The N6-methyladenosine demethylase ALKBH5 regulates the hypoxic HBV transcriptome.

Chronic hepatitis B is a global health problem and current treatments only suppress hepatitis B virus (HBV) infection, highlighting the need for new curative treatments. Oxygen levels influence HBV replication and we previously reported that hypoxia inducible factors (HIFs) activate the basal core promoter (BCP). Here we show that the hypoxic-dependent increase in BCP-derived transcripts is dependent on N6-methyladenosine (m6A) modifications in the 5' stem loop that regulate RNA half-life. Application of a probe-enriched long-read sequencing method to accurately map the HBV transcriptome showed an increased abundance of pre-genomic RNA under hypoxic conditions. Mapping the transcription start sites of BCP-RNAs identified a role for hypoxia to regulate pre-genomic RNA splicing that is dependent on m6A modification. Bioinformatic analysis of published single cell RNA-seq of murine liver showed an increased expression of the RNA demethylase ALKBH5 in the peri-central low oxygen region. In vitro studies with a human hepatocyte derived HepG2-NTCP cell line showed increased ALKBH5 gene expression under hypoxic conditions and a concomitant reduction in m6A-modified HBV BCP-RNA and host RNAs. Silencing the demethylase reduced the level of BCP-RNAs and host gene (CA9, NDRG1, VEGFA, BNIP3, FUT11, GAP and P4HA1) transcripts and this was mediated via reduced HIFα expression. In summary, our study highlights a previously unrecognized role for ALKBH5 in orchestrating viral and cellular transcriptional responses to low oxygen.

The N6-methyladenosine demethylase ALKBH5 regulates the hypoxic HBV transcriptome.

Chronic hepatitis B is a global health problem and current treatments only suppress hepatitis B virus (HBV) infection, highlighting the need for new curative treatments. Oxygen levels influence HBV replication and we previously reported that hypoxia inducible factors (HIFs) activate the basal core promoter to transcribe pre-genomic RNA. Application of a probe-enriched long-read sequencing method to map the HBV transcriptome showed an increased abundance of all viral RNAs under low oxygen or hypoxic conditions. Importantly, the hypoxic-associated increase in HBV transcripts was dependent on N6-methyladenosine (m6A) modifications and an m6A DRACH motif in the 5' stem loop of pre-genomic RNA defined transcript half-life under hypoxic conditions. Given the essential role of m6A modifications in the viral transcriptome we assessed the oxygen-dependent expression of RNA demethylases and bioinformatic analysis of published single cell RNA-seq of murine liver showed an increased expression of the RNA demethylase ALKBH5 in the peri-central low oxygen region. In vitro studies with a human hepatocyte derived HepG2 cell line showed increased ALKBH5 gene expression under hypoxic conditions. Silencing the demethylase reduced the levels of HBV pre-genomic RNA and host gene (CA9, NDRG1, VEGFA, BNIP3, FUT11, GAP and P4HA1) transcripts and this was mediated via reduced HIFα expression. In summary, our study highlights a previously unrecognized role for ALKBH5 in orchestrating viral and cellular transcriptional responses to low oxygen.

Improved epitaxial growth and multiferroic properties of Bi3Fe2Mn2Ox using CeO2 re-seeding layers.

In ferroelectric and multiferroic-based devices, it is often necessary to grow thicker films for enhanced properties. For certain phases that rely on substrate strain for growth, such thicker film growths beyond the typical thin film regime could be challenging. As an example, the Bi3Fe2Mn2Ox (BFMO) Aurivillius supercell (SC) phase possesses highly desirable multiferroic (i.e., ferromagnetic and ferroelectric) properties and a unique layered structure but relies heavily on substrate strain. Beyond the thin film regime (approximately 100 nm), a less desirable pseudo-cubic (PC) phase is formed. In this work, a novel heterogeneous re-seeding method is applied to maintain the strained growth in this SC phase beyond the thin film regime, thus enabling the growth of thick BFMO SC phase films. The insertion of periodic CeO2 interlayers reintroduces the heteroepitaxial strain and effectively re-initiates the growth of the SC phase. The thick BFMO SC phase maintains the overall multiferroic and interesting anisotropic optical properties, even exceeding those of the typical 100 nm SC film. This re-seeding method can be effectively adopted with other SC systems or strain-dependent thin films, thus introducing practical applications of the new SC phases without thickness limitations.

