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

2016 ◽  
Vol 90 (8) ◽  
pp. 3994-4004 ◽  
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.

scholarly journals Hepatitis B Virus-Specific miRNAs and Argonaute2 Play a Role in the Viral Life Cycle

PLoS ONE ◽  
2012 ◽  
Vol 7 (10) ◽  
pp. e47490 ◽  
Author(s):  
C. Nelson Hayes ◽  
Sakura Akamatsu ◽  
Masataka Tsuge ◽  
Daiki Miki ◽  
Rie Akiyama ◽  
...  
Keyword(s):  
Life Cycle ◽  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Viral Life Cycle ◽  
B Virus

scholarly journals Antiviral Properties and Mechanism of Action Studies of the Hepatitis B Virus Capsid Assembly Modulator JNJ-56136379

2020 ◽  
Vol 64 (5) ◽  
Author(s):  
Jan Martin Berke ◽  
Pascale Dehertogh ◽  
Karen Vergauwen ◽  
Wendy Mostmans ◽  
Koen Vandyck ◽  
...  
Keyword(s):  
Life Cycle ◽  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Core Protein ◽  
Hbv Dna ◽  
Size Exclusion ◽  
Capsid Assembly ◽  
Phase Ii Trials ◽  
B Virus

ABSTRACT Capsid assembly is a critical step in the hepatitis B virus (HBV) life cycle, mediated by the core protein. Core is a potential target for new antiviral therapies, the capsid assembly modulators (CAMs). JNJ-56136379 (JNJ-6379) is a novel and potent CAM currently in phase II trials. We evaluated the mechanisms of action (MOAs) and antiviral properties of JNJ-6379 in vitro. Size exclusion chromatography and electron microscopy studies demonstrated that JNJ-6379 induced the formation of morphologically intact viral capsids devoid of genomic material (primary MOA). JNJ-6379 accelerated the rate and extent of HBV capsid assembly in vitro. JNJ-6379 specifically and potently inhibited HBV replication; its median 50% effective concentration (EC50) was 54 nM (HepG2.117 cells). In HBV-infected primary human hepatocytes (PHHs), JNJ-6379, when added with the viral inoculum, dose-dependently reduced extracellular HBV DNA levels (median EC50 of 93 nM) and prevented covalently closed circular DNA (cccDNA) formation, leading to a dose-dependent reduction of intracellular HBV RNA levels (median EC50 of 876 nM) and reduced antigen levels (secondary MOA). Adding JNJ-6379 to PHHs 4 or 5 days postinfection reduced extracellular HBV DNA and did not prevent cccDNA formation. Time-of-addition PHH studies revealed that JNJ-6379 most likely interfered with postentry processes. Collectively, these data demonstrate that JNJ-6379 has dual MOAs in the early and late steps of the HBV life cycle, which is different from the MOA of nucleos(t)ide analogues. JNJ-6379 is in development for chronic hepatitis B treatment and may translate into higher HBV functional cure rates.

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

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

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

2018 ◽  
Vol 92 (20) ◽  
Author(s):  
Lu Ruan ◽  
Jodi A. Hadden ◽  
Adam Zlotnick
Keyword(s):  
Molecular Dynamics ◽  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Core Protein ◽  
Dynamics Simulation ◽  
Single Atom ◽  
Allosteric Modulators ◽  
Hydrophobic Contact ◽  
B Virus ◽  
Capsid Stability

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.

scholarly journals The RNA binding proteins YTHDC1 and FMRP regulate the nuclear export of N6-methyladenosine modified Hepatitis B Virus transcripts and affect the viral life cycle

2021 ◽  
Author(s):  
Geon-Woo Kim ◽  
Hasan Imam ◽  
Aleem Siddiqui
Keyword(s):  
Life Cycle ◽  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Reverse Transcription ◽  
Nuclear Export ◽  
Binding Proteins ◽  
Rna Binding ◽  
Viral Life Cycle ◽  
Circular Dna ◽  
B Virus

