Harris C E, Boden R A, Astell C R
Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada.
J Virol. 1999 Jan;73(1):72-80. doi: 10.1128/JVI.73.1.72-80.1999.
NS1, the major nonstructural parvovirus protein of the minute virus of mice, is a multifunctional protein responsible for several aspects of viral replication. NS1 transactivates the P38 promoter (used to express the structural proteins), as well as its own strong promoter, P4. To study the mechanism of activation and to map regions of NS1 responsible for transactivation, NS1 and various deletions of NS1 were cloned in frame with the GAL4DB and cotransfected into COS-7 and LA9 cells with a synthetic GAL4-responsive reporter plasmid. These studies showed NS1 can directly activate transcription through its 129 carboxyl-terminal amino acid residues. Any deletion from this region of the C terminus, even as few as 8 amino acids, completely abolishes transactivation. A yeast two-hybrid system used to identify protein-protein interactions demonstrated that NS1 is able to dimerize when expressed in yeast cells. However, only an almost complete NS11-638 bait was able to interact with the full-length NS1. A two-hybrid screen identified a HeLa cell cDNA clone (NS1-associated protein 1 [NSAP1]) that interacts with NS11-276 and NS11-638. An additional sequence was predicted from human EST (expressed sequence tag) data, and the cDNA was estimated to be at least 2,221 bp long, potentially encoding a 562-amino-acid protein product. A polyclonal antibody raised to a synthetic peptide within NSAP1 recognizes an approximately 65-kDa cellular protein. This NSAP1 cDNA has not previously been characterized, but the predicted protein sequence is 80% identical to the recently identified heterogeneous nuclear ribonucleoprotein (hnRNP) R (W. Hassfeld et al., Nucleic Acids Res. 26:439-445, 1998). NSAP1 contains four ribonucleoprotein domains, as well as a highly repetitive C-terminal region. A closely related mouse cDNA (deduced from murine EST data) encodes a protein with only a single amino acid residue change from the human protein. NSAP1 is predicted to be a 65-kDa polynucleotide binding protein, and it likely functions in the regulation of splicing and/or transport of mRNAs from the nucleus.
NS1是小鼠微小病毒的主要非结构细小病毒蛋白，是一种多功能蛋白，负责病毒复制的多个方面。NS1可反式激活P38启动子（用于表达结构蛋白）及其自身的强启动子P4。为了研究激活机制并定位NS1负责反式激活的区域，将NS1及其各种缺失片段与GAL4DB框内克隆，并与合成的GAL4反应性报告质粒共转染到COS-7和LA9细胞中。这些研究表明，NS1可通过其129个羧基末端氨基酸残基直接激活转录。从C末端的这个区域进行任何缺失，即使只有8个氨基酸，也会完全消除反式激活。用于鉴定蛋白质-蛋白质相互作用的酵母双杂交系统表明，NS1在酵母细胞中表达时能够二聚化。然而，只有几乎完整的NS1 1-638诱饵能够与全长NS1相互作用。双杂交筛选鉴定出一个与NS1 1-276和NS1 1-638相互作用的HeLa细胞cDNA克隆（NS1相关蛋白1 [NSAP1]）。根据人类EST（表达序列标签）数据预测了一个额外的序列，该cDNA估计至少有2221 bp长，可能编码一个562个氨基酸的蛋白质产物。针对NSAP1内的合成肽产生的多克隆抗体识别一种约65 kDa的细胞蛋白。该NSAP1 cDNA以前尚未被表征，但预测的蛋白质序列与最近鉴定的异质性核糖核蛋白（hnRNP）R有80%的同一性（W. Hassfeld等人，《核酸研究》26:439-445，1998）。NSAP1包含四个核糖核蛋白结构域以及一个高度重复的C末端区域。一个密切相关的小鼠cDNA（从鼠EST数据推导）编码的蛋白质与人类蛋白质只有一个氨基酸残基的变化。NSAP1预计是一种65 kDa的多核苷酸结合蛋白，它可能在mRNA从细胞核的剪接和/或转运调控中发挥作用。
