Engineering Metastability into a Virus-like Particle to Enable Triggered Dissociation
Engineering Metastability into a Virus-like Particle to Enable Triggered Dissociation
复制标题
DOI：
10.1021/jacs.2c10937
复制
发表时间：
2023-01-18
影响因子：
15
通讯作者：
Zlotnick，Adam
中科院分区：
文献类型：
--
作者：
Starr，Caleb A.;Nair，Smita;Zlotnick，Adam
对于作为递送平台的病毒样颗粒（VLP），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破坏自组装衣壳的稳定性，以便随后触发拆卸。我们重新设计了二聚体的B型肝炎病毒（HBV）衣壳蛋白（Cp）与两个差异寻址的半胱氨酸，C150可逆交联的衣壳和C124反应的不稳定部分。所得的构建体Cp 150-V124 C组装成二十面体的120-二聚体VLP，其通过C-末端C150自发交联，使C124埋在二聚体-二聚体界面处。当C124与大体积荧光团马来酰亚胺基BoDIPY-FL反应时，VLP被驱动进入亚稳态。产生的VLP在暴露于适度的生理相关浓度的还原剂之前是稳定的。我们通过FRET极化弛豫、尺寸排阻色谱和电阻脉冲传感观察到解离。解离是缓慢的，几分钟到几小时，具有特征性的滞后期。数学建模的基础上存在的成核步骤预测拆卸动力学与实验观察是一致的。转染到肝癌细胞中的VLP表现出类似的解离行为。这些结果表明，一个可推广的策略，设计一个VLP，可以释放其内容在一个环境响应反应。
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.