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 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 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 Polymerase Localizes to the Mitochondria, and Its Terminal Protein Domain Contains the Mitochondrial Targeting Signal

2016 ◽  
Vol 90 (19) ◽  
pp. 8705-8719 ◽  
Author(s):  
Nuruddin Unchwaniwala ◽  
Nathan M. Sherer ◽  
Daniel D. Loeb
Keyword(s):  
Amino Acids ◽  
Life Cycle ◽  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Dna Synthesis ◽  
Viral Life Cycle ◽  
Mitochondrial Targeting ◽  
Mitochondrial Localization ◽  
B Virus ◽  
Targeting Signal

ABSTRACTTo understand subcellular sites of hepatitis B virus (HBV) replication, we visualized core (Cp), polymerase (Pol), and pregenomic RNA (pgRNA) in infected cells. Interestingly, we found that the majority of Pol localized to the mitochondria in cells undergoing viral replication. The mitochondrial localization of Pol was independent of both the cell type and other viral components, indicating that Pol contains an intrinsic mitochondrial targeting signal (MTS). Neither Cp nor pgRNA localized to the mitochondria during active replication, suggesting a role other than DNA synthesis for Pol at the mitochondria. The Pol of duck hepatitis B virus (DHBV) also localized to the mitochondria. This result indicates that localization of Pol to mitochondria is likely a feature of all hepadnaviruses. To map the MTS within HBV Pol, we generated a series of Pol-green fluorescent protein (Pol-GFP) fusions and found that a stretch spanning amino acids (aa) 141 to 160 of Pol was sufficient to target GFP to the mitochondria. Surprisingly, deleting aa 141 to 160 in full-length Pol did not fully ablate Pol's mitochondrial localization, suggesting that additional sequences are involved in mitochondrial targeting. Only by deleting the N-terminal 160 amino acids in full-length Pol was mitochondrial localization ablated. Crucial residues for pgRNA packaging are contained within aa 141 to 160, indicating a multifunctional role of this region of Pol in the viral life cycle. Our studies show an unexpected Pol trafficking behavior that is uncoupled from its role in viral DNA synthesis.IMPORTANCEChronic infection by HBV is a serious health concern. Existing therapies for chronically infected individuals are not curative, underscoring the need for a better understanding of the viral life cycle to develop better antiviral therapies. To date, the most thoroughly studied function of Pol is to package the pgRNA and reverse transcribe it to double-stranded DNA within capsids. This study provides evidence for mitochondrial localization of Pol and defines the MTS. Recent findings have implicated a non-reverse transcription role for Pol in evading host innate immune responses. Mitochondria play an important role in controlling cellular metabolism, apoptosis, and innate immunity. Pol may alter one or more of these host mitochondrial functions to gain a replicative advantage and persist in chronically infected individuals.

scholarly journals Hepatitis B virus X protein is not central to the viral life cycle in vitro.

1992 ◽  
Vol 66 (2) ◽  
pp. 1223-1227 ◽  
Author(s):  
H E Blum ◽  
Z S Zhang ◽  
E Galun ◽  
F von Weizsäcker ◽  
B Garner ◽  
...  
Keyword(s):  
Life Cycle ◽  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Viral Life Cycle ◽  
X Protein ◽  
B Virus

scholarly journals Novel reporter system to monitor early stages of the hepatitis B virus life cycle

2015 ◽  
Vol 106 (11) ◽  
pp. 1616-1624 ◽  
Author(s):  
Hironori Nishitsuji ◽  
Saneyuki Ujino ◽  
Yuko Shimizu ◽  
Keisuke Harada ◽  
Jing Zhang ◽  
...  
Keyword(s):  
Life Cycle ◽  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Reporter System ◽  
Early Stages ◽  
Virus Life Cycle ◽  
B Virus

scholarly journals N6-methyladenosine modification of hepatitis B virus RNA differentially regulates the viral life cycle

2018 ◽  
Vol 115 (35) ◽  
pp. 8829-8834 ◽  
Author(s):  
Hasan Imam ◽  
Mohsin Khan ◽  
Nandan S. Gokhale ◽  
Alexa B. R. McIntyre ◽  
Geon-Woo Kim ◽  
...  
Keyword(s):  
Life Cycle ◽  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Reverse Transcription ◽  
Mutational Analysis ◽  
Regulatory Function ◽  
Messenger Rnas ◽  
Stem Loop ◽  
B Virus ◽  
A Site

N6-methyladenosine (m6A) RNA methylation is the most abundant epitranscriptomic modification of eukaryotic messenger RNAs (mRNAs). Previous reports have found m6A on both cellular and viral transcripts and defined its role in regulating numerous biological processes, including viral infection. Here, we show that m6A and its associated machinery regulate the life cycle of hepatitis B virus (HBV). HBV is a DNA virus that completes its life cycle via an RNA intermediate, termed pregenomic RNA (pgRNA). Silencing of enzymes that catalyze the addition of m6A to RNA resulted in increased HBV protein expression, but overall reduced reverse transcription of the pgRNA. We mapped the m6A site in the HBV RNA and found that a conserved m6A consensus motif situated within the epsilon stem loop structure, is the site for m6A modification. The epsilon stem loop is located in the 3′ terminus of all HBV mRNAs and at both the 5′ and 3′ termini of the pgRNA. Mutational analysis of the identified m6A site in the 5′ epsilon stem loop of pgRNA revealed that m6A at this site is required for efficient reverse transcription of pgRNA, while m6A methylation of the 3′ epsilon stem loop results in destabilization of all HBV transcripts, suggesting that m6A has dual regulatory function for HBV RNA. Overall, this study reveals molecular insights into how m6A regulates HBV gene expression and reverse transcription, leading to an increased level of understanding of the HBV life cycle.

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