Targeting retroviral vectors to CD34-expressing cells: Binding to CD34 does not catalyze virus-cell fusion
Targeting retroviral vectors to CD34-expressing cells: Binding to CD34 does not catalyze virus-cell fusion
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DOI：
10.1089/10430349950018625
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发表时间：
1999-03-01
影响因子：
4.2
通讯作者：
Anderson, WF
中科院分区：
文献类型：
--
作者：
Benedict, CA;Tun, RYM;Anderson, WF
我们尝试设计基于鼠白血病病毒 (MuLV) 的逆转录病毒载体，以特异性转导表达人 CD34（一种存在于未分化造血干细胞表面的抗原）的细胞。构建了许多嵌合共亲性 MuLV 包膜 (Env) 蛋白，其中含有抗 CD34 单链抗体可变片段 (scFv)。 scFv-Env 蛋白是通过用 scFv 替换 Env 的受体结合结构域或通过将 scFv 插入 Env 蛋白的 N 末端来生成的。只有在氨基酸 6 和 7 之间插入有 scFv 的嵌合 Env 蛋白才会被整合到病毒颗粒中，并且天然 MuLV Env 的共表达并不能挽救整合缺陷的蛋白质。此外，掺入效率随所使用的特异性抗CD34 scFv而变化，含有scFv-Env蛋白的逆转录病毒载体与CD34(+)细胞结合并转导表达人CD34的NIH 3T3细胞(3T3-CD34细胞)，效率约为亲代NIH 3T3细胞效率的两倍。然而，突变 D84K 的引入阻止了与亲嗜性 MuLV 受体 eat-1 的结合，从而阻止了 NIH 3T3 和 3T3-CD34 细胞的转导。互补细胞-细胞融合测定[Zhao et al, (1997)。 J.维罗尔。 [71, 6967-6972] 在 3T3-CD34 细胞中的研究表明，虽然 scFv-Env 蛋白在与mea-1结合时可以提供结合后进入功能，但在与 CD34 结合时却无法做到这一点。总而言之，这些数据表明，尽管与 CD34 的相互作用有效地增加了 3T3-CD34 细胞上病毒的浓度，但只有通过与 Meat-1 的相互作用才能进入； CD34 单独不能触发导致病毒进入的适当的结合后变化。
We have attempted to engineer murine leukemia virus (MuLV)-based retroviral vectors to specifically transduce cells expressing human CD34, an antigen present on the surface of undifferentiated hematopoietic stem cells. A number of chimeric ecotropic MuLV envelope (Env) proteins were constructed that contained anti-CD34 single-chain antibody variable fragments (scFvs). The scFv-Env proteins were generated either by replacing the receptor-binding domain of Env with the scFv or by inserting the scFv into the N terminus of the Env protein. Only chimeric Env proteins with scFv insertions between amino acids 6 and 7 were incorporated into viral particles, and coexpression of native MuLV Env did not rescue incorporation-defective proteins. In addition, the efficiency of incorporation varied with the specific anti-CD34 scFv that was used, Retroviral vectors containing the scFv-Env proteins bound to CD34(+) cells and transduced NIH 3T3 cells expressing human CD34 (3T3-CD34 cells) at approximately twice the efficiency of the parental NIH 3T3 cells. However, the introduction of the mutation D84K, which prevents binding to the ecotropic MuLV receptor meat-1, prevented transduction of both NIH 3T3 and 3T3-CD34 cells. Complementation cell-cell fusion assays [Zhao et al, (1997). J. Virol. 71, 6967-6972] in 3T3-CD34 cells revealed that although the scFv-Env proteins could contribute postbinding entry functions when bound to meat-1, they were unable to do so when bound to CD34. Taken together, these data suggest that although the interaction with CD34 effectively increased the concentration of virus on 3T3-CD34 cells, entry could occur only through an interaction with meat-1; CD34 alone was not capable of triggering the appropriate postbinding changes that lead to viral entry.