Assembly-Directed Antivirals Differentially Bind Quasiequivalent Pockets to Modify Hepatitis B Virus Capsid Tertiary and Quaternary Structure

Katen, Sarah P.; Tan, Zhenning; Chirapu, Srinivas Reddy; Finn, M. G.; Zlotnick, Adam

doi:10.1016/j.str.2013.06.013

Cited by 124 publications

(177 citation statements)

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“…HBV assembly is driven by hydrophobic interactions at the interdimer interface, where about 75% of the buried surface area is hydrophobic (31,41). CpAMs bind a hydrophobic pocket at the interface and strengthen protein-protein interactions by filling that gap (41,44). Previously, by replacing V124 at the HAP binding pocket with tryptophan, we designed a Cp mutant with enhanced assembly kinetics and association energy (45).…”

Section: Resultsmentioning

confidence: 99%

“…Small antiviral molecules, called core protein allosteric modulators (CpAMs), can target the interdimer interface and modulate assembly. For example, heteroaryldihydropyrimidines (HAPs) and phenylpropenamides bind a hydrophobic pocket at the interdimer interface and enhance assembly kinetics and thermodynamics (40)(41)(42)(43)(44). The phenylpropenamides notably affect the Cp tertiary structure (44).…”

mentioning

confidence: 99%

“…For example, heteroaryldihydropyrimidines (HAPs) and phenylpropenamides bind a hydrophobic pocket at the interdimer interface and enhance assembly kinetics and thermodynamics (40)(41)(42)(43)(44). The phenylpropenamides notably affect the Cp tertiary structure (44). Remarkably, a hydrophobic substitution at the HAP binding pocket of Cp demonstrated a correlation between enhancing assembly and inhibiting virus replication (45).…”

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confidence: 99%

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

Tan

Pionek

Unchwaniwala

et al. 2015

J Virol

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101

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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...

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Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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

Tan

Pionek

Unchwaniwala

et al. 2015

J Virol

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101

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“…In contrast, the fluctuations at the bases of the spikes were small, in agreement with reported in the previous crystallographic studies. [6][7][8] When AT-130 was replaced with C13, this distribution did not change and the fluctuations at the bases of the spikes remained small, as shown in Fig. 3 (B4g93C13).…”

Section: Resultsmentioning

confidence: 88%

“…The large part of the structural change in the protein units, was accounted for by translation and rotation of the chains, as has been reported previously. 7,8) The A/B and C/D dimers were also fairly rigid. The rigidity of the chains and the dimers was observed in all other simulations.…”

Section: Simulationmentioning

confidence: 99%

Molecular Dynamics Simulations to Determine the Structure and Dynamics of Hepatitis B Virus Capsid Bound to a Novel Anti-viral Drug

et al. 2016

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Hepatitis B virus (HBV) chronically infects millions of people worldwide and is a major cause of serious liver diseases, including liver cirrhosis and liver cancer. In our previous study, in silico screening was used to isolate new anti-viral compounds predicted to bind to the HBV capsid. Four of the isolated compounds have been reported to suppress the cellular multiplication of HBV experimentally. In the present study, molecular dynamics simulations of the HBV capsid were performed under rotational symmetry boundary conditions, to clarify how the structure and dynamics of the capsid are affected at the atomic level by the binding of one of the isolated compounds, C13. Two simulations of the free HBV capsid, two further simulations of the capsid-C13 complex, and one simulation of the capsid-AT-130 complex were performed. For statistical confidence, each set of simulations was repeated by five times, changing the simulation conditions. C13 continued to bind at the predicted binding site during the simulations, supporting the hypothesis that C13 is a capsid-binding compound. The structure and dynamics of the HBV capsid were greatly influenced by the binding and release of C13, and these effects were essentially identical to those seen for AT-130, indicating that C13 likely inhibits the function of the HBV capsid.Key words rotational symmetry boundary condition; in silico screening; hepatitis B virus; capsid Hepatitis B virus (HBV) chronically infects millions of people worldwide and is a major cause of serious liver diseases, including liver cirrhosis and liver cancer. 1) Interferon-based inhibitors and nucleic acid analogs have been approved for the treatment of HBV-related diseases in the U.S.A. However, the interferon-based inhibitors fall short of delivering ideal clinical outcomes, and the nucleic acid analogs need to be administered over a long term. Therefore, more effective and fast acting treatments targeting HBV are required. In our previous study, in silico screening 2) was used to isolate 16 anti-viral candidate compounds, which are predicted to be bound to the HBV capsid.3) The candidate compounds were tested in an in vitro HBV infection system. The levels of HBV DNA and HBV surface antigens were significantly reduced in the PXB cells (PhoenixBio, Hiroshima, Japan) which were treated separately with four of the previously identified compounds, namely C9, C10, C13, and C16. However, it has not been established whether the compounds actually inhibit the function of the HBV capsid at the atomic level. Thus, we performed molecular dynamics (MD) simulations of the HBV capsid to model how the structure and dynamics of the capsid are likely to be affected by the binding of C13, one of the most effective compounds found in the previous study. The chemical name of C13 is 4-(4-acetylpiperazin-1-yl)-N-[1-(cyclopropylamino)-1-oxo-3-phenylpropan-2-yl]-3-nitrobenzamide.The structure of HBV capsid has been reported by cryoelectron microscopy. 4,5) Later, the atomic coordinates usable for the initial coordinates of ...

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Clinical Implications of the Molecular Biology of Hepatitis B Virus

2009

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Assembly-Directed Antivirals Differentially Bind Quasiequivalent Pockets to Modify Hepatitis B Virus Capsid Tertiary and Quaternary Structure

Cited by 124 publications

References 60 publications

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

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

Molecular Dynamics Simulations to Determine the Structure and Dynamics of Hepatitis B Virus Capsid Bound to a Novel Anti-viral Drug

Clinical Implications of the Molecular Biology of Hepatitis B Virus

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