Feuerman M H, Davis B R, Pattengale P K, Fan H
J Virol. 1985 Jun;54(3):804-16. doi: 10.1128/JVI.54.3.804-816.1985.
A Moloney murine leukemia virus (M-MuLV) recombinant carrying the v-src gene of avian sarcoma virus was generated by the introduction of a cloned portion of v-src from Schmidt-Ruppin A avian sarcoma virus into a molecular clone of M-MuLV provirus at the recombinant DNA level. The v-src sequences (lacking a portion of the 5' end of v-src) were inserted into the p30 region of the M-MulV gag gene so that M-MuLV gag and v-src were in the same reading frame. Transfection of this chimeric clone, pMLV(src), into NIH 3T3 cells which were constitutively producing M-MuLV gag and pol protein resulted in the formation of foci of transformed cells. Infectious and transforming virus could be recovered from the transformed cells. This virus was designated M-MuLV(src). M-MuLV(src)-transformed cells contained two novel proteins of 78 and 90 kilodaltons. The 78-kilodalton protein, p78gag-src, contained both gag and src determinants, exhibited kinase activity in an immune kinase assay, and is probably a fusion of Pr65gag and src. The 90-kilodalton protein, which is of the appropriate size to be the gPr80gag fused to src, contained gag determinants as well as a V8 protease cleavage fragment typical of the carboxy terminus of avian sarcoma virus pp60src. However, it could not be immunoprecipitated with an anti-v-src serum. M-MuLV(src)-transformed cells showed elevated levels of intracellular phosphotyrosine in proteins, although the elevation was intermediate compared with cells transformed with wild-type v-src. M-MuLV and amphotropic murine leukemia virus pseudotypes of M-MuLV(src) were inoculated into newborn NIH Swiss mice. Inoculated mice developed solid tumors at the site of inoculation after 3 to 6 weeks, with most animals dying by 14 weeks. Histopathological analysis indicated that the solid tumors were mesenchymally derived fibrosarcomas that were both invasive and metastatic.
通过在重组 DNA 水平上，将来自施密特 - 鲁平 A 型禽肉瘤病毒的 v-src 克隆片段引入莫洛尼鼠白血病病毒（M-MuLV）前病毒的分子克隆中，构建了携带禽肉瘤病毒 v-src 基因的 M-MuLV 重组体。v-src 序列（缺少 v-src 5' 端的一部分）被插入到 M-MuLV gag 基因的 p30 区域，使得 M-MuLV gag 和 v-src 处于同一阅读框中。将这种嵌合克隆 pMLV(src) 转染到组成型产生 M-MuLV gag 和 pol 蛋白的 NIH 3T3 细胞中，导致转化细胞灶的形成。可从转化细胞中回收具有感染性和转化能力的病毒。这种病毒被命名为 M-MuLV(src)。M-MuLV(src) 转化的细胞含有两种新的蛋白质，分子量分别为 78 和 90 千道尔顿。78 千道尔顿的蛋白质 p78gag-src 同时含有 gag 和 src 决定簇，在免疫激酶试验中表现出激酶活性，可能是 Pr65gag 和 src 的融合蛋白。90 千道尔顿的蛋白质，其大小适合作为与 src 融合的 gPr80gag，含有 gag 决定簇以及禽肉瘤病毒 pp60src 羧基末端典型的 V8 蛋白酶裂解片段。然而，它不能被抗 v-src 血清免疫沉淀。M-MuLV(src) 转化的细胞中蛋白质的细胞内磷酸酪氨酸水平升高，尽管与用野生型 v-src 转化的细胞相比，升高程度处于中等水平。将 M-MuLV(src) 的 M-MuLV 和嗜异性鼠白血病病毒假型接种到新生 NIH 瑞士小鼠中。接种后 3 至 6 周，接种小鼠在接种部位出现实体瘤，大多数动物在 14 周内死亡。组织病理学分析表明，实体瘤是间充质来源的纤维肉瘤，具有侵袭性和转移性。
