Separation and crystallization of T = 3 and T = 4 icosahedral complexes of the hepatitis B virus core protein

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Though 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 ϳ3 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. Hepatitis B virus (HBV) causes degenerative liver disease and is the leading cause of liver cancer, with 240 million chronically infected individuals (1, 2). Current antiviral therapies control the progression of the disease but fail to eliminate the virus (3, 4). There is a need for improved therapies to combat chronic infections. One approach is to target virus assembly (5-7).HBV is an enveloped, double-stranded DNA virus with an icosahedral nucleoprotein core. The HBV capsid is the protein shell of the core. Beyond genome protection, the capsid is involved in intracellular trafficking, interaction with nuclear import machinery, regulation of reverse transcription, signaling, completion of reverse transcription, RNA chaperoning, and envelope acquisition (8). Capsid assembly is central to HBV replication. In vivo HBV capsids assemble around an RNA copy of the viral g...

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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

Venkatakrishnan

Katen

Francis

et al. 2016

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“…A single line fit both data sets, indicating that the wild-type protein and Cp149-F97L do not have significant differences in stability. (45). At 21°C and 150 mM NaCl, a concentration greater than 15 M Cp149-wt is required for appreciable accumulation of capsid (7).…”

Section: Fig 3 CD Spectroscopy Demonstrates That Cp149-f97l and -Wtmentioning

confidence: 99%

Hepatitis B Virus Capsid Assembly Is Enhanced by Naturally Occurring Mutation F97L

Ceres

Stray

Zlotnick

2004

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In chronic hepatitis B virus (HBV) infections, one of the most common mutations to the virus occurs at amino acid 97 of the core protein, where leucine replaces either phenylalanine or isoleucine, depending on strain. This mutation correlates with changes in viral nucleic acid metabolism and/or secretion. We hypothesize that this phenotype is due in part to altered core assembly, a process required for DNA synthesis. We examined in vitro assembly of empty HBV capsids from wild-type and F97L core protein assembly domains. The mutation enhanced both the rate and extent of assembly relative to those for the wild-type protein. The difference between the two proteins was most obvious in the temperature dependence of assembly, which was dramatically stronger for the mutant protein, indicating a much more positive enthalpy. Since the structures of the mutant and wild-type capsids are essentially the same and the mutation is not involved in the contact between dimers, we suggest that the F97L mutation affects the dynamic behavior of dimer and capsid.

“…The HBV capsid is in Tϭ4 or Tϭ3 icosahedral symmetry, and Tϭ4 capsids account for a major population in the natural HBV pool (19)(20)(21)(22)(23). The basic building block of HBV capsid is Cp, which is 183 amino acids (aa) in length and exists as a homodimer in solution (24).…”

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confidence: 99%

The Interface between Hepatitis B Virus Capsid Proteins Affects Self-Assembly, Pregenomic RNA Packaging, and Reverse Transcription

Tan

Pionek

Unchwaniwala

et al. 2015

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101

Hepatitis B virus (HBV) capsid proteins (Cps) assemble around the pregenomic RNA (pgRNA) and viral reverse transcriptase (P). pgRNA is then reverse transcribed to double-stranded DNA (dsDNA) within the capsid. The Cp assembly domain, which forms the shell of the capsid, regulates assembly kinetics and capsid stability. The Cp, via its nucleic acid-binding C-terminal domain, also affects nucleic acid organization. We hypothesize that the structure of the capsid may also have a direct effect on nucleic acid processing. Using structure-guided design, we made a series of mutations at the interface between Cp subunits that change capsid assembly kinetics and thermodynamics in a predictable manner. Assembly in cell culture mirrored in vitro activity. However, all of these mutations led to defects in pgRNA packaging. The amount of first-strand DNA synthesized was roughly proportional to the amount of RNA packaged. However, the synthesis of second-strand DNA, which requires two template switches, was not supported by any of the substitutions. These data demonstrate that the HBV capsid is far more than an inert container, as mutations in the assembly domain, distant from packaged nucleic acid, affect reverse transcription. We suggest that capsid molecular motion plays a role in regulating genome replication. IMPORTANCE The hepatitis B virus (HBV) capsid plays a central role in the virus life cycle and has been studied as a potential antiviral target.The capsid protein (Cp) packages the viral pregenomic RNA (pgRNA) and polymerase to form the HBV core. The role of the capsid in subsequent nucleic acid metabolism is unknown. Here, guided by the structure of the capsid with bound antiviral molecules, we designed Cp mutants that enhanced or attenuated the assembly of purified Cp in vitro. In cell culture, assembly of mutants was consistent with their in vitro biophysical properties. However, all of these mutations inhibited HBV replication. Specifically, changing the biophysical chemistry of Cp caused defects in pgRNA packaging and synthesis of the second strand of DNA. These results suggest that the HBV Cp assembly domain potentially regulates reverse transcription, extending the activities of the capsid protein beyond its presumed role as an inert compartment. Hepatitis B virus (HBV) chronically infects more than 240 million people worldwide and contributes to 780,000 deaths per year (1). Currently there is no reliable cure for chronic hepatitis (2, 3). HBV is an enveloped double-stranded DNA (dsDNA) virus with an icosahedral core. HBV replicates its genome through an RNA intermediate. During replication, the capsid protein (Cp; also called the core protein) assembles around pregenomic RNA (pgRNA) and reverse transcriptase (the P protein) to form RNAfilled cores (4-8). Unlike HIV, reverse transcription of the HBV genome is a prerequisite for the progeny viruses to leave the host cell (5, 9).HBV reverse transcription is a complex reaction involving three template switches. P protein is incorporated into capsids durin...