Ishii N, Minami N, Chen E Y, Medina A L, Chico M M, Kasamatsu H
Department of Molecular, Cell, and Developmental Biology, University of California, Los Angeles 90024, USA.
J Virol. 1996 Feb;70(2):1317-22. doi: 10.1128/JVI.70.2.1317-1322.1996.
The nuclear localization signal of the major structural protein, Vp1, of simian virus 40 was further defined by mutagenesis. The targeting activity was examined in cells microinjected with SV-Vp1 variant viral DNAs bearing either an initiation codon mutation of the agnoprotein or mutations in the Vp1 coding sequence or microinjected with pSG5-Vp1 and pSG5-Vp1 mutant DNAs in which Vp1 or mutant Vp1 is expressed from simian virus 40 early promoter. The Vp1 nuclear localization signal functioned autonomously without agno-protein once the Vp1 protein was synthesized in the cytoplasm. The targeting activity was localized to the amino-terminal 19 residues. While replacement of cysteine 10 with glycine, alanine, or serine did not affect the activity, replacement of arginine 6 with glycine caused the cytoplasmic phenotype. When multiple mutations were introduced among residue 5, 6, 7, 16, 17, or 19, the targeting activity was found to reside in two clusters of basic residues, a cluster of lysine 5, arginine 6, and lysine 7 and a cluster of lysine 16, lysine 17, and lysine 19. The clusters are independently important for nuclear localization activity.
通过诱变进一步确定了猴病毒40主要结构蛋白Vp1的核定位信号。在显微注射携带agnoprotein起始密码子突变或Vp1编码序列突变的SV - Vp1变异病毒DNA的细胞中，或显微注射从猴病毒40早期启动子表达Vp1或突变Vp1的pSG5 - Vp1和pSG5 - Vp1突变DNA的细胞中检测靶向活性。一旦Vp1蛋白在细胞质中合成，Vp1核定位信号无需agnoprotein即可自主发挥作用。靶向活性定位于氨基末端的19个残基。用甘氨酸、丙氨酸或丝氨酸取代第10位的半胱氨酸不影响活性，但用甘氨酸取代第6位的精氨酸会导致细胞质表型。当在第5、6、7、16、17或19位残基中引入多个突变时，发现靶向活性存在于两个碱性残基簇中，一个是赖氨酸5、精氨酸6和赖氨酸7簇，另一个是赖氨酸16、赖氨酸17和赖氨酸19簇。这些簇对核定位活性具有独立的重要性。
