Structural Differences between the Woodchuck Hepatitis Virus Core Protein in the Dimer and Capsid States Are Consistent with Entropic and Conformational Regulation of Assembly.

Hepadnaviruses are hepatotropic enveloped DNA viruses with an icosahedral capsid. Hepatitis B virus (HBV) causes chronic infection in an estimated 240 million people; woodchuck hepatitis virus (WHV), an HBV homologue, has been an important model system for drug development. The dimeric capsid protein (Cp) has multiple functions during the viral life cycle and thus has become an important target for a new generation of antivirals. Purified HBV and WHV Cp spontaneously assemble into 120-dimer capsids. Though they have 65% identity, WHV Cp has error-prone assembly with stronger protein-protein association. We have taken advantage of the differences in assemblies to investigate the basis of assembly regulation. We determined the structures of the WHV capsid to 4.5-Å resolution by cryo-electron microscopy (cryo-EM) and of the WHV Cp dimer to 2.9-Å resolution by crystallography and examined the biophysical properties of the dimer. We found, in dimer, that the subdomain that makes protein-protein interactions is partially disordered and rotated 21° from its position in capsid. This subdomain is susceptible to proteolysis, consistent with local disorder. WHV assembly shows similar susceptibility to HBV antiviral molecules, suggesting that HBV assembly follows similar transitions. These data show that there is an entropic cost for assembly that is compensated for by the energetic gain of burying hydrophobic interprotein contacts. We propose a series of stages in assembly that incorporate a disorder-to-order transition and structural shifts. We suggest that a cascade of structural changes may be a common mechanism for regulating high-fidelity capsid assembly in HBV and other viruses.IMPORTANCE Virus capsids assemble spontaneously with surprisingly high fidelity. This requires strict geometry and a narrow range of association energies for these protein-protein interactions. It was hypothesized that requiring subunits to undergo a conformational change to become assembly active could regulate assembly by creating an energetic barrier and attenuating association. We found that woodchuck hepatitis virus capsid protein undergoes structural transitions between its dimeric and its 120-dimer capsid states. It is likely that the closely related hepatitis B virus capsid protein undergoes similar structural changes, which has implications for drug design. Regulation of assembly by structural transition may be a common mechanism for many viruses.

Charge Detection Mass Spectrometry Identifies Preferred Non-Icosahedral Polymorphs in the Self-Assembly of Woodchuck Hepatitis Virus Capsids.

Woodchuck hepatitis virus (WHV) is prone to aberrant assembly in vitro and can form a broad distribution of oversized particles. Characterizing aberrant assembly products is challenging because they are both large and heterogeneous. In this work, charge detection mass spectrometry (CDMS) is used to measure the distribution of WHV assembly products. CDMS is a single-particle technique where the masses of individual ions are determined from simultaneous measurement of each ion's charge and m/z (mass-to-charge) ratio. Under relatively aggressive, assembly promoting conditions, roughly half of the WHV assembly products are T=4 capsids composed of exactly 120 dimers while the other half are a broad distribution of larger species that extends to beyond 210 dimers. There are prominent peaks at around 132 dimers and at 150 dimers. In part, the 150 dimer complex can be attributed to elongating a T=4 capsid along its 5-fold axis by adding a ring of hexamers. However, most of the other features cannot be explained by existing models for hexameric defects. Cryo-electron microscopy provides evidence of elongated capsids. However, image analysis reveals that many of them are not closed but have "spiral-like" morphologies. The CDMS data indicate that oversized capsids have a preference for growth by addition of 3 or 4 dimers, probably by completion of hexameric vertices.

Combinatorial approach to hepadnavirus-like particle vaccine design.