Vertical Etching of Scandium Aluminum Nitride Thin Films Using TMAH Solution.

A wide bandgap, an enhanced piezoelectric coefficient, and low dielectric permittivity are some of the outstanding properties that have made ScxAl1-xN a promising material in numerous MEMS and optoelectronics applications. One of the substantial challenges of fabricating ScxAl1-xN devices is its difficulty in etching, specifically with higher scandium concentration. In this work, we have developed an experimental approach with high temperature annealing followed by a wet etching process using tetramethyl ammonium hydroxide (TMAH), which maintains etching uniformity across various Sc compositions. The experimental results of etching approximately 730 nm of ScxAl1-xN (x = 0.125, 0.20, 0.40) thin films show that the etch rate decreases with increasing scandium content. Nevertheless, sidewall verticality of 85°~90° (±0.2°) was maintained for all Sc compositions. Based on these experimental outcomes, it is anticipated that this etching procedure will be advantageous in the fabrication of acoustic, photonic, and piezoelectric devices.

N6-Methyladenine Modification of Hepatitis Delta Virus Regulates Its Virion Assembly by Recruiting YTHDF1.

Hepatitis delta virus (HDV) is a defective satellite virus that uses hepatitis B virus (HBV) envelope proteins to form its virions and infect hepatocytes via the HBV receptors. Concomitant HDV/HBV infection continues to be a major health problem, with at least 25 million people chronically infected worldwide. N6-methyladenine (m6A) modification of cellular and viral RNAs is the most prevalent internal modification that occurs cotranscriptionally, and this modification regulates various biological processes. We have previously described a wider range of functional roles of m6A methylation of HBV RNAs, including its imminent regulatory role in the encapsidation of pregenomic RNA. In this study, we present evidence that m6A methylation also plays an important role in the HDV life cycle. Using the methylated RNA immunoprecipitation (MeRIP) assay, we identified that the intracellular HDV genome and antigenome are m6A methylated in HDV- and HBV-coinfected primary human hepatocytes and HepG2 cell expressing sodium taurocholate cotransporting polypeptide (NTCP), while the extracellular HDV genome is not m6A methylated. We observed that HDV genome and delta antigen levels are significantly decreased in the absence of METTL3/14, while the extracellular HDV genome levels are increased by depletion of METTL3/14. Importantly, YTHDF1, an m6A reader protein, interacts with the m6A-methylated HDV genome and inhibits the interaction between the HDV genome and antigens. Thus, m6A of the HDV genome negatively regulates virion production by inhibiting the interaction of the HDV genome with delta antigens through the recruitment of YTHDF1. This is the first study that provides insight into the functional roles of m6A in the HDV life cycle. IMPORTANCE The functional roles of N6-methyladenine (m6A) modifications in the HBV life cycle have been recently highlighted. Here, we investigated the functional role of m6A modification in the HDV life cycle. HDV is a subviral agent of HBV, as it uses HBV envelope proteins to form its virions. We found that m6A methylation also occurs in the intracellular HDV genome and antigenome but not in the extracellular HDV genome. The m6A modification of the HDV genome recruits m6A reader protein (YTHDF1) onto the viral genome. The association of YTHDF1 with the HDV genome abrogates the interaction of delta antigens with the HDV genome and inhibits virion assembly. This study describes the unique effects of m6A on regulation of the HDV life cycle.

High-Quality Dry Etching of LiNbO3 Assisted by Proton Substitution through H2-Plasma Surface Treatment.

