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HBV - How Capsid Works - Allosteric conformational changes of human HBV core protein transform its assembly
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Scientific Reports 7, Article number: 1404 (2017)
Chuang Liu1, Guizhen Fan1, Zhao Wang1, Hong-Song Chen2 & Chang-Cheng Yin1
Hepatitis B Virus core protein (HBc) has multiple roles in the viral lifecycle: viral assembly, compartment for reverse transcription, intracellular trafficking, and nuclear functions. HBc displays assembly polymorphism - it can assemble into icosahedral capsid and aberrant non-capsid structures. It has been hypothesized that the assembly polymorphism is due to allosteric conformational changes of HBc dimer, the smallest assembly unit, however, the mechanism governing the polymorphic assembly of the HBc dimer is still elusive. By using the experimental antiviral drug BAY 41-4109, we successfully transformed the HBc assembly from icosahedral capsid to helical tube. Structural analyses of HBc dimers from helical tubes, T = 4 icosahedral capsid, and sheet-like HBc ensemble revealed differences within the inter-dimer interface. Disruption of the HBc inter-dimer interface may likely promote the various assembly forms of HBc. Our work provides new structural insights into the HBV assembly mechanism and strategic guide for anti-HBV drug design.
Biological processes, such as gene replication, transcription and translation, are performed by macromolecular complexes. The correct assembly of macromolecules into a structurally and functionally relevant form is a key step for a biological process to be carried out successfully. Viruses, such as hepatitis B virus (HBV), present as ideal candidates for the study of a range of biological processes, including macromolecular assembly1. HBc (or named Cp2), is the capsid-forming "core protein" of human HBV, a major pathogen that kills 600,000 people annually3. Although excellent vaccines exist, there are no effective cures for extant chronic infections3, 4. In addition to capsid formation, HBc plays many essential roles in HBV lifecycle5,6,7, making it an attractive drug target8,9,10,11,12,13.
Wild-type HBc is a 183-residue polypeptide comprising a structured capsid-forming region (residues 1-149) and a basic, nucleic acid-binding domain (residues 150-183)14,15,16. The structured N-terminal region (hereafter referred as HBc) spontaneously self-assembles in vitro and in vivo to form icosahedral capsid-like particles identical to nucleo-capsids isolated from patient serum17, 18. The structure of HBc 1-149 has been characterized within the context of icosahedral capsids, virions, and sheet-like ensembles14, 17,18,19,20,21,22,23,24. HBc homodimers comprise two structural domains (Figure S1): helices 3 and 4 from opposing monomers pack together and form a four-helix bundle dimerization interface, whereas helices 1, 2 and 5 pack together around the base of the four-helix bundle to create the hydrophobic core of "contact" domains17. Weak inter-dimer interactions between contact domains stabilize HBV capsids17, 25.
Multiple thermodynamic and kinetic studies suggest that HBc has a very malleable structure, with this structural plasticity argued to be functionally important20, 21. Studies of HBV capsid assembly have inferred the existence of capsid assembly-active (HBcAct) and capsid assembly-incompetent (HBcInc) or aberrant (HBcAbb) conformations, where HBcAct assemble into the icosahedral capsid and HBcInc/HBcAbb assemble into non-icosahedral forms10, 11, 20, 22, 23. As HBc assembly into icosahedral capsid is essential for HBV lifecycle, it is desirable to understand the structural mechanism of HBc assembly. While structural analyses have proposed hydrophobic interactions between HBc dimers may be responsible for HBc assembly17, 19,20,21,22,23, insights into the polymorphic nature of HBc assembly remains elusive.
Bay 41-4109, a heteroaryldihydropyrimidine (HAP) derivative, interacts with HBc altering its assembly, making it potentially useful as an antiviral drug26, 27. Previously, it has been shown that HAP drugs at stoichiometric levels can facilitate HBc assembly into capsids, while at higher concentrations HAP drugs can induce HBc assembly into aberrant forms, e.g. tube-like structures10, 24, 27. It is noteworthy that BAY 41-4109 can exert its effects on assembly polymorphism both before and after assembly either by misdirecting HBc assembly or by transforming the pre-assembled icosahedral particle into aberrant forms28. Using BAY 41-4109, we successfully transformed the HBc assembly from an icosahedral capsid ensemble to a helical tube ensemble. Cryo-electron microscopy and helical reconstruction of the tubular HBc ensemble reveals a unique quaternary structure of HBc that differs from the icosahedral capsid and sheet-like ensembles. Intriguingly, the conformational differences of HBc dimers of the tubular ensemble from the icosahedral capsid and the sheet-like ensemble are subtle, with only lateral and rotational helix movement. These subtle conformational changes alter the inter-dimer interactions, leading to a smaller contact area between HBc dimers. This renders the icosahedral capsid thermodynamically unfavourable due to the weakened inter-dimer interactions and drives the HBc assembly into larger ensemble, the helical tube.
