Expression of functional parvoviral NS1 from recombinant vaccinia virus: effects of mutations in the nucleotide-binding motif.
Expression of functional parvoviral NS1 from recombinant vaccinia virus: effects of mutations in the nucleotide-binding motif.
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DOI：
10.1016/0042-6822(92)90202-z
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发表时间：
1992-11
期刊：
影响因子：
3.7
通讯作者：
Jürg P.F. Noesch;S. Cotmore;P. Tattersall
中科院分区：
文献类型：
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作者：
Jürg P.F. Noesch;S. Cotmore;P. Tattersall
将编码小鼠微小病毒(MVM)主要复制蛋白NS1的基因转移到重组痘苗病毒载体中，取代痘苗病毒胸苷激酶基因。NS1基因被置于T7噬菌体启动子的控制下，并在与另一种编码T7RNA聚合酶的重组痘苗病毒vTF7-3共感染的细胞中表达。来自脑心肌炎病毒的5‘非翻译区的存在进一步增强了NS1的表达，这使得高效的帽子无关翻译成为可能。利用该系统对野生型NS1及其两种突变形式NSI K405R和NS1K405M进行了分析，其中位于NS1三磷酸嘌呤结合位点的高度保守的赖氨酸密码子分别被改变为精氨酸和蛋氨酸。在感染了这三种重组病毒的人和小鼠细胞中，全长NS1都得到了有效的表达，并且在每种情况下，NSI都被快速而有效地移位到细胞核中。用这种方法表达的野生型NS1具有生物学活性。它能够反式激活位于宿主染色体上的MVM P38启动子，而NS1的两个突变形式在本实验中没有显著活性，并且它能够解析从多聚体MVM复制形式DNA分子中克隆的回文连接片段。这些代表MVM基因组左端：左端和右端：右端融合的底物，在依赖DNA合成的体外反应中被分解，并辅以含有重组野生型NS1的核提取物。这两种突变形式的多肽在本实验中都没有任何可检测到的活性。
The gene encoding the major replicative protein, NS1, of minute virus of mice (MVM) was transferred into a recombinant vaccinia virus vector in place of the vaccinia thymidine kinase gene. The NS1 gene was placed under control of a bacteriophage T7 promoter and expressed in cells coinfected with another recombinant vaccinia virus, vTF7-3, which encodes the T7 RNA polymerase. Expression of NS1 was further enhanced by the presence of a 5′ untranslated region, derived from encephalomyocarditis virus, which allows efficient cap-independent translation. This system was used to produce and analyze wild-type NS1 and two mutant forms of the protein, NSi K405R and NS1 K405M, in which the highly conserved lysine codon located in the putative purine triphosphate binding site of NS1 was changed to arginine and methionine, respectively. Full-length NS1 was expressed efficiently in both human and mouse cells infected with each of the three recombinant viruses, and in each case the NSi was rapidly and efficiently translocated into the nucleus. Wild-type NS1 expressed in this way was biologically active. It was able to trans-activate an MVM P38 promoter located in a host chromosomal site, whereas the two mutant forms of NS1 showed no significant activity in this assay, and it was capable of resolving palindromic junction fragments cloned from multimeric MVM replicative form DNA molecules. These substrates, representing MVM genomic left-end:left-end and right-end:right-end fusions, were resolved in a DNA synthesis-dependentin vitroreaction supplemented with nuclear extracts containing recombinant wild-type NS1. Neither of the two mutant forms of the polypeptide had any detectable activity in this assay.