This project was under the spotlight of ESRF.
In collaboration with Adam Zlotnick's lab, we are investigating Hepatitis B virus (HBV) capsid assembly reactions, using modern synchrotron time-resolved solution X-ray scatteirng.
Hepatitis B Virus (HBV), is a well-characterized endemic pathogen and a promising target for antiviral drug development. In-vivo, 90% of the HBV particles are empty. HBV is an experimentally tractable system and our analyses are equally applicable to describing bottom up assembly and to understanding a medically relevant endemic and deadly pathogen. Antiviral agents (including molecules that are now in clinical trials) act by manipulating the mechanism of capsid assembly reaction. The assembly reaction of HBV capsid can be recapitulated in vitro.
Our biophysical and computational analyses are determining the paths and conditions that allow virus capsid subunits to successfully work together and form stable 120-component complex capsids with icosahedral symmetry; the analyses are also showing where assembly is likely to go irretrievably off-path, which has implications for development of antivirals. The icosahedral geometry can lead to an immense (about 1030) set of possible intermediates. This set is impossible to explore and realize both physically and computationally. We show that the selection of a very limited number of assembly reaction products, both physically and computationally, is made by the Boltzmann weighting indicated by the association free energy between subunits. These observations are a realization of a solution to the Levinthal paradox.
Similarly, to successfully form capsids, the association energy should be within a narrow window of allowed values. Below a critical value, no assembly takes place. Above a certain threshold, kinetically trapped states rapidly form, deplete the available free subunits, and limit the possibility to attain equilibrium.
By plotting the grand canonical free energy landscape for the onset of the reaction, calibrated by our experiments and analyses, we found that in reaction conditions that successfully led to capsids, the most compact and stable intermediates are likely to form. Outside the successful assembly conditions, the grand canonical free energy landscapes clearly shows why assembly does not start or lead to kinetically trapped states.
Our experiments include state-of-the-art synchrotron solution X-ray scattering under a wide range of reaction conditions. The theoretical analyses include, our home developed solution scattering data analysis software, D+, umbrella sampling Monte Carlo simulations, based on graph representations of assembly intermediates, maximum entropy optimization analysis, and thermodynamic analysis of macromolecular assemblies.
Because new antivirals directed against HBV capsid protein strengthen association between subunits, we can provide new insight into their mechanisms and means for simplifying assembly models and identifying new targets for antiviral intervention (read more in our ACS Nano 2019 paper).
To explore the pathways that virus capsid subunits follow to form stable 120-subunit HBV empty capsids, we have used state-of-the-art Time-Resolved Small Angle X-ray Scattering and data analysis methods, developed in our lab in the past decade. From rigorous analyses of our data, and examination of the free energy landscape, we find that an increase of 1 kBT in the interaction strength between subunits can dramatically affect the reaction rates, accumulation of intermediates, and assembly mechanism. Remarkably, under the conditions that we tested, the path of assembly was determined in less than a second.
Under mild assembly conditions, after a 10 sec lag phase, the reaction appeared two-state from dimer to 120-dimer capsid. The energy landscape directs the reaction to follow a narrow minimum free energy path through the most compact and stable intermediates. There is a relatively high and broad energy barrier, facilitating multiple reversible steps, following which the energy decreases towards the full capsid with no local minima, consistent with a heterogeneous nucleation mechanism. At aggressive assembly conditions (in which the interaction energy between subunits is stronger by only 1 kBT), a diverse array of small to mid-size intermediates accumulated within the first 250 msec. Capsids then assembled by either slow elongation of the mid-size intermediates or by establishing new 'capsid assembly lines'. (read more in our JACS 2020 paper).
