Molecular and Cellular Biochemistry, Indiana University, Bloomington, Indiana 47405, United States.
Department of Chemistry, Indiana University, Bloomington, Indiana 47405, United States.
J Am Chem Soc. 2023 Feb 1;145(4):2322-2331. doi: 10.1021/jacs.2c10937. Epub 2023 Jan 18.
For a virus-like particle (VLP) to serve as a delivery platform, the VLP must be able to release its cargo in response to a trigger. Here, we use a chemical biology approach to destabilize a self-assembling capsid for a subsequent triggered disassembly. We redesigned the dimeric hepatitis B virus (HBV) capsid protein (Cp) with two differentially addressable cysteines, C150 for reversibly crosslinking the capsid and C124 to react with a destabilizing moiety. The resulting construct, Cp150-V124C, assembles into icosahedral, 120-dimer VLPs that spontaneously crosslink via the C-terminal C150, leaving C124 buried at a dimer-dimer interface. The VLP is driven into a metastable state when C124 is reacted with the bulky fluorophore, maleimidyl BoDIPY-FL. The resulting VLP is stable until exposed to modest, physiologically relevant concentrations of reducing agent. We observe dissociation with FRET relaxation of polarization, size exclusion chromatography, and resistive-pulse sensing. Dissociation is slow, minutes to hours, with a characteristic lag phase. Mathematical modeling based on the presence of a nucleation step predicts disassembly dynamics that are consistent with experimental observations. VLPs transfected into hepatoma cells show similar dissociation behavior. These results suggest a generalizable strategy for designing a VLP that can release its contents in an environmentally responsive reaction.
为了使病毒样颗粒（VLP）能够作为递送平台，VLP 必须能够响应触发而释放其货物。在这里，我们使用化学生物学方法来使自组装衣壳不稳定，以进行随后的触发式解体。我们重新设计了具有两个可区分的半胱氨酸的二聚体乙型肝炎病毒（HBV）衣壳蛋白（Cp），C150 用于可逆交联衣壳，C124 用于与不稳定部分反应。所得的构建体 Cp150-V124C 组装成二十面体、120 二聚体 VLPs，通过 C 末端 C150 自发交联，使 C124 埋藏在二聚体-二聚体界面上。当 C124 与大体积荧光团马来酰亚胺 BoDIPY-FL 反应时，VLP 被驱动进入亚稳态。所得的 VLP 在暴露于适度的、生理相关浓度的还原剂之前是稳定的。我们观察到荧光各向异性松弛、尺寸排阻色谱和电阻脉冲感应的解离。解离缓慢，需要几分钟到几个小时，具有特征的滞后期。基于成核步骤存在的数学模型预测了与实验观察一致的解组装动力学。转染到肝癌细胞中的 VLPs 显示出相似的解离行为。这些结果表明，设计能够在环境响应反应中释放其内容物的 VLP 的策略具有通用性。