The exceptional material properties of Lithium Niobate (LiNbO3) make it an excellent material platform for a wide range of RF, MEMS, phononic and photonic applications; however, nano-micro scale device concepts require high fidelity processing of LN films. Here, we reported a highly optimized processing methodology that achieves a deep etch with nearly vertical and smooth sidewalls. We demonstrated that Ti/Al/Cr stack works perfectly as a hard mask material during long plasma dry etching, where periodically pausing the etching and chemical cleaning between cycles were leveraged to avoid thermal effects and byproduct redeposition. To improve mask quality on X- and Y-cut substrates, a H2-plasma treatment was implemented to relieve surface tension by modifying the top surface atoms. Structures with etch depths as deep as 3.4 µm were obtained in our process across a range of crystallographic orientations with a smooth sidewall and perfect verticality on several crystallographic facets.

ZnO-AuxCu1-x Alloy and ZnO-AuxAl1-x Alloy Vertically Aligned Nanocomposites for Low-Loss Plasmonic Metamaterials.

Hyperbolic metamaterials are a class of materials exhibiting anisotropic dielectric function owing to the morphology of the nanostructures. In these structures, one direction behaves as a metal, and the orthogonal direction behaves as a dielectric material. Applications include subdiffraction imaging and hyperlenses. However, key limiting factors include energy losses of noble metals and challenging fabrication methods. In this work, self-assembled plasmonic metamaterials consisting of anisotropic nanoalloy pillars embedded into the ZnO matrix are developed using a seed-layer approach. Alloys of AuxAl1-x or AuxCu1-x are explored due to their lower losses and higher stability. Optical and microstructural properties were explored. The ZnO-AuxCu1-x system demonstrated excellent epitaxial quality and optical properties compared with the ZnO-AuxAl1-x system. Both nanocomposite systems demonstrate plasmonic resonance, hyperbolic dispersion, low losses, and epsilon-near-zero permittivity, making them promising candidates towards direct photonic integration.

N6-methyladenosine modification of the 5' epsilon structure of the HBV pregenome RNA regulates its encapsidation by the viral core protein.

Hepatitis B virus (HBV) contains a partially double-stranded DNA genome. During infection, its replication is mediated by reverse transcription (RT) of an RNA intermediate termed pregenomic RNA (pgRNA) within core particles in the cytoplasm. An epsilon structural element located in the 5' end of the pgRNA primes the RT activity. We have previously identified the N6-methyladenosine (m6A)-modified DRACH motif at 1905 to 1909 nucleotides in the epsilon structure that affects myriad functions of the viral life cycle. In this study, we investigated the functional role of m6A modification of the 5' ε (epsilon) structural element of the HBV pgRNA in the nucleocapsid assembly. Using the m6A site mutant in the HBV 5' epsilon, we present evidence that m6A methylation of 5' epsilon is necessary for its encapsidation. The m6A modification of 5' epsilon increased the efficiency of viral RNA packaging, whereas the m6A of 3' epsilon is dispensable for encapsidation. Similarly, depletion of methyltransferases (METTL3/14) decreased pgRNA and viral DNA levels within the core particles. Furthermore, the m6A modification at 5' epsilon of HBV pgRNA promoted the interaction with core proteins, whereas the 5' epsilon m6A site-mutated pgRNA failed to interact. HBV polymerase interaction with 5' epsilon was independent of m6A modification of 5' epsilon. This study highlights yet another pivotal role of m6A modification in dictating the key events of the HBV life cycle and provides avenues for investigating RNA-protein interactions in various biological processes, including viral RNA genome encapsidation in the context of m6A modification.

ZnO-ferromagnetic metal vertically aligned nanocomposite thin films for magnetic, optical and acoustic metamaterials.

Magnetoacoustic waves generated in piezoelectric and ferromagnetic coupled nanocomposite films through magnetically driven surface acoustic waves present great promise of loss-less data transmission. In this work, ferromagnetic metals of Ni, Co and Co x Ni1-x are coupled with a piezoelectric ZnO matrix in a vertically-aligned nanocomposite (VAN) thin film platform. Oxidation was found to occur in the cases of ZnO-Co, forming a ZnO-CoO VAN, while only very minor oxidation was found in the case of ZnO-Ni VAN. An alloy approach of Co x Ni1-x has been explored to overcome the oxidation during growth. Detailed microstructural analysis reveals limited oxidation of both metals and distinct phase separation between the ZnO and the metallic phases. Highly anisotropic properties including anisotropic ferromagnetic properties and hyperbolic dielectric functions are found in the ZnO-Ni and ZnO-Co x Ni1-x systems. The magnetic metal-ZnO-based hybrid metamaterials in this report present great potential in coupling of optical, magnetic, and piezoelectric properties towards future magnetoacoustic wave devices.

