Department of Biological Sciences.
J Virol. 2013 Oct;87(20):11231-43. doi: 10.1128/JVI.01054-13. Epub 2013 Aug 7.
Rational design and directed evolution are powerful tools to generate and improve protein function; however, their uses are mostly limited to enzyme and antibody engineering. Here we describe a directed-evolution strategy, named the tandem selection and enrichment system (TSES), and its use in generating virus with exclusive specificity for a particular cellular receptor. In TSES, evolving viruses are sequentially and iteratively transferred between two different host cells, one for selection of receptor specificity and the other for enrichment of the fittest virus. By combining rational design and TSES, we generated human epidermal growth factor receptor (EGFR)-specific virus 1 (ESV1). ESV1 has the backbone of Sindbis virus (SINV) and displays an EGF domain engrafted onto structural protein E2 after residue Pro192, together with eight amino acid changes stabilizing the E2-EGF chimera. ESV1 uses EGFR to initiate infection and has lost the capacity to interact with all known SINV receptors. A 12.2-Å cryoelectron microscopic (cryoEM) reconstruction of ESV1 reveals that the E2-EGF fusion adopts a fixed conformation, with EGF sitting at the top of the E2 spike; The EGFR binding interface faces outward, and the EGF domain completely masks SINV receptor binding. The cryoEM structure of ESV1 explains the desirable properties of ESV1 and provides insights for its further modification. TSES expands the scope of directed evolution and can be easily extended to other targeting molecules and viral systems.
理性设计和定向进化是产生和改善蛋白质功能的有力工具；然而，它们的用途大多仅限于酶和抗体工程。在这里，我们描述了一种定向进化策略，称为串联选择和富集系统（TSES），并将其用于产生对特定细胞受体具有独特特异性的病毒。在 TSES 中，进化病毒在两种不同的宿主细胞之间顺序和迭代地转移，一种用于选择受体特异性，另一种用于富集最适合的病毒。通过将理性设计与 TSES 相结合，我们生成了具有表皮生长因子受体（EGFR）特异性的病毒 1（ESV1）。ESV1 的骨架为辛德毕斯病毒（SINV），并在结构蛋白 E2 的残基 Pro192 之后展示了一个 EGF 结构域，同时还有八个稳定 E2-EGF 嵌合体的氨基酸变化。ESV1 使用 EGFR 启动感染，并失去了与所有已知 SINV 受体相互作用的能力。ESV1 的 12.2-Å 冷冻电镜（cryoEM）重建揭示了 E2-EGF 融合采用固定构象，EGF 位于 E2 刺突的顶部；EGFR 结合界面面向外部，EGF 结构域完全掩盖了 SINV 受体结合。ESV1 的 cryoEM 结构解释了 ESV1 的理想特性，并为其进一步修饰提供了思路。TSES 扩展了定向进化的范围，可以很容易地扩展到其他靶向分子和病毒系统。
