Assembly Properties of Hepatitis B Virus Core Protein Mutants Correlate with Their Resistance to Assembly-Directed Antivirals

ABSTRACTThe hepatitis B virus (HBV) capsid or core protein (Cp) can self-assemble to form an icosahedral capsid. It is now being pursued as a target for small-molecule antivirals that enhance the rate and extent of its assembly to yield empty and/or aberrant capsids. These small molecules are thus called core protein allosteric modulators (CpAMs). We sought to understand the physical basis of CpAM-resistant mutants and how CpAMs might overcome them. We examined the effects of two closely related CpAMs, HAP12 and HAP13, which differ by a single atom but have drastically different antiviral activities, on the assembly of wild-type Cp and three T109 mutants (T109M, T109I, and T109S) that display a range of resistances. The T109 side chain forms part of the mouth of the CpAM binding pocket. A T109 mutant that has substantial resistance even to a highly active CpAM strongly promotes normal assembly. Conversely, a mutant that weakens assembly is more susceptible to CpAMs. In crystal and cryo-electron microscopy (cryo-EM) structures of T=4 capsids with bound CpAMs, the CpAMs preferentially fit into two of four quasi-equivalent sites. In these static representations of capsid structures, T109 does not interact with the neighboring subunit. However, all-atom molecular dynamics simulations of an intact capsid show that T109 of one of the four classes of CpAM site has a hydrophobic contact with the neighboring subunit at least 40% of the time, providing a physical explanation for the mutation's ability to affect capsid stability, assembly, and sensitivity to CpAMs.IMPORTANCEThe HBV core protein and its assembly into capsids have become important targets for development of core protein allosteric modulators (CpAMs) as antivirals. Naturally occurring T109 mutants have been shown to be resistant to some of these CpAMs. We found that mutation of T109 led to changes in capsid stability and recapitulated resistance to a weak CpAM, but much less so than to a strong CpAM. Examination of HBV capsid structures, determined by cryo-EM and crystallography, could not explain how T109 mutations change capsid stability and resistance. However, by mining data from a microsecond-long all-atom molecular dynamics simulation, we found that the capsid was extraordinarily flexible and that T109 can impede entry to the CpAM binding site. In short, HBV capsids are incredibly dynamic and molecular mobility must be considered in discussions of antiviral mechanisms.

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Hepatitis B Virus Capsids Have Diverse Structural Responses to Small-Molecule Ligands Bound to the Heteroaryldihydropyrimidine Pocket

Journal of Virology ◽

10.1128/jvi.03058-15 ◽

2016 ◽

Vol 90 (8) ◽

pp. 3994-4004 ◽

Cited By ~ 40

Author(s):

Balasubramanian Venkatakrishnan ◽

Sarah P. Katen ◽

Samson Francis ◽

Srinivas Chirapu ◽

M. G. Finn ◽

...

Keyword(s):

Life Cycle ◽

Hepatitis B Virus ◽

Hepatitis B ◽

Drug Design ◽

Quaternary Structure ◽

Core Protein ◽

Viral Life Cycle ◽

Structural Basis ◽

Allosteric Modulators ◽

B Virus

ABSTRACTThough the hepatitis B virus (HBV) core protein is an important participant in many aspects of the viral life cycle, its best-characterized activity is self-assembly into 240-monomer capsids. Small molecules that target core protein (core protein allosteric modulators [CpAMs]) represent a promising antiviral strategy. To better understand the structural basis of the CpAM mechanism, we determined the crystal structure of the HBV capsid in complex with HAP18. HAP18 accelerates assembly, increases protein-protein association more than 100-fold, and induces assembly of nonicosahedral macrostructures. In a preformed capsid, HAP18 is found at quasiequivalent subunit-subunit interfaces. In a detailed comparison to the two other extant CpAM structures, we find that the HAP18-capsid structure presents a paradox. Whereas the two other structures expanded the capsid diameter by up to 10 Å, HAP18 caused only minor changes in quaternary structure and actually decreased the capsid diameter by ∼3 Å. These results indicate that CpAMs do not have a single allosteric effect on capsid structure. We suggest that HBV capsids present an ensemble of states that can be trapped by CpAMs, indicating a more complex basis for antiviral drug design.IMPORTANCEHepatitis B virus core protein has multiple roles in the viral life cycle—assembly, compartment for reverse transcription, intracellular trafficking, and nuclear functions—making it an attractive antiviral target. Core protein allosteric modulators (CpAMs) are an experimental class of antivirals that bind core protein. The most recognized CpAM activity is that they accelerate core protein assembly and strengthen interactions between subunits. In this study, we observe that the CpAM-binding pocket has multiple conformations. We compare structures of capsids cocrystallized with different CpAMs and find that they also affect quaternary structure in different ways. These results suggest that the capsid “breathes” and is trapped in different states by the drug and crystallization. Understanding that the capsid is a moving target will aid drug design and improve our understanding of HBV interaction with its environment.

