We investigated the ultrastructural dynamics of HBV (nucleo)capsids and identified an evolutionary conserved disulfide switch involved in regulating the interaction of the capsid proteins with the enclosed nucleic acids. In addition, we successfully generated stable cell lines for the production of HBV nucleocapsids arrested at different states of genome maturation. Ultrastructural dete-minations of these particles are currently ongoing and will be finalized early in the next funding period. Importantly, these cell lines are not only a tool to generate highly concentrated particle preparations, but will also be used to image the intracellular steps of the viral life cycle via in situ cryo-electron microscopy (CEM) techniques in close collaboration with Bartenschlager.
We now shift our focus towards studying enveloped virions. Previously, we described a distinct maturation step in virions circulating in the bloodstream that slowly renders them infectious. Briefly, virions are released from cells in an inactive form, in which the PreS domains of the large envelope protein (L) are hidden internally. The subsequent translocation of PreS across the viral membrane onto the particles’ surface is prerequisite for receptor binding and entry into hepatocytes. We showed that this stochastic conversion proceeds within the virion population with a half-time of about 5 hours. Immature and mature virions can be physically separated by their heparin binding competence. Indeed, we demonstrated in a mouse model that this protracted maturation process drastically increases the probability for transmitted virions to reach the liver without prior loss due to abortive extrahepatic binding to ubiquitous heparan sulfate proteoglycans. Considering these results, in the next funding period we will pursue three complementary aims. First, in order to resolve virion maturation at the ultrastructural level, we will use CEM to reconstruct at subnanometer level the virions’ envelope proteins in their relation to the nucleocapsids and the lipid bilayer of the membrane, with a particular interest in the two topological variants of L. To this end, we will utilize highly purified virions from culture supernatants of HBV producer cell lines and from sera, provided by Urban/Nkongolo, of chronically infected patients. Deco-ration of mature virions with monovalent recombinant single-chain antibodies will enable tracing the position of translocated PreS and unveil any asymmetric distribution on the particle surface indicative of a “domino effect” by which the topological switch is supposed to proceed through an individual virion. In parallel, we will investigate the determinants of nucleocapsid envelopment by envelope-protein derived pep-tides in vitro using magnetic resonance and scattering techniques to detect binding. Our second aim is to identify liver-specific local triggers prompting virion maturation. First, we will investigate the role of temperature, pH and the redox milieu on the development of heparin binding competence as key characteristic of virion conversion. Second, we will fractionate culture supernatants and cell lysates from human hepatocyte cell lines. Incubation of immature virions with these fractions will reveal any (positive or negative) effect on maturation. In particular, we will focus on factors that could be involved in modifying the lipid composition in the viral membrane, e.g. diverse apolipoproteins (with Bartenschlager and Protzer/Wettengel). Our third aim is based on the assumption that this peculiar maturation mechanism might represent an ideal target for therapeutic intervention. Thus, we will screen in close collaboration with Urban/Nkongolo small molecule libraries for compounds that efficiently interfere with this essential step in the viral life cycle. We anticipate that locking HBV in the immature state might contribute another promising approach towards a sustainable cure for chronic hepatitis B.