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.

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.

Expression of an enzymatically active polymerase of human hepatitis B virus in a coupled transcription-translation system

1999 ◽  
Vol 77 (2) ◽  
pp. 119-126 ◽  
Author(s):  
Zhi Li ◽  
D Lorne J. Tyrrell
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Reverse Transcription ◽  
Translation System ◽  
Stem Loop ◽  
Human Hepatitis ◽  
B Virus ◽  
Reticulocyte Lysate ◽  
Protein Priming

Genome replication of hepadnavirus proceeds by reverse transcription from a viral pregenomic RNA template by a virally encoded polymerase that possesses protein-priming, reverse transcriptase, DNA polymerase, and RNase H activities. Characterization of this enzyme has been hampered by failure to purify an active enzyme from virions and difficulties in expressing an active polymerase in heterologous systems. In this study, we constructed human hepatitis B virus polymerase cDNA under the control of a phage T7 promoter and expressed it in a rabbit reticulocyte lysate-coupled transcription-translation system. In vitro site-directed mutagenesis confirmed that the recombinant polymerase cDNA produced three products: a full-length protein (~94 kDa), an internally initiated protein (~81 kDa), and an N-terminal protein (~40 kDa). The in vitro expressed polymerase possessed protein priming activity, as demonstrated by32P-dGTP-labeling of the full size polymerase and the N-terminal portion of the molecule in an in vitro priming assay. The polymerase also exhibited polymerization activity, as detected in an in vitro polymerase assay by incorporation of radionucleotides into acid-precipitable polynucleotides and by synthesis of human hepatitis B virus (HBV) specific DNA with product lengths between 100 and 500 nucleotides. In addition, the polymerase was found to use an RNA sequence bearing HBV DR1/epsilon stem-loop motif as a template for DNA synthesis. Both the protein-priming and the reverse transcription activities of this recombinant polymerase are dependent on the RNA fragment containing the HBV DR1/epsilon stem-loop sequence known to be required for the polymerase activities. The in vitro systems used in this study will be applicable to further functional and biochemical studies of this enzyme.Key words: hepadnavirus, HBV polymerase, protein priming, reverse transcription, rabbit reticulocyte lysate.

Regulatory Function of Interferon‐Inducible 44 Like for Hepatitis B Virus cccDNA in Primary Human Hepatocytes

2021 ◽  
Author(s):  
Takuto Nosaka ◽  
Tatsushi Naito ◽  
Yosuke Murata ◽  
Hidetaka Matsuda ◽  
Masahiro Ohtani ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Human Hepatocytes ◽  
Regulatory Function ◽  
Primary Human Hepatocytes ◽  
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 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 Complete Genome Sequence of a Precore-Defective Hepatitis B Virus Genotype D2 Strain Isolated in Bangladesh

2020 ◽  
Vol 9 (11) ◽  
Author(s):  
Md Golzar Hossain ◽  
Md Muket Mahmud ◽  
Md Arifur Rahman ◽  
Sharmin Akter ◽  
K. H. M. Nazmul Hussain Nazir ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Nucleotide Sequence ◽  
Hepatitis B ◽  
Complete Genome Sequence ◽  
Complete Nucleotide Sequence ◽  
Mutational Analysis ◽  
Limited Information ◽  
Virus Genotype ◽  
Hbv Genotype ◽  
B Virus

Hepatitis B virus (HBV) genomic mutations affect viral replication, disease progression, and diagnostic and vaccination efficiency. There is limited information regarding characterization and mutational analysis of HBV isolated in Bangladesh. Here, we report the complete nucleotide sequence of a precore-defective HBV genotype D2 strain isolated in Bangladesh.

Sign in / Sign up

Export Citation Format

Share Document