Characterization of Virus Capsids and Their Assembly Intermediates by Multicycle Resistive-Pulse Sensing with Four Pores in Series.
Characterization of Virus Capsids and Their Assembly Intermediates by Multicycle Resistive-Pulse Sensing with Four Pores in Series.
复制标题
通过四孔串联多周期电阻脉冲传感表征病毒衣壳及其组装中间体。
DOI：
10.1021/acs.analchem.8b00452
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发表时间：
2018
影响因子：
7.4
通讯作者：
Jacobson,StephenC
中科院分区：
文献类型：
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作者：
Zhou,Jinsheng;Kondylis,Panagiotis;Haywood,DanielG;Harms,ZacharyD;Lee,LyeSiang;Zlotnick,Adam;Jacobson,StephenC
病毒自组装是病毒生命周期中的关键步骤。了解病毒是如何组装和分解的，为开发抗病毒药物提供了必要的见解。很少有工具能提供足够的分辨率来研究大小相差几个二聚体的组装中间体。我们的目标是改进纳米流体设备上的阻性脉冲传感，以提供更好的颗粒大小和时间分辨率来研究组装过程中产生的中间体和衣壳。为了提高阻性脉冲技术的颗粒尺寸分辨率，我们测量了相同的单个病毒颗粒多达1000次，通过切换施加电势的极性，即病毒乒乓球，使它们在一系列纳米孔中来回循环。串联的多个气孔在每个周期中提供独特的多脉冲信号，从而改进了颗粒跟踪，从而识别单个颗粒，并减少了进行必要数量测量所需的周期数。对于T=3和T=4的乙肝病毒衣壳，我们发现颗粒大小分布的标准差随测量次数的平方根而减小，并接近用单一二聚体区分不同大小的颗粒。然后，我们研究了乙肝病毒衣壳的体外组装，并观察到在2天的退火期中，中间产物的集合向更大的尺寸移动。相反，组装反应在启动后1小时稀释到较低的二聚体浓度时，在2天孵育后持续存在的中间体较少，且具有较高的FT=4 TOT=3衣壳比率。这些反应表明，LabileT=4中间体的形成速度很快，根据条件的不同，中间体可能会以亚稳态物种的形式被捕获，或者被捕获以生成完整的衣壳。
Virus self-assembly is a critical step in the virus lifecycle. Understanding how viruses assemble and disassemble provides needed insight into developing antiviral pharmaceuticals. Few tools offer sufficient resolution to study assembly intermediates that differ in size by a few dimers. Our goal is to improve resistive-pulse sensing on nanofluidic devices to offer better particle-size and temporal resolution to study intermediates and capsids generated along the assembly pathway. To increase the particle-size resolution of the resistive-pulse technique, we measured the same, single virus particles up to a thousand times, cycling them back and forth across a series of nanopores by switching the polarity of the applied potential, i.e., virus ping-pong. Multiple pores in series provide a unique multipulse signature during each cycle that improves particle tracking and, therefore, identification of a single particle and reduces the number of cycles needed to make the requisite number of measurements. WithT= 3 andT= 4 hepatitis B virus (HBV) capsids, we showed the standard deviation of the particle-size distribution decreased with the square root of the number of measurements and approached discriminating particles differing in size by single dimers. We then studied in vitro assembly of HBV capsids and observed that the ensemble of intermediates shift to larger sizes over 2 days of annealing. On the contrary, assembly reactions diluted to lower dimer concentrations an hour after initiation had fewer intermediates that persisted after the 2 day incubation and had a higher ratio ofT= 4 toT= 3 capsids. These reactions indicate that labileT= 4 intermediates are formed rapidly, and dependent on conditions, intermediates may be trapped as metastable species or progress to yield complete capsids.