PATHWAYS OF HEPATITIS B VIRUS CAPSID ASSEMBLY The assembly pathways of Hepatitis B virus capsid were resolved using time-resolved small-angle X-ray scattering (SAXS), simulations, thermodynamic analysis and maximum information entropy optimisation. The earliest steps of the reaction, controlled by the association free energy between capsid proteins, dictate the assembly pathway.

COMPLEX SYSTEMS AND BIOMEDICAL SCIENCES

60 ESRF

Viruses are the most abundant biological entities on our planet. Their structure, composed of only a few components, presents an ideal example of a biologically functional, self- assembled structure. Viruses encapsulate and protect their genetic materials in a protein shell called a capsid. Minimising the amount of viral genome coding structural proteins, capsids are comprised of many copies of a small number of proteins, often only one, usually in a spherical or helical arrangement. This minimalistic design supports an extremely efficient assembly process, resulting in a monodisperse, stable structure.

Half of the known virus families have icosahedral capsids. While the structures of many capsids are known, their assembly and disassembly processes are still poorly understood. Capsids can have a hundred or even thousands of subunits, consequently there are a huge number of possible intermediates, and many more potential assembly pathways. Yet, assembly is rapid and with high fidelity. Recently, the assembly pathways of the Hepatitis B Virus (HBV) capsid were observed. HBV causes about 270 million cases of chronic infection, leading to about 880,000 deaths each year from liver disease and cancer. HBV has an icosahedral capsid composed of homodimeric core protein (Cp). In-vivo assembly involves spontaneous

nucleation to form empty particles, comprising 90% of the particles present during infection. Recombinant capsid protein assembly domain, Cp149 (the first 149 residues of Cp), assembles in vitro into empty capsids, identical to the capsids isolated from virus-expressing cells.

HBV capsids are composed of 90 (capsid symmetry T = 3) or 120 (T = 4) Cp149 dimers. There are an estimated 1030 intermediates on the assembly path from dimer to complete capsid. Hence, resolving the HBV capsid assembly mechanism is an ill-posed problem. To identify assembly pathways, graph theory was used to represent the structure of HBV intermediates. Umbrella sampling of Monte Carlo simulations was then performed to create a comprehensive library of unique assembly intermediates [1]. Using the software D+ (https://scholars.huji. ac.il/uriraviv/book/d-0) [2], the atomic model of each member in the library was generated and its solution SAXS curve was computed. Intermediates were selected from this library based on a grand canonical free energy landscape of HBV capsid that had been calibrated with experimental SAXS data HBV capsid assembly reactions at equilibrium [1,3].

Using the time-resolved SAXS setup of ID02, the real-time assembly of HBV capsids was tracked (Figure 44). To rigorously analyse the data with minimal bias, maximum informational entropy

optimisation analysis was applied with prior distributions, derived from the calibrated grand canonical free energy landscape at the onset of the assembly reactions (Figure 45) [3]. Equilibrium- based prior knowledge filtered out the most stable intermediates that are likely to make a significant contribution to the assembly reaction.

Fig. 44: Kinetics of Cp149 dimer assembly. a) Mild assembly conditions (163 mM ammonium acetate). b) Aggressive conditions (513 mM). (Left panel) TR-SAXS data (grey) at selected early times, fitted to a library of intermediates by maximum informational entropy optimisation (black curves). (Right panel) Mass fraction as a function of intermediate size (s - number of dimers), and time. Illustrations show the major components along the assembly path.