Valsesia-Wittmann S, Drynda A, Deléage G, Aumailley M, Heard J M, Danos O, Verdier G, Cosset F L
CGMC, CNRS UMR106, Université Claude Bernard Lyon-I, Villeurbanne.
J Virol. 1994 Jul;68(7):4609-19. doi: 10.1128/JVI.68.7.4609-4619.1994.
On the basis of theoretical structural and comparative studies of various avian leukosis virus SU (surface) envelope proteins, we have identified four small regions (I, II, III, and IV) in their receptor-binding domains that could potentially be involved in binding to receptors. From the envelope gene of an avian leukosis virus of subgroup A, we have constructed a set of SU mutants in which these regions were replaced by the coding sequence of FLA16, a 16-amino-acid RGD-containing peptide known to be the target for several cellular integrin receptors. Helper-free retroviral particles carrying a neo-lacZ retroviral vector were produced with the mutant envelopes. SU mutants in which regions III and IV were substituted yielded normal levels of envelope precursors but were not detectably processed or incorporated in viral particles. In contrast, substitutions in regions I and II did not affect the processing and the viral incorporation of SU mutants. When FLA16 was inserted in region II, it could be detected with antibodies against FLA16 synthetic peptide, but only when viral particles were deglycosylated. Viral particles with envelopes mutated in region I or II were able to infect avian cells through the subgroup A receptor at levels similar to those of the wild type. When viruses with envelopes containing FLA16 peptide in region II were applied to plastic dishes, they were found to promote binding of mammalian cells resistant to infection by subgroup A avian leukosis viruses but expressing the integrins recognized by FLA16. Deglycosylated helper-free viruses obtained by mild treatment with N-glycosidase F have been used to infect these mammalian cells, and infections have been monitored by neomycin selection. No neomycin-resistant clones could be obtained after infection by viruses with wild-type envelopes. Conversely, colonies were obtained after infection by viruses with envelopes bearing FLA16 in region II, and the genome of the retroviral vector was found correctly integrated in cell DNA of these colonies. By using a blocking peptide containing the minimal adhesive RGD sequence contained in FLA16, we have shown that preincubation of target cells could specifically inhibit infection by viruses with FLA16.
基于对各种禽白血病病毒SU（表面）包膜蛋白的理论结构和比较研究，我们在其受体结合域中确定了四个小区域（I、II、III和IV），这些区域可能参与与受体的结合。从A亚群禽白血病病毒的包膜基因中，我们构建了一组SU突变体，其中这些区域被FLA16的编码序列取代，FLA16是一种含16个氨基酸的含RGD肽，已知是几种细胞整合素受体的靶点。携带neo-lacZ逆转录病毒载体的无辅助逆转录病毒颗粒用突变包膜产生。区域III和IV被取代的SU突变体产生正常水平的包膜前体，但未检测到被加工或整合到病毒颗粒中。相反，区域I和II的取代不影响SU突变体的加工和病毒整合。当FLA16插入区域II时，可用抗FLA16合成肽的抗体检测到，但仅在病毒颗粒去糖基化时。区域I或II中包膜发生突变的病毒颗粒能够通过A亚群受体感染禽细胞，其水平与野生型相似。当将区域II中含有FLA16肽的包膜病毒应用于塑料培养皿时，发现它们能促进对A亚群禽白血病病毒感染有抗性但表达FLA16识别的整合素的哺乳动物细胞的结合。用N-糖苷酶F轻度处理获得的去糖基化无辅助病毒已用于感染这些哺乳动物细胞，并通过新霉素选择监测感染情况。用野生型包膜病毒感染后未获得新霉素抗性克隆。相反，用区域II中带有FLA16的包膜病毒感染后获得了菌落，并且发现逆转录病毒载体的基因组正确整合到这些菌落的细胞DNA中。通过使用含有FLA16中最小粘附RGD序列的封闭肽，我们表明靶细胞的预孵育可以特异性抑制含FLA16病毒的感染。
