Nagel H, Maag S, Tassis A, Nestlé F O, Greber U F, Hemmi S
Institute of Molecular Biology, University of Zürich, Zürich, Switzerland.
Gene Ther. 2003 Sep;10(19):1643-53. doi: 10.1038/sj.gt.3302058.
Epithelial and endothelial cells expressing the primary Coxsackie virus B adenovirus (Ad) receptor (CAR) and integrin coreceptors are natural targets of human Ad infections. The fiber knob of species A, C, D, E and F Ad serotypes binds CAR by mimicking the CAR-homodimer interface, and the penton base containing arginine-glycine-aspartate (RGD) motifs binds with low affinity to alphav integrins inducing cell activation. Here, we generated seven different genetically modified Ad vectors with RGD sequences inserted into the HI loop of fiber knob. All mutants bound and infected CAR and alphav integrin-positive epithelial cells with equal efficiencies. However, the Ads containing two additional cysteines, both N and C terminals of the RGD sequence (RGD-4C), were uniquely capable of transducing CAR-less hematopoietic and nonhematopoietic human tumor cell lines and primary melanoma cells. Both binding and transduction of RGD-4C Ad were blocked by soluble RGD peptides. Flow cytometry of cell surface integrins and virus binding to CAR-less cells in the presence of function-blocking anti-integrin antibodies indicated that the alphavbeta5 integrin, but not alphavbeta3, alphaIIbbeta3 or beta1,alpha5 or alpha6-containing integrins served as a functional transduction receptor of the RGD-4C Ads. However, in cells with low levels of alphavbeta5 integrin, the function-blocking anti-alphavbeta5 antibodies were not effective, unlike soluble RGD peptides. Collectively, our data demonstrate that the alphavbeta5 integrin is a functional transduction receptor of RGD-4C Ads in the absence of CAR, and that additional RGD receptors are targets of these viruses. The RGD-4C vectors further extend the tropism of Ads towards potential human therapies.
表达柯萨奇病毒B型腺病毒（Ad）主要受体（CAR）和整合素共受体的上皮细胞和内皮细胞是人类Ad感染的天然靶标。A、C、D、E和F型Ad血清型的纤维结通过模拟CAR-同型二聚体界面与CAR结合，而含有精氨酸-甘氨酸-天冬氨酸（RGD）基序的五聚体基底以低亲和力与αv整合素结合，诱导细胞活化。在此，我们构建了七种不同的基因修饰Ad载体，将RGD序列插入纤维结的HI环中。所有突变体以相同效率结合并感染CAR和αv整合素阳性上皮细胞。然而，含有两个额外半胱氨酸的Ad，即RGD序列的N端和C端（RGD-4C），能够独特地转导缺乏CAR的造血和非造血人类肿瘤细胞系以及原发性黑色素瘤细胞。RGD-4C Ad的结合和转导均被可溶性RGD肽阻断。在存在功能阻断性抗整合素抗体的情况下，对细胞表面整合素进行流式细胞术分析以及病毒与缺乏CAR的细胞的结合表明，αvβ5整合素而非αvβ3、αIIbβ3或含β1、α5或α6的整合素作为RGD-4C Ad的功能性转导受体。然而，在αvβ5整合素水平较低的细胞中，与可溶性RGD肽不同，功能阻断性抗αvβ5抗体无效。总体而言，我们的数据表明，在缺乏CAR的情况下，αvβ5整合素是RGD-4C Ad的功能性转导受体，并且其他RGD受体是这些病毒的靶标。RGD-4C载体进一步扩展了Ad对潜在人类治疗的趋向性。
