Hepatitis B virus X (HBx) Protein Expression is Tightly Regulated by N6-methyladenosine Modification of its mRNA

2021 ◽  
Author(s):  
Geon-Woo Kim ◽  
Aleem Siddiqui
Keyword(s):  
Life Cycle ◽  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Protein Expression ◽  
Regulatory Protein ◽  
Coding Region ◽  
Structural Element ◽  
Viral Mrnas ◽  
B Virus ◽  
Protein Expressions

Hepatitis B virus (HBV) encodes a regulatory protein termed HBx, that has been intensely studied in the past and shown to play a key role(s) in viral transcription and replication. In addition, a huge body of work exists in the literature related to signal transduction and possible mechanism(s) leading to hepatocarcinogenesis associated with infection. We have previously reported that HBV transcripts are modified by N6-methyladenosine (m6A) at the single consensus DRACH motif at 1905-1909 nucleotide (nt) in the epsilon structural element and this m6A modification affects the HBV life cycle. In this study, we present evidence that additional variants of m6A (DRACH) motifs are located within 1606 to 1809 nt correspond on the coding region of HBx mRNA and 3′ untranslated region (UTR) of other viral mRNAs. Using the mutants of additional m6A site in 1606 to 1809 nt and a depletion strategy of m6A methyltransferases (METTL3/14) and reader proteins (YTHDFs), we show that m6A modification at 1616 nt, located in HBx coding region, regulates HBx protein expression. The HBx RNA and protein expressions were notably increased by the silencing of m6A reader YTHDF2 and methyltransferases as well as the mutation of m6A sites in the HBx coding region. However, other viral protein expressions were not affected by the m6A modification at 1616 nt. Thus, m6A modifications in the HBx open reading frame (ORF), downregulate HBx protein expression, commonly seen during HBV transfections, transgenic mice, and natural infections of human hepatocytes. These studies identify the functional role of m6A modification in the subtle regulation of HBx protein expression consistent with its possible role in establishing chronic hepatitis. Importance N6-methyladenosien (m6A) modifications have been recently implicated in the HBV life cycle. Previously, we observed that m6A modification occurs in the adenosine at 1907 nt of HBV genome and this modification regulates the viral life cycle. Here, we identified an additional m6A site located in 1616 nt of the HBV genome. This modification negatively affects HBx RNA and protein expression. In the absence of m6A methyltransferases (METTL3/14) and reader protein (YTHDF2), the HBx RNA and protein expression were increased. Using HBV mutants that lack m6A in the HBx coding region, we present the unique positional effects of m 6 A in the regulation of HBx protein expression.

scholarly journals Role of NF-κB and Myc Proteins in Apoptosis Induced by Hepatitis B Virus HBx Protein

2001 ◽  
Vol 75 (1) ◽  
pp. 215-225 ◽  
Author(s):  
Fei Su ◽  
Christian N. Theodosis ◽  
Robert J. Schneider
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Apoptotic Cell ◽  
Regulatory Protein ◽  
Family Members ◽  
Necrosis Factor Alpha ◽  
Tnf Α ◽  
B Virus ◽  
High Level

ABSTRACT Chronic infection with hepatitis B virus (HBV) promotes a high level of liver disease and cancer in humans. The HBV HBx gene encodes a small regulatory protein that is essential for viral replication and is suspected to play a role in viral pathogenesis. HBx stimulates cytoplasmic signal transduction pathways, moderately stimulates a number of transcription factors, including several nuclear factors, and in certain settings sensitizes cells to apoptosis by proapoptotic stimuli, including tumor necrosis factor alpha (TNF-α) and etopocide. Paradoxically, HBx activates members of the NF-κB transcription factor family, some of which are antiapoptotic in function. HBx induces expression of Myc protein family members in certain settings, and Myc can sensitize cells to killing by TNF-α. We therefore examined the roles of NF-κB, c-Myc, and TNF-α in apoptotic killing of cells by HBx. RelA/NF-κB is shown to be induced by HBx and to suppress HBx-mediated apoptosis. HBx also induces c-Rel/NF-κB, which can promote apoptotic cell death in some contexts or block it in others. Induction of c-Rel by HBx was found to inhibit its ability to directly mediate apoptotic killing of cells. Thus, HBx induction of NF-κB family members masks its ability to directly mediate apoptosis, whereas ablation of NF-κB reveals it. Investigation of the role of Myc protein demonstrates that overexpression of Myc is essential for acute sensitization of cells to killing by HBx plus TNF-α. This study therefore defines a specific set of parameters which must be met for HBx to possibly contribute to HBV pathogenesis.

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 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 Inhibition of interferon-inducible MxA protein expression by hepatitis B virus capsid protein.

1999 ◽  
Vol 80 (5) ◽  
pp. 1253-1262 ◽  
Author(s):  
O Rosmorduc ◽  
H Sirma ◽  
P Soussan ◽  
E Gordien ◽  
P Lebon ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Protein Expression ◽  
Capsid Protein ◽  
Virus Capsid ◽  
B Virus ◽  
Virus Capsid Protein

Infectious Hepatitis B Virus Variant Defective in Pre-S2 Protein Expression in a Chronic Carrier

Virology ◽  
1993 ◽  
Vol 194 (1) ◽  
pp. 137-148 ◽  
Author(s):  
Doris Fernholz ◽  
Peter R. Galle ◽  
Marietta Stemler ◽  
Maurizia Brunetto ◽  
Ferruccio Bonino ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Protein Expression ◽  
Infectious Hepatitis ◽  
Virus Variant ◽  
B Virus ◽  
Chronic Carrier
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