Structural Evolution of the Dimer interface in Hepadnaviridae

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Hepadnaviridae are an ancient family of enveloped viruses that has emerged more than 400 million years ago. Hepadnaviridae infect most vertebrates including fish (meta- and parahepdanavirusses), amphibians, reptiles (herpetohepadnaviruses), birds (avihepadnaviruses) and mammals (orthohepadnaviruses). The most prominent family member is the human Hepatitis B virus with some 250 million chronic carriers world-wide. All virions of hepadnaviridae have capsids that are formed by multiple copies of a single type of core proteins (HBc). The capsids are surrounded by a pleiomorphic lipidic envelope that is densely packed with surface proteins (HBs). Within capsids, HBc forms dimers with protruding spikes at the inner-dimer interface. These spikes interact with HBs in the envelope. Phylogenetic analysis and current structural knowledge show that only the contact between HBc dimers in capsids is conserved while the inner-dimer interface and thus the protruding spike is not. This suggests that capsid formation is evolutionary maintained, while sites for envelopment and secretion adapt to or co-evolve with their respective hosts. While many structures of human HBc capsids are known, structures from capsids of other genera are rare and are unknown for para-, meta- and herpetohepadnaviruses. The comparison of capsids from human HBc (orthohepadnavirus) and duck HBc (avihepadnavirus) show that avihepadnaviruses have additional domains that fold at both sides of the spike. It is likely that these domains interact with the envelope. In contrast, the interaction site in orthohepadnaviruses maps to the inner dimer interface with contributions from both monomers. Such altered binding to HBs is concomitant with a reorganization of the dimeric interface. This raises the question how the dimerization and HBs binding have co-evolved. To address this question, we will determine representative structures of capsids from the different genera of Hepadnaviridae by electron cryo microscopy and image processing. The analysis will include capsids of paleo-viruses, which have been reconstructed from endogenous fragments in the genomes of crocodile, zebra finch and Melopsittacus. The structures of the paleo-capsids will show how the spike architecture has changed over geological ages. Finally, the binding sites between HBs and HBc in different genera will be mapped with peptide spot array libraries and validated by mutations that pinpoint the interaction sites on the capsids. The proposed work addresses a fundamental aspect of the structural evolution in hepadnaviridae that is currently inaccessible to modelling due to a lack of reference structures and sequence conservation in the spike region.