RNA-STIMULATED NTPASE ACTIVITY ASSOCIATED WITH THE P80 PROTEIN OF THE PESTIVIRUS BOVINE VIRAL DIARRHEA VIRUS
RNA-STIMULATED NTPASE ACTIVITY ASSOCIATED WITH THE P80 PROTEIN OF THE PESTIVIRUS BOVINE VIRAL DIARRHEA VIRUS
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DOI：
10.1006/viro.1993.1097
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发表时间：
1993-03-01
期刊：
影响因子：
3.7
通讯作者：
COLLETT, MS
中科院分区：
文献类型：
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作者：
TAMURA, JK;WARRENER, P;COLLETT, MS
瘟病毒的基因组RNA含有编码病毒体结构蛋白和病毒非结构多肽的单个大的开放框架。基于特定氨基酸序列基序的存在，预测鼠疫病毒非结构蛋白p80既是一种丝氨酸型蛋白酶，又是一种核苷三磷酸酶（NTPase）/RNA解旋酶。我们先前证明p80具有前一种活性（Wisherchen和Collett，Virology 184，341-350，1991）。在这里，我们提供的实验证据表明，这种蛋白质也是一种RNA刺激的NTR。采用免疫亲和层析，我们部分纯化的p80蛋白类似物（p87）从重组杆状病毒感染的昆虫细胞。我们表明该制剂含有特定的NTR活性。在从不表达p87蛋白的杆状病毒感染的昆虫细胞的裂解物中类似地纯化的材料中未发现这种活性。NTR活性与p87多肽的相关性以两种方式证明。首先，NTR的活性被p80多肽特异性单克隆抗体完全抑制，但不受无关抗原单克隆抗体的影响。第二，放射性标记的ATP可以特异性地与p87多肽交联。p87蛋白的NTP水解受到特定单链RNA分子的刺激。初步酶的表征瘟病毒p80 NTR，并讨论了这种活动在瘟病毒复制的假定作用。
The genomic RNA of pestiviruses contains a single large open frame coding for virion structural proteins and viral nonstructural polypeptides. Based on the presence of specific amino acid sequence motifs, pestivirus nonstructural protein p80 was predicted to be both a serine-type proteinase and a nucleoside triphosphatase (NTPase)/RNA helicase. We previously demonstrated p80 possesses the former activity (Wisherchen and Collett,Virology184, 341-350, 1991). Here, we provide experimental evidence that this protein is also an RNA-stimulated NTPase. Employing immunoaffinity chromatography, we partially purified a p80 protein analog (p87) from recombinant baculovirus-infected insect cells. We show this preparation contained a specific NTPase activity. This activity was not found in material similarly purified from lysates of baculovirus-infected insect cells not expressing the p87 protein. That the NTPase activity was associated with the p87 polypeptide was demonstrated in two ways. First, the NTPase activity was shown to be completely inhibited by monoclonal antibodies specific to the p80 polypeptide, but was unaffected by monoclonal antibodies to unrelated antigens. Second, radiolabeled ATP could be specifically cross-linked to the p87 polypeptide. NTP hydrolysis by the p87 protein was stimulated by the presence of particular single-strand RNA molecules. Initial enzymologic characterization of the pestivirus p80 NTPase is presented, and the presumptive role of this activity in pestivirus replication is discussed.