Department of Immunology and Microbial Sciences, The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, CA 92037, USA.
J Virol. 2011 Mar;85(6):2631-41. doi: 10.1128/JVI.02321-10. Epub 2011 Jan 5.
The identification of the adenovirus (AdV) protein that mediates endosome penetration during infection has remained elusive. Several lines of evidence from previous studies suggest that the membrane lytic factor of AdV is the internal capsid protein VI. While these earlier results imply a role for protein VI in endosome disruption, direct evidence during cell entry has not been demonstrated. To acquire more definitive proof, we engineered random mutations in a critical N-terminal amphipathic α-helix of VI in an attempt to generate AdV mutants that lack efficient membrane penetration and infection. Random mutagenesis within the context of the AdV genome was achieved via the development of a novel technique that incorporates both error-prone PCR and recombineering. Using this system, we identified a single mutation, L40Q, that significantly reduced infectivity and selectively impaired endosome penetration. Furthermore, we obtained biophysical data showing that the lack of efficient endosomalysis is associated with reduced insertion of the L40Q mutation in protein VI (VI-L40Q) into membranes. Our studies indicate that protein VI is the critical membrane lytic factor of AdV during cellular entry and reveal the biochemical basis for its membrane interactions.
在感染过程中介导内体穿透的腺病毒 (AdV) 蛋白的鉴定一直难以捉摸。来自先前研究的几条证据表明，AdV 的膜裂解因子是内部衣壳蛋白 VI。虽然这些早期结果暗示蛋白 VI 在破坏内体方面发挥作用，但在细胞进入过程中尚未证明直接证据。为了获得更明确的证据，我们在 VI 的关键 N 端两性 α 螺旋中设计了随机突变，试图生成缺乏有效膜穿透和感染能力的 AdV 突变体。通过开发一种新的技术，在 AdV 基因组的背景下实现了随机诱变，该技术结合易错 PCR 和重组酶工程。使用该系统，我们鉴定出一个单一突变 L40Q，它显著降低了感染力，并选择性地损害了内体穿透。此外，我们获得了生物物理数据，表明缺乏有效的内体溶解与 VI 中的 L40Q 突变（VI-L40Q）插入到膜中的效率降低有关。我们的研究表明，蛋白 VI 是 AdV 在细胞进入过程中的关键膜裂解因子，并揭示了其膜相互作用的生化基础。