BAY 41-4109 transforms HBc assembly

HAP drugs can affect HBc assembly in a dose-dependent manner10, 26,27,28. HAPs induce assembly-active states (HBcAct) at stoichiometric levels but stabilize non-capsid polymers at higher concentrations10, 26,27,28. Consistent with these findings, we observed that BAY 41-4109 also affects HBc assembly in a dose-dependent manner: (1) in the absence of BAY 41-4109, HBc dimers assemble normally into icosahedral particles with diameters of 31 nm and 28 nm, indicating that free HBc dimers could assemble into both T = 4 and T = 3 icosahedral capsids (Figure S2a). (2) With a BAY41-4109/HBc dimer molar ratio of 1:6, while intact icosahedral particles were still observed, some icosahedral particles with nicks and larger disk-like particles were present, indicating that at this HAP concentration the icosahedral assembly has begun to be less favoured (Figure S2b); (3) With a BAY 41-4109/HBc dimer molar ratio of 1:5, most icosahedral particles disappear and large disk-like particles and sheet-like ensembles are observed (Figure S2c); (4) With a BAY 41-4109/HBc dimer molar ratio of ≥1:3, cone-shaped and tubular structures of various diameters dominate; the length of the tubular structures varies from 0.6 μm to 1.5 μm, and the width of the tubular structures varies from 30 nm to 50 nm (Figure S2d).
Cryo-electron microscopy of the HBc tubular ensemble reveals the structure of HBc dimers in the tubular ensemble
Previous studies of HBV capsid assembly have proposed the existence of capsid assembly-active (HBcAct) and capsid assembly-incompetent (HBcInc) conformations of HBc, in which HBc dimers either assemble into icosahedral capsid or aberrant structures. The HBc in the icosahedral capsid represents the HBcAct form, whereas the HBc in the aberrant structures represents the HBcInc form. To obtain structural insights into the assembly polymorphism of HBc, it is necessary to compare the structures of HBcAct and HBcInc. However, due to lack of order, the aberrant structures are not amenable for structural analyses. To overcome this difficulty, we targeted the experimental conditions that would favour HBc tubular ensembles (see Methods, Fig. 1a). Cryo-electron microscopy (cryo-EM) of frozen-hydrated HBc tubular ensembles reveal well-ordered helical structures, as manifested by the Fourier transform of the cryo-EM images, which exhibit typical helical diffraction layer lines extending to 12.1 Å: resolution (Fig. 1b). A number of helical families with different tube diameters, ranging from 460 to 530 Å:, are obtained. We selected 11 of the highest quality images recorded at different defocus values from tubes belonging to helical family (-16, 17) for further structural analyses. Real space three-dimensional (3D) reconstruction was performed by iterative helical real-space reconstruction (IHRSR29) and refined with helical constraints and full contrast transfer function (CTF) correction. The final density maps are displayed in Fig. 1c-e. The resolution of the EM density map is estimated as 9.2 Å: according to Fourier shell correlation (FSC) 0.5 criterion (Figure S3). At such resolution, secondary structural features, such as α-helices, are clearly identifiable (Figs 1c-e and 2a,b). At sub-nanometer resolution, Cα structural model building is feasible using molecular dynamics flexible fitting (MDFF) approach30. We therefore constructed a Cα structural model of the tubular ensemble by MDFF using the crystal structure of the HBc dimer (PDB ID 1QGT) in the T = 4 icosahedral capsid as an initial model.
The HBc tubular ensemble is a single-walled tube with a wall thickness of 47 Å:, corresponding to the height of the HBc dimer in the crystal structure of the T = 4 icosahedron; the diameter of the tube is 520 Å:; and the dimensions of the plane unit cell are a = 102 Å:, b = 104 Å:, γ = 62° (Fig. 2a). In our HBc helical ensemble, the unit cell of helical family (-16, 17) is composed of a triangle of three dimers or trimer of dimers (Fig. 2a, inset). The arrangement of trimers of dimers in the lattice displays a network of hexameric rings arrayed with quasi-p6 symmetry (Fig. 2a), consistent with the proposal by Stray et al. that the HAP drug-induced tube/sheet probably has p6 symmetry10.
The EM densities of three HBc dimers (AB, CD, and EF) in the unit cell of the tube ensemble were segmented and the Cα model for each HBc dimer was built by MDFF (Fig. 2b,c). Although the overall structures of dimers AB, CD, and EF are similar, subtle conformational differences exist between them, particularly in helix α5 (Fig. 3, indicated by arrows) and helix α1 (Fig. 3, indicated by stars); dimers AB and EF have similar conformation but they have significant different conformation from dimer CD in the contact helix α5 (Fig. 3).