Hepatitis B Virus X Protein Expression Is Tightly Regulated by N6-Methyladenosine Modification of Its mRNA.

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 nucleotides (nt) 1905 to 1909 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 located within nt 1606 to 1809 correspond to the coding region of HBx mRNA and 3' untranslated region (UTR) of other viral mRNAs. Using the mutants of additional m6A sites in nt 1606 to 1809 and a depletion strategy of m6A methyltransferases (METTL3/14) and reader proteins (YTHDFs), we show that m6A modification at nt 1616, located in the HBx coding region, regulates HBx protein expression. The HBx RNA and protein expression levels 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 expression levels were not affected by the m6A modification at nt 1616. 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 recently have been implicated in the HBV life cycle. Previously, we observed that m6A modification occurs in the adenosine at nt 1907 of the HBV genome, and this modification regulates the viral life cycle. Here, we identified an additional m6A site located in nt 1616 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 m6A in the regulation of HBx protein expression.

Monitoring Mitochondrial Function in Aedes albopictus C6/36 Cell Line during Dengue Virus Infection.

Aedes aegypti and Aedes albopictus mosquitoes are responsible for dengue virus (DENV) transmission in tropical and subtropical areas worldwide, where an estimated 3 billion people live at risk of DENV exposure. DENV-infected individuals show symptoms ranging from sub-clinical or mild to hemorrhagic fever. Infected mosquitoes do not show detectable signs of disease, even though the virus maintains a lifelong persistent infection. The interactions between viruses and host mitochondria are crucial for virus replication and pathogenicity. DENV infection in vertebrate cells modulates mitochondrial function and dynamics to facilitate viral proliferation. Here, we describe that DENV also regulates mitochondrial function and morphology in infected C6/36 mosquito cells (derived from Aedes albopictus). Our results showed that DENV infection increased ROS (reactive oxygen species) production, modulated mitochondrial transmembrane potential and induced changes in mitochondrial respiration. Furthermore, we offer the first evidence that DENV causes translocation of mitofusins to mitochondria in the C6/36 mosquito cell line. Another protein Drp-1 (Dynamin-related protein 1) did not localize to mitochondria in DENV-infected cells. This observation therefore ruled out the possibility that the abovementioned alterations in mitochondrial function are associated with mitochondrial fission. In summary, this report provides some key insights into the virus-mitochondria crosstalk in DENV infected mosquito cells.

Bandwidth extension and conversion efficiency improvements beyond phase matching limitations using cavity-enhanced OPCPA.

The conversion efficiency and phase matching bandwidth of ultrafast optical parametric amplification (OPA) are constrained by the dispersion and nonlinear coefficient of the employed crystal as well as pulse shaping effects. In our work we show that an enhancement cavity resonant with the pump seeded at the full repetition rate of the pump laser can automatically reshape the small-signal gain in optical parametric chirped-pulse amplification (OPCPA) to achieve close-to-optimal operation. This new method termed cavity-enhanced OPCPA or C-OPCPA significantly increases both the gain bandwidth and the conversion efficiency, in addition to boosting gain for high-repetition-rate amplification. The goal in C-OPCPA is to arrive at a condition of impedance matching at all temporal coordinates, such that, in the absence of linear losses, all the incident pump power is dissipated in the nonlinear loss element, i.e., converted to signal and idler. The use of a low finesse enhancement cavity resonant with a low average power (<1W) and a high repetition rate (78MHz) pump source is shown to achieve more than 50% conversion efficiency into signal and idler from the coupled pump in an optical parametric process, whereas an equivalent amount of pump power in a single-pass configuration leads to negligible conversion. Additionally, the gain bandwidth is extended by a factor of 3-4 beyond the phase-matching limit. Our empirical observations are corroborated by a numerical analysis of depletion optimizing the single-pass case, which assesses the underlying impedance matching that is responsible for the observed performance improvements.