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Phosphorylation of the Arginine-Rich C-Terminal Domains of the Hepatitis B Virus (HBV) Core Protein as a Fine Regulator of the Interaction between HBc and Nucleic Acid

Viruses ◽

10.3390/v12070738 ◽

2020 ◽

Vol 12 (7) ◽

pp. 738 ◽

Cited By ~ 2

Author(s):

Hugues de Rocquigny ◽

Virgile Rat ◽

Florentin Pastor ◽

Jean Luc Darlix ◽

Christophe Hourioux ◽

...

Keyword(s):

Hepatitis B Virus ◽

Hepatitis B ◽

Nucleic Acids ◽

Core Protein ◽

Cell Trafficking ◽

Terminal Domain ◽

Capsid Shell ◽

B Virus ◽

Capsid Stability ◽

Terminal Domains

The morphogenesis of Hepatitis B Virus (HBV) viral particles is nucleated by the oligomerization of HBc protein molecules, resulting in the formation of an icosahedral capsid shell containing the replication-competent nucleoprotein complex made of the viral polymerase and the pre-genomic RNA (pgRNA). HBc is a phospho-protein containing two distinct domains acting together throughout the viral replication cycle. The N-terminal domain, (residues 1–140), shown to self-assemble, is linked by a short flexible domain to the basic C-terminal domain (residues 150–183) that interacts with nucleic acids (NAs). In addition, the C-terminal domain contains a series of phospho-acceptor residues that undergo partial phosphorylation and de-phosphorylation during virus replication. This highly dynamic process governs the homeostatic charge that is essential for capsid stability, pgRNA packaging and to expose the C-terminal domain at the surface of the particles for cell trafficking. In this review, we discuss the roles of the N-terminal and C-terminal domains of HBc protein during HBV morphogenesis, focusing on how the C-terminal domain phosphorylation dynamics regulate its interaction with nucleic acids throughout the assembly and maturation of HBV particles.

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Author response: Hepatitis B virus core protein allosteric modulators can distort and disrupt intact capsids

10.7554/elife.31473.028 ◽

2017 ◽

Author(s):

Christopher John Schlicksup ◽

Joseph Che-Yen Wang ◽

Samson Francis ◽

Balasubramanian Venkatakrishnan ◽

William W Turner ◽

...

Keyword(s):

Hepatitis B Virus ◽

Hepatitis B ◽

Core Protein ◽

Author Response ◽

Allosteric Modulators ◽

Virus Core ◽

B Virus

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Hepatitis B virus core protein allosteric modulators can distort and disrupt intact capsids

eLife ◽

10.7554/elife.31473 ◽

2018 ◽

Vol 7 ◽

Cited By ~ 41

Author(s):

Christopher John Schlicksup ◽

Joseph Che-Yen Wang ◽

Samson Francis ◽

Balasubramanian Venkatakrishnan ◽

William W Turner ◽

...

Keyword(s):

Hepatitis B Virus ◽

Hepatitis B ◽

Binding Sites ◽

Core Protein ◽

Spectroscopic Studies ◽

Allosteric Modulators ◽

Virus Core ◽

B Virus ◽

High Resolution Reconstruction ◽

Direct Acting

Defining mechanisms of direct-acting antivirals facilitates drug development and our understanding of virus function. Heteroaryldihydropyrimidines (HAPs) inappropriately activate assembly of hepatitis B virus (HBV) core protein (Cp), suppressing formation of virions. We examined a fluorophore-labeled HAP, HAP-TAMRA. HAP-TAMRA induced Cp assembly and also bound pre-assembled capsids. Kinetic and spectroscopic studies imply that HAP-binding sites are usually not available but are bound cooperatively. Using cryo-EM, we observed that HAP-TAMRA asymmetrically deformed capsids, creating a heterogeneous array of sharp angles, flat regions, and outright breaks. To achieve high resolution reconstruction (<4 Å), we introduced a disulfide crosslink that rescued particle symmetry. We deduced that HAP-TAMRA caused quasi-sixfold vertices to become flatter and fivefold more angular. This transition led to asymmetric faceting. That a disordered crosslink could rescue symmetry implies that capsids have tensegrity properties. Capsid distortion and disruption is a new mechanism by which molecules like the HAPs can block HBV infection.

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