ATPASE AND GTPASE ACTIVITIES ASSOCIATED WITH SEMLIKI FOREST VIRUS NONSTRUCTURAL PROTEIN NSP2
ATPASE AND GTPASE ACTIVITIES ASSOCIATED WITH SEMLIKI FOREST VIRUS NONSTRUCTURAL PROTEIN NSP2
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DOI：
10.1128/jvi.68.9.5804-5810.1994
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发表时间：
1994-09-01
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：
RIKKONEN, M;PERANEN, J;KAARIAINEN, L
塞姆利基森林病毒的复制需要四种非结构蛋白（nsP1 至 nsP4），全部源自相同的多蛋白。其中之一，nsP2，是 RNA 复制和非结构多蛋白加工所需的多功能蛋白。根据氨基酸序列同源性，预测nsP2具有核苷三磷酸酶和RNA解旋酶活性。在这里，我们报告了使用高效 T7 表达系统在大肠杆菌中工程化表达 nsP2 及其氨基末端片段。两种多肽均以氨基末端带有组氨酸标签的融合蛋白形式产生，并通过固定化金属亲和层析纯化。这两种重组蛋白表现出 ATPase 和 GTPase 活性，单链 RNA 的存在进一步刺激了这些活性。在来自未诱导的对照细胞或表达不相关多肽的细胞的类似制备的级分中很容易发现这些活性。放射性标记的核糖核苷三磷酸可以与全长和羧基末端截短的 nsP2 蛋白交联，并且两种多肽都具有 RNA 结合能力。我们还表达并纯化了 nsP2 变体，该变体在核苷酸结合基序中具有单个氨基酸取代（Lys-192-->Asn）。没有核苷三磷酸酶活性与该突变蛋白相关，
The replication of Semliki Forest virus requires four nonstructural proteins (nsP1 to nsP4), all derived from the same polyprotein. One of these, nsP2, is a multifunctional protein needed in RNA replication and in the processing of the nonstructural polyprotein. On the basis of amino acid sequence homologies, nsP2 was predicted to possess nucleoside triphosphatase and RNA helicase activities. Here, we report the engineered expression in Escherichia coli of nsP2 and of an amino-terminal fragment of it by use of the highly efficient T7 expression system. Both polypeptides were produced as fusion proteins with a histidine tag at the amino terminus and purified by immobilized-metal affinity chromatography. The two recombinant proteins exhibited ATPase and GTPase activities, which were further stimulated by the presence of single-stranded RNA. The activities were hot found in similarly prepared fractions from uninduced control cells or cells expressing an unrelated polypeptide. Radiolabeled ribonucleoside triphosphates could be cross-linked to both the full-length and the carboxy-terminally truncated nsP2 protein, and both polypeptides had RNA-binding capacity. We also expressed and purified an nsP2 variant which had a single amino acid substitution in the nucleotide-binding motif (Lys-192-->Asn). No nucleoside triphosphatase activity was associated with this mutant protein,