Disassembly of Single Virus Capsids Monitored in Real Time with Multicycle Resistive-Pulse Sensing

Abstract

Virus assembly and disassembly are critical steps in the virus lifecycle; however, virus disassembly is much less well understood than assembly. For hepatitis B virus (HBV) capsids, disassembly of the virus capsid in the presence of guanidine hydrochloride (GuHCl) exhibits strong hysteresis that requires additional chemical energy to initiate disassembly and disrupt the capsid structure. To study disassembly of HBV capsids, we mixed T = 4 HBV capsids with 1.0-3.0 M GuHCl, monitored the reaction over time by randomly selecting particles, and measured their size with resistive-pulse sensing. Particles were cycled forward and backward multiple times to increase the observation time and likelihood of observing a disassembly event. The four-pore device used for resistive-pulse sensing produces four current pulses for each particle during translocation that improves tracking and identification of single particles and increases the precision of particle-size measurements when pulses are averaged. We studied disassembly at GuHCl concentrations below and above denaturing conditions of the dimer, the fundamental unit of HBV capsid assembly. As expected, capsids showed little disassembly at low GuHCl concentrations (e.g., 1.0 M GuHCl), whereas at higher GuHCl concentrations (≥1.5 M), capsids exhibited disassembly, sometimes as a complex series of events. In all cases, disassembly was an accelerating process, where capsids catastrophically disassembled within a few 100 ms of reaching critical stability; disassembly rates reached tens of dimers per second just before capsids fell apart. Some disassembly events exhibited metastable intermediates that appeared to lose one or more trimers of dimers in a stepwise fashion.

Figure 1.

(a) Schematic of the nanofluidic device and scanning electron microscope image of the nanopore region (inset) for the single particle disassembly experiments. The four nanopores are 60 nm wide, 60 nm deep, and 300 nm long and are labeled p1, p2, p3, and p4. The nanochannels between adjacent nanopores are 300 nm wide, 130 nm deep, and 500 nm long. (b) Schematic of device operation. The potential is applied along the nanochannel and controlled by a LabVIEW program, and potential switching is triggered after four current pulses are detected that correspond to a particle translocating through pores p1, p2, p3, and p4. The particle is cycled forward and backward (i.e., ping pong) until a specified time of 60 s (8004 current pulses; 1000.5 cycles) is achieved, the particle disassembles, or the particle is lost.

Figure 2.

(a) Current trace of a T = 4 hepatitis B virus (HBV) capsid being cycled forward and backward during the final four cycles before disassembly in 2.0 M guanidine hydrochloride (GuHCl). The blue and red rectangles highlight current pulses of typical amplitude for a T = 4 HBV capsid in the forward and reverse directions, respectively, and the green rectangle highlights current pulses of lower amplitude as the particle disassembles. (b)-(d) Enlarged regions of panel (a) that show four current pulses associated with the T = 4 capsid translocating sequentially through (b) pores p1, p2, p3, and p4 in the forward direction and (d) pores p4, p3, p2, and p1 in the reverse direction. (d) Final four current pulses measured for the T = 4 capsid before complete disassembly. Scale bars for panels (b)-(d) are the same.

Figure 3.

Variation of particle size in dimers with trapping time of single T = 4 HBV capsids during disassembly experiments in 2.0 M GuHCl. (a) Trapping of six T = 4 capsids that do not change in size and do not disassemble before they are lost by the cycling program or the 60-s monitoring period ends (8004 current pulses; 1000.5 cycles). (b) Trapping of six T = 4 capsids that exhibit a rapid downturn in size and disassemble (marked with arrows). Full-size T = 4 HBV capsids are 120 dimers (dashed gray lines), and the detection limit is ~40 dimers.

Figure 4.

(a) Probabilities of all disassembly events and multi-level disassembly events of T = 4 HBV capsids in 1.0 to 3.0 M GuHCl. (b) Variation of fraction of counts with start time of particle trapping and time to disassemble for 2.0 and 2.5 M GuHCl. (c)-(f) Scatter plots of particle starting and ending sizes in dimers for HBV capsids trapped in 1.0, 1.5, 2.0, and 2.5 M GuHCl. Blue lines set the cutoff (90% of the T = 4 capsid size) below which particles are counted as disassembled and reported in panel (a). Vertical and horizontal red lines show the T = 4 capsid size (120 dimers), and the 45° red lines indicate where the starting and ending sizes are the same.

Figure 5.

(a) Variation of average disassembly rate with particle size for HBV capsids disassembled in 1.5, 2.0, 2.2, and 2.5 M GuHCl. The average disassembly rate was calculated from at least five particles having the same size (dimers). (b) Slope analysis data for all disassembly events at 2.0 M GuHCl and average disassembly rate for those data. Heat map of slope data ranges from most frequent (red) to least frequent (purple), and the yellow line with black outline designates the average disassembly rate.

Figure 6.

(a) Examples of two T = 4 HBV capsids exhibiting multi-level disassembly in 2.0 M GuHCl. Points are the average of 4 pulses (0.5 cycle), and lines are the average of 20 current pulses (2.5 cycles). The dashed red line corresponds to the size of a T = 4 capsid (120 dimers). (b) Variation of particle counts with particle size for the line data in panel (a) for particles 1 and 2 projected onto the y-axis. (c) Variation of particle counts with particle size for all metastable intermediates measured in 2.0 M GuHCl. The peaks in panels (b) and (c) denote common particle sizes observed during stepwise disassembly events.