YTHDC1 and fragile X mental retardation protein (FMRP) bind N6-methyladenosine (m6A) modified RNAs and facilitate their transport to the cytoplasm. Here, we investigated the role of these proteins in Hepatitis B virus (HBV) gene expression and life cycle. We have previously reported that HBV transcripts are m6A-methylated and this modification regulates the viral life cycle. HBV is particularly interesting as its DNA genome upon transcription gives rise to a pregenomic RNA (pgRNA), which serves as a template for reverse transcription to produce the relaxed circular DNA that transforms into a covalently closed circular DNA (cccDNA). While m6A modification negatively affects RNA stability and translation of viral transcripts, our current results revealed the possibility that it may positively affect pgRNA encapsidation in the cytoplasm. Thus, it plays a differential dual role in the viral life cycle. YTHDC1 as well as FMRP recognize m6A-methylated HBV transcripts and facilitate their transport to the cytoplasm. In cells depleted with YTHDC1 or FMRP, viral transcripts accumulate in the nucleus to affect the viral life cycle. Most importantly, the core-associated DNA and subsequent cccDNA syntheses are dramatically affected in FMRP or YTHDC1-silenced cells. This study highlights the functional relevance of YTHDC1 and FMRP in the HBV life cycle with the potential to arrest liver disease pathogenesis. IMPORTANCE YTHDC1 and FMRP have been recently implicated in the nuclear export of m6A modified mRNAs. Here, we show that FMRP and YTHDC1 proteins bind with m6A-modified HBV transcripts and facilitate their nuclear export. In the absence of FMRP and YTHDC1, HBV transcripts accumulate inside the nucleus to reduce reverse transcription inside HBV core particles and subsequently the cccDNA synthesis. Our study shows how m6A binding proteins can regulate the HBV life cycle by facilitating the nuclear export of m6A-modified HBV RNA.

Viral life cycle of hepatitis B virus: Host factors and druggable targets

2016 ◽  
Vol 46 (9) ◽  
pp. 871-877 ◽  
Author(s):  
Kenichi Morikawa ◽  
Goki Suda ◽  
Naoya Sakamoto
Keyword(s):  
Life Cycle ◽  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Viral Life Cycle ◽  
Host Factors ◽  
B Virus ◽  
Druggable Targets

scholarly journals Capsid Assembly Modulators Have a Dual Mechanism of Action in Primary Human Hepatocytes Infected with Hepatitis B Virus

2017 ◽  
Vol 61 (8) ◽  
Author(s):  
Jan Martin Berke ◽  
Pascale Dehertogh ◽  
Karen Vergauwen ◽  
Ellen Van Damme ◽  
Wendy Mostmans ◽  
...  
Keyword(s):  
Life Cycle ◽  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Mechanism Of Action ◽  
Viral Life Cycle ◽  
Human Hepatocytes ◽  
Capsid Assembly ◽  
B Virus ◽  
Dual Mechanism ◽  
Dose Dependent

ABSTRACT Hepatitis B virus (HBV) capsid assembly is a critical step in the propagation of the virus and is mediated by the core protein. Due to its multiple functions in the viral life cycle, core became an attractive target for new antiviral therapies. Capsid assembly modulators (CAMs) accelerate the kinetics of capsid assembly and prevent encapsidation of the polymerase-pregenomic RNA (Pol-pgRNA) complex, thereby blocking viral replication. CAM JNJ-632 is a novel and potent inhibitor of HBV replication in vitro across genotypes A to D. It induces the formation of morphologically intact viral capsids, as demonstrated by size exclusion chromatography and electron microscopy studies. Antiviral profiling in primary human hepatocytes revealed that CAMs prevented formation of covalently closed circular DNA in a dose-dependent fashion when the compound was added together with the viral inoculum, whereas nucleos(t)ide analogues (NAs) did not. This protective effect translated into a dose-dependent reduction of intracellular HBV RNA levels as well as reduced HBe/cAg and HBsAg levels in the cell culture supernatant. The same observation was made with another CAM (BAY41-4109), suggesting that mechanistic rather than compound-specific effects play a role. Our data show that CAMs have a dual mechanism of action, inhibiting early and late steps of the viral life cycle. These effects clearly differentiate CAMs from NAs and may translate into higher functional cure rates in a clinical setting when given alone or in combination with the current standard of care.

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

eLife ◽  
2018 ◽  
Vol 7 ◽  
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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