# Single-Particle Analysis of Virus Capsid Assembly and Disassembly by Resistive-Pulse Sensing

> **NIH NIH R01** · TRUSTEES OF INDIANA UNIVERSITY · 2020 · $300,254

## Abstract

Project Summary
 A typical virus capsid consists of hundreds of copies of capsid protein that act as the protective package
of the viral genome. Therefore, the typical capsid assembly reaction will have hundreds of steps, each one of
which can go wrong. Yet, even in vitro, self-assembly of virus capsids can occur spontaneously and with high
fidelity. This reaction is of fundamental interest to virologists, a focus of antiviral development, and a general
model of self-assembly with implications for harnessing viruses for nano- and biotechnology.
 Understanding the mechanism of virus assembly requires not only knowledge of precursors and final
products, but also access to intermediates. Where many rare intermediates are involved, ensemble methods
obscure them so that virus assembly resembles a two-state reaction, i.e., only subunits and capsids are
observed. Computational models of assembly suggest that the observed kinetics reflect establishment of early
intermediates needed to support capsid formation. The nucleation step and these early intermediates are
believed to play a role in recruiting viral components in vivo. In our previous work, we established hepatitis B
virus (HBV) assembly as a well-defined and robust experimental system for interrogating assembly reactions.
HBV capsids, composed of 120 homodimers, self-assemble in response to buffer conditions. Surprisingly, we
found metastable intermediates in assembly and disassembly. These results are timely as HBV capsid
assembly has become an important target for development of antiviral assembly effectors which over-
stimulate nucleation, distorting the distribution of intermediates and often their structure.
 Resistive-pulse sensing on in-plane nanofluidic devices is a unique platform and permits a label-free,
single-particle approach to monitor assembly in real time at biologically relevant concentrations (nM to µM).
Our resistive-pulse measurements have provided highly complementary data to other state-of-the-art
techniques, e.g., time-resolved small angle x-ray scattering, light scattering, charge detection mass
spectrometry, and transmission electron microscopy. All of these approaches require much higher protein
concentrations than resistive-pulse sensing and, thus, obscure many features of these complex reactions.
We have developed needed fabrication methods, characterized individual HBV capsids, and monitored their
assembly below, near, and above the pseudo-critical dimer concentration. Because of our ability to probe
single particles in real time and over a range of assembly conditions, we are now poised to address a number
of questions, previously thought unanswerable. The specific aims for this application are to: (1) integrate on-
device mixing and multiplexed detection to probe early time points of assembly; (2) compare assembly of
virus capsids with and without assembly effectors; (3) evaluate capsid assembly and disassembly in the
presence of chaotropes; (4) monitor the evoluti...

## Key facts

- **NIH application ID:** 9959448
- **Project number:** 5R01GM129354-03
- **Recipient organization:** TRUSTEES OF INDIANA UNIVERSITY
- **Principal Investigator:** Stephen C Jacobson
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $300,254
- **Award type:** 5
- **Project period:** 2018-08-01 → 2022-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9959448

## Citation

> US National Institutes of Health, RePORTER application 9959448, Single-Particle Analysis of Virus Capsid Assembly and Disassembly by Resistive-Pulse Sensing (5R01GM129354-03). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/9959448. Licensed CC0.

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