Serotype 5 Adenovirus fiber (F7F41S) chimeric vectors incur packaging deficiencies when targeting peptides are inserted into Ad41 short fiber.
Serotype 5 Adenovirus fiber (F7F41S) chimeric vectors incur packaging deficiencies when targeting peptides are inserted into Ad41 short fiber.
复制标题
当靶向肽插入 Ad41 短纤维时，血清型 5 腺病毒纤维 (F7F41S) 嵌合载体会出现包装缺陷。
DOI：
10.1016/j.virol.2009.08.041
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发表时间：
2009
期刊：
影响因子：
3.7
通讯作者：
Falck-Pedersen,Erik
中科院分区：
文献类型：
--
作者：
Schoggins,JohnW;Falck-Pedersen,Erik
腺病毒是一种成熟的病毒基因转移模型系统，在考虑用于细胞特异性靶向应用时存在两个主要障碍。首先是需要使载体从固有的宿主结合机制中去靶向，其次是需要建立一种生产性和稳定的方法来使载体重新靶向所需的细胞受体。在以前的研究中，我们已经产生了一种腺病毒载体平台，其缺乏来自纤维和五邻体衣壳蛋白的正常靶向属性。在本研究中，我们将我们的去靶向Ad 5载体（Ad5.F7F41S和Ad5.F7F41SΔRGD）作为新型重靶向病毒的平台。实验策略依赖于将小肽配体掺入Ad 41短纤维球结构域的几个位点（AB、CD、HI、G和Cterm）。Ad 41短纤维的再工程导致纤维7使用的旁路，或显性负包装/生产缺陷表型。在特定的生长条件下，我们可以弥补一些衣壳缺陷，并产生高滴度的病毒。然而，当通过蛋白质印迹分析检查时，所得病毒的衣壳内容物仍有缺陷。串联纤维F7 F41 S平台显示腺病毒包装对纤维41短结构修饰的非预期敏感性。这些研究表明纤维组装成完整的病毒体或纤维影响衣壳稳定性作为有效颗粒生产的瓶颈。我们还表明，CsCl显带后的成熟病毒粒子的病毒颗粒的衣壳蛋白含量可以显着变化。考虑到病毒进入靶细胞的复杂性，修饰的“成熟病毒体”可能不仅通过预期的修饰，而且由于二级结构包装冲突而在转导水平上受到损害。
Adenovirus is a well-established viral gene transfer model system that presents two major hurdles when being considered for cell-specific targeting applications. First is the need to detarget the vector from inherent host binding mechanisms, and second is the need to establish a productive and stable method to retarget the vector to a desired cell receptor. In previous studies we had generated an adenovirus vector platform that lacks the normal targeting attributes derived from the fiber and penton capsid proteins. In the current study we characterized our detargeted Ad5-based vectors (Ad5.F7F41S and Ad5.F7F41SΔRGD) as platforms for novel retargeted viruses. The experimental strategy relied on incorporating small peptide ligands into several sites of the Ad 41short fiber knob domain (AB, CD, HI, G and Cterm). Reengineering of Ad41 short fiber resulted either in a bypass to fiber 7 usage, or in a dominant negative packaging/production deficiency phenotype. Under specific growth conditions we could remedy some of the capsid deficiencies and generate high titer viruses. However when examined by Western blot analysis, the resulting viruses were still defective in capsid content. The tandem fiber F7F41S platform has revealed an unanticipated sensitivity of Adenovirus packaging to fiber 41short structural modifications. These studies indicate fiber assembly into an intact virion or fiber influenced capsid stability as a bottleneck to efficient particle production. We also demonstrate that virus particles characterized as mature virions following CsCl banding can vary significantly in capsid protein content. Considering the complexity of virus entry into a target cell, modified “mature virions” may be compromised at the level of transduction not only through the intended modification, but also by virtue of secondary structural packaging conflicts.