts4, ts6, ts11, and ts13a mutations, and a specific defect appeared in ts9. All mutations except ts13a were associated with a large reduction in the production of the subgenomic 26S mRNA, indicating that both protease and helicase domains influence the recognition of the subgenomic promoter during virus replication.