Kukolj G, Katz R A, Skalka A M
Institute for Cancer Research, Fox Chase Cancer Center, 7701 Burholme Avenue, Philadelphia, PA 19111, USA.
Gene. 1998 Nov 26;223(1-2):157-63. doi: 10.1016/s0378-1119(98)00169-3.
A sequence of 21 amino acids (aa) in the C-terminal region of the 286-aa avian sarcoma virus (ASV) integrase (IN) protein has been shown previously to mediate nuclear localization of both IN and beta-galactosidase (betaGal) protein fused to it. This karyophilic sequence includes a high proportion of prolines and residues with basic side chains. In this report, site-directed mutagenesis was used to introduce single aa substitutions of several of these residues. Indirect immunofluorescence showed that IN-betaGal fusion constructs with Ala substitutions for sequence constituents K206, P215, K225 or R227 had lost the exclusive nuclear localization capability of the wild-type fusion. A fusion protein with the conservative substitution K206R retained the nuclear localization capacity. The site-specific substitutions that reduced karyophilic activity had no effect on the processing or joining activities of IN in vitro. However, the introduction of three of the four Ala codon substitutions into viral DNA clones caused a significant delay in viral replication following transfection of cycling chicken embryo fibroblasts. These results are consistent with a possible role for ASV IN in nuclear targeting.
先前已表明，286个氨基酸的禽肉瘤病毒（ASV）整合酶（IN）蛋白C端区域的一段21个氨基酸（aa）序列可介导与其融合的IN和β-半乳糖苷酶（βGal）蛋白的核定位。这段亲核序列包含高比例的脯氨酸和带有碱性侧链的残基。在本报告中，使用定点诱变对其中几个残基进行单个氨基酸替换。间接免疫荧光显示，用丙氨酸替换序列成分K206、P215、K225或R227的IN-βGal融合构建体失去了野生型融合体独有的核定位能力。具有保守替换K206R的融合蛋白保留了核定位能力。降低亲核活性的位点特异性替换对IN在体外的加工或连接活性没有影响。然而，将四个丙氨酸密码子替换中的三个引入病毒DNA克隆，在转染增殖的鸡胚成纤维细胞后导致病毒复制显著延迟。这些结果与ASV IN在核靶向中可能发挥的作用一致。
