During FY10, we focussed on three subprojects relating respectively to hepatitis B virus, bacteriophage HK97, and bacteriophage phi6. (1) Hepatitis B Virus Capsid Assembly. We study the HBV capsid protein which presents two of the three clinically important antigens - core antigen (capsids) and e-antigen (unassembled protein) - of this major human pathogen. After first showing that capsid protein self-assembles from dimers into shells of two different sizes, we obtained, in 1997, a cryo-EM density map in which we visualized the 4-helix bundle that forms the dimerization motif. This was the first time that such detailed information had been achieved by cryo-EM. Our subsequent research helped delineate the path of the polypeptide chain. We went on to investigate the antigenic diversity of HBV by using cryo-EM to characterize the conformational epitopes of seven different monoclonal antibodies raised against capsids. In FY10, we followed three main lines of investigation. (i) We completed and published a study in which surface plasmon resonance was used to measure the binding affinities of a set of murine monoclonal antibodies commonly used to discriminate between core-antigen and e-antigen, including several whose epitopes we previously identified by cryo-EM. Unexpectedly, most antibodies bind to both antigens with high affinity. The exceptions are antibody e6 which detects an epitope accessible only on dimers and occluded on capsids, and antibody 3120 which detects an epitope presented only on capsids because its epitope spans an inter-dimer interface. (ii) We followed up on the """"""""native"""""""" high resolution mass spectrometry experiments reported in FY08 in which the masses of both size variants of the capsid were determined to within 0.1%. Specifically, we exploited this high mass discrimination to measure the rates at which both capsids exchange dimers with the unassembled pool, and found this rate to be slow but significant (10% exchange in 3 months) for the smaller T=3 capsid and undetectably slow for the T=4 capsid. (iii) We also pursued a collaborative project to measure the mechanical properties of both capsids by nanoindentation methods carried out by atomic force microscopy. The two capsids have similar overall stability and elasticity (Young's modulus of 0.4 GPa) and their mechanical properties can be successfully modelled using both finite element and molecular dynamics formalisms. (2) Assembly and Maturation of Bacteriophage Capsids. Our interest in capsid assembly lies in the massive conformational changes that accompany their maturation. These transitions afford unique insights into allosteric regulation. We study maturation of several phages to exploit expedient aspects of each system. The tailed phages afford an excellent model for herpesvirus capsids, reflecting common evolutionary origins. In FY10, we focussed mainly on two projects. (2a) All genomes cycle between a condensed state assumed during replication of the organism and a decondensed states assumed during gene expression, etc. The encapsidated state of phage DNA represents an extreme case of genome condensation. We used a combination of scanning calorimetry and cryo-EM to investigate this phenomenon in the phage HK97 system which is particularly well suited for study in view of detailed knowledge of its capsid structure. We found that, as filled capsids are heated, their DNA is released at relatively low temperatures (40 to 50 degrees). Heating increases the internal pressure, causing the capsid to rupture, releasing the DNA. DNA packaging also induces a change in the capsid structure that is reflected both in an earlier onset of thermal denaturation than empty capsids and in subtle morphological differences. (Previously, we detected a similar effect in herpesvirus capsids). We envisage that this transition in the capsid shell is transmitted to the portal, altering its interactions with the packaging enzyme and thus signaling that packaging is complete. This project was completed and published in FY10. (2b) The capsids of double-stranded RNA viruses serve as specialized compartments for the replication and transcription of the viral genomes. We investigate the structural basis of this remarkable phenomenon in the phage phi6 system, which has a tripartite genome. In FY08, we published a paper describing the location of the P2 polymerase in the interior of the viral procapsid, as determined by cryo-EM of wild type and mutant particles. P2 is substoichiometric, occupying only 3 - 10 (depending on the mutant) of 20 potential sites. In FY10 we completed and published an extension of these studies by using cryo-electron tomography to map the internal sites occupied by P2 and the external sites occupied by P4, the packaging ATPase. The observed distributions disclosed that both proteins are randomly distributed and therefore there is no direct coupling between the activities of these two viral enzymes that respectively conduct RNA packaging, and replication and transcription. Our ongoing effort on this system targets the maturational expansion of the procapsid which has been hypothesized to proceed sequentially according to the successive packagings of the three segments of genomic RNA. We are attempting to induce the corresponding transitions in vitro by exposing procapsids to differing solution conditions (pH, ionic strength, temperature, etc). To date, we have succeeded in identifying and characterizing one intermediate state.
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