Structural comparison of HBc dimers in different assembly forms
It has been proposed that HBc assembly is allosterically controlled20. This implies that different assembly forms, such as icosahedral capsid, tubular ensemble and sheet-like ensemble, may have different conformations. To determine the critical components of the structural polymorphisms we compared the structures of HBc dimers from the tubular ensemble to dimers from the T = 4 icosahedral capsid (PDB ID 1QGT) and sheet-like ensemble (PDB ID 3KXS20). To distinguish the different HBc conformations of the three assembly forms, we name the conformation in icosahedral capsid as HBcAct, the conformation in sheet-like ensemble as HBcInc, and the conformation in helical tube as HBcAbb. Structural overlays of each dimer show considerable differences, indicating their distinct conformations (Figs 4, S4 and S5). In the AB dimer, significant conformational differences are observed for helix α4a when comparing HBcAbb and HBcAct (Fig. 4a) and HBcAbb and HBcInc (Fig. 4b). Additionally, subtle conformational differences are also observed in the contact helix α5 in the AB dimer. For CD dimer, significant conformational differences are observed in the contact helix α5 between HBcAbb and HBcAct (Fig. 4c), and between HBcAbb and HBcInc (Fig. 4d); significant conformational difference also exists in helix α4a between HBcAbb and HBcInc (Fig. 4d). For EF dimer, significant conformational differences exist in the contact helix α5 and helix α4a between HBcAbb and HBcInc (Fig. 4e). Apart from these significant conformational differences, subtle conformational differences exist in other structural elements throughout the dimers amongst three different assembly forms (Figs 4, S4 and S5).
To determine the critical interfaces between the different assembly forms, we conducted structural analyses of the HBc dimers from capsid, sheet-like and tubular ensembles. We observed that (1) HBcAct dimers within the same ensemble (i.e., apo- or drug-induced capsid) have subtle differences displaying an upward <4° displacement of helix α5 (Figure S6a); (2) HBcAct dimers from different ensembles (i.e., apo- and drug-induced capsid) also have subtle differences, with <4° upward displacement of helix α5 (Figure S6b, Table S1); (3) dimers of the HBcInc or HBcAbb form within the same ensemble (i.e., tubular or sheet-like ensemble) have significant differences, with a helix α5 upward displacement of >7° in the tubular form and 13° in the sheet-like form (Figs 3 and S6c); (4) the same type of HBc dimers from different assembly forms (i.e., capsid, sheet, tube) also have subtle but major differences, with a helix α5 upward displacement of >6.7° (Figs 4, S4 and S6d) with the largest upward displacement of >15.3° (Fig. 4e). We conclude that subtle but major structural change of helix α5 transforms the HBc assembly from capsid to aberrant forms (sheet or tube).
To see how these structural differences affect the assembly of HBc, we then analysed the inter-dimer interactions in these different assembly forms. Compared with the dimers in icosahedral capsid and sheet-like ensemble, the most significant structural change of the dimer in tubular ensemble is an up-ward rotation movement of the contact helix α5 (Figs 4 and S4). Such movement drives helix α5 moving closer to helices α1 and α2a, thus making the individual dimers more compact than the other two forms (Fig. 5a,b); meanwhile, such movement decreases the contact area between neighbouring dimers in the tubular form than in the other two forms, thus making the interaction between neighbouring dimers in tube weaker than the other two forms (Figs 5c and S7).
HBc displays assembly polymorphism but the structural mechanism is elusive. It has been hypothesized that the assembly polymorphism is due to allosteric conformational changes of HBc dimer, the smallest assembly unit20. In this work, we analysed the structures of HBc tubular ensemble and the HBc dimers in the context of the tubular ensemble. Structural comparison with other type of high-order assembly forms, including the T = 4 icosahedral capsid and a sheet-like ensemble, revealed that the conformations of HBc dimers in these different HBc assembly forms contain important structural differences that alter the inter-dimer interactions allowing for the formation of alternate assembly forms. Based on structural analyses, we propose a mechanism how BAY 41-4109 transforms the HBc assembly from capsid into tube (Fig. 6). In the presence of BAY 41-4109, the drug induces a conformational change within the HBc dimer to promote a structural transfer from HBcAct to HBcAbb; by weakening the inter-dimer interactions, and destabilizing the capsid thereby driving the assembly into the helical tube ensemble. Alternatively, BAY 41-4109, which is structurally similar to HAP-126, could bind like HAP-1 at the hydrophobic pocket formed by helices α2, α4b and α5 of HBc and helix α5 of neighbour HBc27, 31. Thus BAY 41-4109 induced conformational changes of HBc, particularly the up-ward movement of helix α5, may change the dimer-dimer interaction angle and decrease the curvature of the ensemble, driving the assembly from capsid to tube.
HBc has multiple roles in the HBV viral lifecycle- assembly, compartment for reverse transcription, intracellular trafficking, and nuclear functions1, 2, thus, making it an attractive antiviral target. Zlotnick and colleagues propose that the assembly of HBc could be taken as a target for novel antiviral drug design1, 2. Our work provides structural insights into HBc assembly, and may serve as a strategic guide for antiviral drug design. HBc assembly into icosahedral capsid is essential for HBV lifecycle; therefore drugs that can misdirect the HBc assembly could be potentially effective in anti-HBV targets. Anti-viral drugs could be designed such that they either enhance the interaction between HBc dimers to trap the ensemble intermediates, or weaken the interactions between HBc dimers, thus misdirecting the assembly into aberrant forms could prove beneficial to HBV treatments.

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