Monitoring Assembly of Virus Capsids with Nanofluidic Devices

Abstract

Virus assembly is a coordinated process in which typically hundreds of subunits react to form complex, symmetric particles. We use resistive-pulse sensing to characterize the assembly of Hepatitis B Virus core protein dimers into T = 3 and T = 4 icosahedral capsids. This technique counts and sizes intermediates and capsids in real time, with single particle sensitivity, and at biologically relevant concentrations. Other methods are not able to produce comparable real-time, single-particle observations of assembly reactions below, near, and above the pseudocritical dimer concentration, at which the dimer and capsid concentrations are approximately equal. Assembly reactions across a range of dimer concentrations reveal three distinct patterns. At dimer concentrations as low as 50 nM, well below the pseudo-critical dimer concentration of 0.5 µM, we observe a switch in the ratio of T = 3 to T = 4 capsids, which increases with decreasing dimer concentration. Far above the pseudo-critical dimer concentration, kinetically trapped, incomplete T = 4 particles assemble rapidly, then slowly anneal into T = 4 capsids. At all dimer concentrations tested, T = 3 capsids form more rapidly than T = 4 capsids, suggesting distinct pathways for the two forms.

Single Particle Observation of SV40 VP1 Polyanion-Induced Assembly Shows That Substrate Size and Structure Modulate Capsid Geometry

“…Nanofluidic devices were fabricated as previously described 36 . The microchannels were formed in D263 glass substrates by standard UV photolithography and wet chemical etching.…”

AC Electroosmotic Pumping in Nanofluidic Funnels

“…27–28 With a 30-kV ion beam at 50 pA, the nanofunnel and two nanochannels were milled to depths of 50, 110, 260, and 520 nm with doses of 0.25, 0.5, 1, and 2 nC/µm 2 , respectively. For the nanofunnels, these depths yielded aspect ratios (funnel tip width to funnel depth) of 1.0, 0.5, 0.2, and 0.1, respectively.…”

“…We fabricated in-plane nanofunnels in glass substrates with a focused ion beam (FIB) instrument. 27–28 The all-glass device enabled us to work at higher field strengths compared to our prior work on ion transport in nanofunnels. 20,29 The nanofunnels were milled with a 5° taper along the axis of fluid transport 20 and with aspect ratios (funnel tip width to funnel depth) from 0.1 to 1.0.…”

Nanofluidic Devices with 8 Pores in Series for Real-Time, Resistive-Pulse Analysis of Hepatitis B Virus Capsid Assembly

“…13–14 Experimental observations indicate that in some cases assembly involves specific intermediates (incomplete capsids), suggesting a limited number of assembly paths. 15–19 Thus, a key factor for the development of thermodynamic and kinetic models for virus capsid assembly is the better characterization of the assembly intermediate species.…”

“…22 The HBV capsid assembly reaction can be triggered in vitro by the dilution of the Cp149 dimer (a truncated version of the 183-residue core protein that is missing the RNA-binding domain) into NaCl solutions at room temperature without the presence of nucleic acid or polyanion template. 23 Truncation of the core protein, 23 concentrations of core protein dimer and salt, 15 and degree of dimer oxidization 22 are factors that affect the ratio of T = 3 and T = 4 capsids formed during capsid assembly. The assembly reaction is characterized by a pseudo-critical concentration, where the concentration of dimers in the capsid equals the concentration of the free dimer in the reaction solution.…”