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.

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 positively affects pgRNA encapsidation in the cytoplasm. Thus, it plays a differential dual role in the virus 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 in the nucleus to reduce reverse transcription in 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.

The role of N6-methyladenosine modification in the life cycle and disease pathogenesis of hepatitis B and C viruses.

N6-methyladenosine (m6A) is the most prevalent modification of mammalian cellular RNAs. m6A methylation is linked to epigenetic regulation of several aspects of gene expression, including RNA stability, splicing, nuclear export, RNA folding, and translational activity. m6A modification is reversibly catalyzed by methyltransferases (m6A writers) and demethylases (m6A erasers), and the dynamics of m6A-modified RNA are regulated by m6A-binding proteins (m6A readers). Recently, several studies have shown that m6A methylation sites have been identified in hepatitis B virus (HBV) transcripts and the hepatitis C virus (HCV) RNA genome. Here, we review the role of m6A modification in HBV/HCV replication and its contribution to liver disease pathogenesis. A better understanding of the functions of m6A methylation in the life cycles of HBV and HCV is required to establish the role of these modifications in liver diseases associated with these viral infections.

N6-methyladenosine modification of HCV RNA genome regulates cap-independent IRES-mediated translation via YTHDC2 recognition.

Hepatitis C virus (HCV) infections are associated with the risk of progression to fibrosis, cirrhosis, and hepatocellular carcinoma. The HCV RNA genome is translated by an internal ribosome entry site (IRES)-dependent mechanism. The structure and function of the HCV IRES have been investigated by both biological and biophysical criteria. Recently, the role of N6-methyladenosine (m6A) in cellular RNA and viral transcripts has been intensely investigated. The HCV RNA genome is m6A-methylated, and this modification regulates the viral life cycle. In this study, we investigated the role of m6A modification of the HCV genome in the IRES-dependent translation function by mutating m6A consensus motifs (DRACH) within the IRES element in stem-loop III and IV regions and studied their effect on translation initiation. There are several DRACH motifs within the IRES element. Of these, the DRACH motif at nucleotide (nt) 329-333, located about 7 nt upstream of initiator AUG (iAUG) codon, regulates IRES-mediated translation initiation. Mutational analysis showed that m6A methylation of the adenosine at nt 331 is essential for the IRES-dependent translation. m6A reader protein YTHDC2, containing the RNA helicase domain, recognizes m6A-methylated adenosine at nt 331 and, in concert with the cellular La antigen, supports HCV IRES-dependent translation. The RNA helicase dead YTHDC2 (E332Q) mutant failed to stimulate HCV translation initiation. This report highlights the functional roles of m6A modification and YTHDC2 in the HCV IRES-dependent translation initiation, thus offering alternative therapeutic avenues to interfere with the infectious process.

Hepatitis B virus X protein recruits methyltransferases to affect cotranscriptional N6-methyladenosine modification of viral/host RNAs.

Chronic hepatitis B virus (HBV) infections are one of the leading causes of cirrhosis and hepatocellular carcinoma. N6-methyladenosine (m6A) modification of cellular and viral RNAs is the most prevalent internal modification that occurs cotranscriptionally. Previously, we reported the dual functional role of m6A modification of HBV transcripts in the viral life cycle. Here, we show that viral HBV X (HBx) protein is responsible for the m6A modifications of viral transcripts. HBV genomes defective in HBx failed to induce m6A modifications of HBV RNAs during infection/transfection, while ectopic expression of HBx restores m6A modifications of the viral RNAs but not the mutant HBx carrying the nuclear export signal. Using chromatin immunoprecipitation assays, we provide evidence that HBx and m6A methyltransferase complexes are localized on the HBV minichromosome to achieve cotranscriptional m6A modification of viral RNAs. HBx interacts with METTL3 and 14 to carry out methylation activity and also modestly stimulates their nuclear import. This role of HBx in mediating m6A modification also extends to host phosphatase and tensin homolog (PTEN) mRNA. This study provides insight into how a viral protein recruits RNA methylation machinery to m6A-modify RNAs.

