Hong J S, Engler J A
Department of Biochemistry, School of Medicine, University of Alabama, Birmingham 35294.
Virology. 1991 Dec;185(2):758-67. doi: 10.1016/0042-6822(91)90547-o.
Using a recombinant vaccinia virus vector, the fiber protein from adenovirus serotype 2 has been expressed in human cells; the protein expressed was correctly assembled into trimers, glycosylated, and transported to the nucleus. Deletion of amino acids 2-5 (KRAR) resulted in accumulation of fiber in the cytoplasm; fusion of the sequence TKRVRL, found at the beginning of Ad7 fiber, to the N-terminus of this mutant restored correct targeting. Changing the charge of amino acids 91 and 92 within another potential targeting sequence (LKKTK to LEETK) had little effect on nuclear targeting. When fused to the N-terminus of beta-galactosidase and expressed in recombinant vaccinia virus, neither MKRARP nor MTKRVRL (from Ad2 and Ad7 fibers, respectively), were sufficient for efficient transport of the hybrid protein to the nucleus; on the other hand, fusions of either MKRARPSEDTF (from Ad2 fiber) or of MKRPRP (a known targeting sequence from the C-terminus of Ad2 E1A proteins) to beta-galactosidase were localized to the nucleus. These results suggest that sequences at the N-terminus of Ad2 and Ad7 fiber are required for correct nuclear targeting.
利用重组痘苗病毒载体，2型腺病毒的纤维蛋白已在人细胞中表达；所表达的蛋白正确组装成三聚体，进行了糖基化，并转运至细胞核。缺失氨基酸2 - 5（KRAR）导致纤维蛋白在细胞质中积累；将Ad7纤维蛋白起始处发现的序列TKRVRL与该突变体的N端融合，恢复了正确的靶向定位。在另一个潜在的靶向序列内改变氨基酸91和92的电荷（从LKKTK变为LEETK）对核靶向作用影响不大。当与β - 半乳糖苷酶的N端融合并在重组痘苗病毒中表达时，MKRARP和MTKRVRL（分别来自Ad2和Ad7纤维蛋白）均不足以使杂合蛋白有效转运至细胞核；另一方面，MKRARPSEDTF（来自Ad2纤维蛋白）或MKRPRP（Ad2 E1A蛋白C端的已知靶向序列）与β - 半乳糖苷酶的融合蛋白则定位于细胞核。这些结果表明，Ad2和Ad7纤维蛋白N端的序列对于正确的核靶向是必需的。
