Expression of quasi-equivalence and capsid dimorphism in the Hepadnaviridae

Abstract

Author summary Hepatitis B virus has infected approximately one third of the human population and causes almost 1 million deaths from liver disease annually. The capsid is a defining feature of a virus, distinct from host components, and therefore a target for intervention. Unusually for a virus, Hepatitis B assembles two capsids, with different geometries, from the same dimeric protein. Geometric principles dictate that the subunits in this system occupy seven different environments. From comparing the two capsids by cryo-electron microscopy at high resolution under the exact same conditions we find that the polypeptide chains adopt seven different conformations. We use these structures to calculate potential energies (analogous to elastic deformation or strain) for the individual chains, dimers, and several higher-order groupings discernible in the two lattices. We also calculate the binding energies between chains. We find that some groupings have substantially lower energy and are therefore potentially more stable, allowing us to predict likely intermediates on the two assembly pathways. We also observe such intermediates by electron microscopy of in vitro capsid assembly reactions. This is the first structural characterization of the early assembly intermediates of this important human pathogen. Hepatitis B virus (HBV) is a leading cause of liver disease. The capsid is an essential component of the virion and it is therefore of interest how it assembles and disassembles. The capsid protein is unusual both for its rare fold and that it polymerizes according to two different icosahedral symmetries, causing the polypeptide chain to exist in seven quasi-equivalent environments: A, B, and C in AB and CC dimers in T = 3 capsids, and A, B, C, and D in AB and CD dimers in T = 4 capsids. We have compared the two capsids by cryo-EM at 3.5 angstrom resolution. To ensure a valid comparison, the two capsids were prepared and imaged under identical conditions. We find that the chains have different conformations and potential energies, with the T = 3 C chain having the lowest. Three of the four quasi-equivalent dimers are asymmetric with respect to conformation and potential energy; however, the T = 3 CC dimer is symmetrical and has the lowest potential energy although its intra-dimer interface has the least free energy of formation. Of all the inter-dimer interfaces, the CB interface has the least area and free energy, in both capsids. From the calculated energies of higher-order groupings of dimers discernible in the lattices we predict early assembly intermediates, and indeed we observe such structures by negative stain EM of in vitro assembly reactions. By sequence analysis and computational alanine scanning we identify key residues and motifs involved in capsid assembly. Our results explain several previously reported observations on capsid assembly, disassembly, and dimorphism.