Olspert Allan, Paves Heiti, Toomela Raavo, Tamm Tiina, Truve Erkki
Department of Gene Technology, Tallinn University of Technology, Akadeemia tee 15, 12618 Tallinn, Estonia.
Virus Genes. 2010 Jun;40(3):423-31. doi: 10.1007/s11262-010-0456-9. Epub 2010 Feb 13.
Cocksfoot mottle virus (CfMV) coat protein (CP) localization was studied in plant and mammalian cells. Fusion of the full-length CP with enhanced green fluorescent protein (EGFP) localized to the cell nucleus whereas similar constructs lacking the first 33 N-terminal amino acids of CP localized to the cytoplasm. CP and EGFP fusions containing mutations in the arginine-rich motif of CP localized to the cytoplasm and to the nucleus in plant cells indicating the involvement of the motif in nuclear localization. In mammalian cells, mutations in the arginine-rich region were sufficient to completely abolish nuclear transport. The analysis of deletions of amino acid residues 1-11, 1-22, and 22-33 of CP demonstrated that there were two separate nuclear localization signals (NLS) within the N-terminus--a strong NLS1 in the arginine-rich region (residues 22-33) and a weaker NLS2 within residues 1-22. Analysis of point mutants revealed that the basic amino acid residues in the region of the two NLSs were individually not sufficient to direct CP to the nucleus. Additional microinjection studies with fluorescently labeled RNA and CP purified from CfMV particles demonstrated that the wild-type CP was capable of transporting the RNA to the nucleus. This feature was not sequence-specific in transient assays since both CfMV and GFP mRNA were transported to the cell nucleus by CfMV CP. Together the results suggest that the nucleus may be involved in CfMV infection.
对鸭茅斑驳病毒（CfMV）外壳蛋白（CP）在植物细胞和哺乳动物细胞中的定位进行了研究。全长CP与增强型绿色荧光蛋白（EGFP）融合后定位于细胞核，而缺少CP前33个N端氨基酸的类似构建体则定位于细胞质。含有CP富含精氨酸基序突变的CP与EGFP融合体在植物细胞中定位于细胞质和细胞核，表明该基序参与核定位。在哺乳动物细胞中，富含精氨酸区域的突变足以完全消除核转运。对CP的氨基酸残基1 - 11、1 - 22和22 - 33缺失的分析表明，在N端有两个独立的核定位信号（NLS）——富含精氨酸区域（残基22 - 33）中有一个强NLS1，残基1 - 22中有一个较弱的NLS2。点突变体分析表明，两个NLS区域中的碱性氨基酸残基单独不足以将CP导向细胞核。用从CfMV颗粒中纯化的荧光标记RNA和CP进行的额外显微注射研究表明，野生型CP能够将RNA转运到细胞核。在瞬时试验中，这一特性不具有序列特异性，因为CfMV和GFP mRNA都能被CfMV CP转运到细胞核。这些结果共同表明细胞核可能参与CfMV感染。