Deposition pressure-induced microstructure control and plasmonic property tuning in hybrid ZnO-Ag x Au1-x thin films.

Self-assembled oxide-metallic alloy nanopillars as hybrid plasmonic metamaterials (e.g., ZnO-Ag x Au1-x ) in a thin film form have been grown using a pulsed laser deposition method. The hybrid films were demonstrated to be highly tunable via systematic tuning of the oxygen background pressure during deposition. The pressure effects on morphology and optical properties have been investigated and found to be critical to the overall properties of the hybrid films. Specifically, low background pressure results in the vertically aligned nanocomposite (VAN) form while the high-pressure results in more lateral growth of the nanoalloys. Strong surface plasmon resonance was observed in the UV-vis region and a hyperbolic dielectric function was achieved due to the anisotropic morphology. The oxide-nanoalloy hybrid material grown in this work presents a highly effective approach for tuning the binary nanoalloy morphology and properties through systematic parametric changes, important for their potential applications in integrated photonics and plasmonics such as sensors, energy harvesting devices, and beyond.

Hibernoma Around Shoulder Joint: Diagnosis and Surgical Treatment of a Rare Presentation.

Introduction: Hibernomas are uncommon benign proliferations arising from the adipocytes of brown fat usually occurring over back, neck, shoulder, and thighs. It is an underreported entity often misdiagnosed as a lipoma. Case Report: We report a case of an otherwise healthy thirty-four-year-old male who presented with a painless swelling, with no preceding trauma, involving the right shoulder joint of three months duration. MRI revealed it to be a fatty intense lesion arising within the anterior fibers of the right deltoid. The tumor was removed en-masse with wide local excision through the deltopectoral route. The histopathology of the resected tumor was consistent with the diagnosis of hibernoma. The patient recovered the previously hindered movements at the shoulder joint 2 weeks post-operatively. Conclusion: Hibernoma should be considered as a differential diagnosis for soft tissue tumors occurring in the above-mentioned sites. It can be completely resected surgically and no recurrences post-excision have been reported till date.

HBV-Induced Increased N6 Methyladenosine Modification of PTEN RNA Affects Innate Immunity and Contributes to HCC.

BACKGROUND AND AIMS: Epitranscriptomic modification of RNA has emerged as the most prevalent form of regulation of gene expression that affects development, differentiation, metabolism, viral infections, and most notably cancer. We have previously shown that hepatitis B virus (HBV) transcripts are modified by N6 methyladenosine (m6 A) addition. HBV also affects m6 A modification of several host RNAs, including phosphatase and tensin homolog (PTEN), a well-known tumor suppressor. PTEN plays a critical role in antiviral innate immunity and the development of hepatocellular carcinoma (HCC). Reports have shown that PTEN controlled interferon regulatory factor 3 (IRF-3) nuclear localization by negative phosphorylation of IRF-3 at Ser97, and PTEN reduced carcinogenesis by inhibiting the phosphatidylinositol-3-kinase (PI3K)/AKT pathway. APPROACH AND RESULTS: Here, we show that HBV significantly increases the m6 A modification of PTEN RNA, which contributes to its instability with a corresponding decrease in PTEN protein levels. This is reversed in cells in which the expression of m6 A methyltransferases is silenced. PTEN expression directly increases activated IRF-3 nuclear import and subsequent interferon synthesis. In the absence of PTEN, IRF-3 dephosphorylation at the Ser97 site is decreased and interferon synthesis is crippled. In chronic HBV patient biopsy samples, m6 A-modified PTEN mRNA levels were uniformly up-regulated with a concomitant decrease of PTEN mRNA levels. HBV gene expression also activated the PI3K/AKT pathway by regulating PTEN mRNA stability in HCC cell lines. CONCLUSIONS: The m6 A epitranscriptomic regulation of PTEN by HBV affects innate immunity by inhibiting IRF-3 nuclear import and the development of HCC by activating the PI3K/AKT pathway. Our studies collectively provide new insights into the mechanisms of HBV-directed immune evasion and HBV-associated hepatocarcinogenesis through m6 A modification of the host PTEN mRNAs.