Interaction of Hepatitis B capsid like particles with surface protein fragments and peptides interfering with envelopment

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Hepatitis B Virus (HBV) is a major human pathogen with more than 250 million chronic carriers worldwide. The virions consist of an outer envelope with a membrane that is densely packed with surface proteins (HBs), and an inner capsid, formed by the hepatitis B core protein (HBc). The majority of enveloped particles with packaged nucleic acid contain a mature partly double stranded DNA genome that was generated from a precursor RNA by reverse transcription inside the capsids. However, phosphorylated capsids without a genome are also readily enveloped suggesting that there might be no conformational maturation signal that makes capsids envelopment competent but that preferences for envelopment are driven by the dynamics of the processes upstream of envelopment. Here we want to investigate the structural properties of the capsid that are essential for envelopment. So far, we have shown that in virions the capsid contacts the envelope via the tips of protruding spikes and that peptides that interfere with envelopment bind to these tips in recombinant capsids. This contrasts finding by others who have used mutational screens that identified a hydrophobic pocket in the center of the spikes as being important for the secretion phenotype. This leads to the question which of the two sites is important for HBs-binding. We propose to address this question by determining the structure of recombinant capsids with bound surface protein fragments. For this we will use electron cryo microscopy and image processing, which allows us to study the capsid structure unhindered by crystal contacts with resolutions of up to 2.4 Å. The structures will show whether the tips of the spikes, the pocket in the center of the spikes or both sites bind to the surface protein fragments. Interrogating different HBc mutants with specific envelopment phenotypes and phosphorylated empty capsids as mimics for envelopment competent capsids in such a way will show whether HBs binding is affected by changes in the capsid structure. We will also determine where peptides that interfere with envelopment bind and whether they compete with HBs for binding sites or are more likely to affect processes upstream of envelopment. All structural studies will be complemented by quantifying binding constants using isothermal titration calorimetry and/or surface plasmon resonance measurements to test whether changes in the binding constant are likely to modulate envelopment.Based on our recent structures of WT-capsids and a premature envelopment mutant, we expect that resolutions in the range of 2.4-3 Å can be readily achieved. Therefore, this project will provide a wealth of structural information on clinically relevant HBc mutants and their interaction with Hbs. The structural information will inform on the envelopment mechanism and will serve others as knowledge base for identifying molecules in silico that can interfere with this process and thus with viral maturation.