The particulate hepatitis core protein (HBcAg) represents an efficient carrier platform with many of the characteristics uniquely required for the delivery of weak immunogens to the immune system. Although the HBcAg is highly immunogenic, the existing HBcAg-based platform technology has a number of theoretical and practical limitations, most notably the "preexisting immunity" and "assembly" problems. To address the assembly problem, we have developed the core protein from the woodchuck hepadnavirus (WHcAg) as a new particulate carrier platform system. WHcAg appears to tolerate insertions of foreign epitopes at a greater number of positions than HBcAg. For example, both within the external loop region and outside the loop region a total of 17 insertion sites were identified on WHcAg. Importantly, the identification of an expanded number of insertion sites was dependent on additional modifications to the C terminus that appear to stabilize the various internal insertions. Indeed, 21 separate C-terminal modifications have been generated that can be used in combination with the 17 insertion sites to ensure efficient hybrid WHcAg particle assembly. This combinatorial technology is also dependent on the sequence of the heterologous insert. Therefore, the three variables of insert position, C terminus, and epitope sequence are relevant in the design of hybrid WHcAg particles for vaccine purposes.

UVDDB p127-binding modulates activities and intracellular distribution of hepatitis B virus X protein.

Mammalian hepatitis B viruses encode a unique regulatory protein termed X, which is essential for infection and likely plays a role in the carcinogenic process associated with hepadnaviral infection. Among the numerous properties ascribed to X protein, two have been widely documented: promiscuous transcriptional transactivation and proapoptosis. However, full understanding of the mechanisms underlying these activities requires the identification of the genuine X partners among the multiple X-binding host proteins. Here we show that (i) mutations in X protein, which markedly alter affinity for the host protein UVDDBp127, inactivate both transactivation and proapoptosis; (ii) ectopic fusion of a functional UVDDB-binding domain to a deficient binding X mutant restored its activity; (iii) in contrast to the loss-of-binding mutants, a mutant with a strong gain-of-binding exerted trans-dominant negative effects on wt X activity and localized in the nucleus and (iv) increase in intracellular UVDDB concentration enhanced both wt X-mediated transactivation and apoptosis. Taken together, our data provide strong evidence for a common upstream step in X mode of action, consisting of its productive interaction with UVDDB, via a structurally and functionally autonomous module. In addition, they underscore a nuclear location step of the viral protein that depends on its ability to bind UVDDB.

Fusogenic activity of hepadnavirus peptides corresponding to sequences downstream of the putative cleavage site.

Sequence homology between the amino-terminal region of the S protein of hepatitis B Virus (HBV) and known fusion peptides from retroviruses and paramyxoviruses led us to propose that this region might be equally involved in the initial infective steps of hepadnaviruses. In fact, we showed that a synthetic peptide corresponding to the N-terminus region of the S protein of HBV had membrane-interacting properties and was able to induce liposome fusion adopting an extended (beta-sheet) conformation (Rodríguez-Crespo et al., 1996, 1995). We describe herein studies on the interaction of peptides derived from the N-terminal region of the S protein of duck (DHBV: Met-Ser-Gly-Thr-Phe-Gly-Gly-Ile-Leu-Ala-Gly-Leu-Ile-Gly-Leu-Leu) and woodchuck hepatitis B viruses (WHV: Met-Ser-Pro-Ser-Ser-Leu-Leu-Gly-Leu-Leu-Ala-Gly-Leu-Gln-Val-Val) with liposomes. These peptides were able to induce to a different extent aggregation, lipid mixing, and leakage of internal aqueous contents from both neutral and negatively charged phospholipid vesicles in a concentration-dependent and pH-independent manner. Fluorescence depolarization of 1,6-diphenyl-1,3,5-hexatriene-labeled vesicles indicated that both peptides become inserted into the hydrophobic core of the lipid bilayer. Circular dichroism studies indicated that the DHBV peptide adopts an extended conformation in the presence of lipids, whereas the WHV peptide displays a high content of alpha-helical conformation. Therefore, these results extend our previous findings obtained for human hepatitis B virus to other members of the hepadnavirus family and suggest that this region of the S protein is important in the initial steps of the infective cycle.