N6 methyladenosine (m6A), the methylation of the adenosine base at the nitrogen-6 position, is the most common epitranscriptomic modification of mRNA that affects a wide variety of biological functions. We have previously reported that hepatitis B viral RNAs are m6A modified, displaying a dual functional role in the viral life cycle. Here, we show that cellular m6A machinery regulates host innate immunity against hepatitis B and C viral (HBV/HCV) infections by inducing m6A modification of viral transcripts. The depletion of the m6A writer enzymes (METTL3 and METTL14) leads to an increase in viral RNA recognition by retinoic acid-inducible gene I (RIG-I), thereby stimulating type-I interferon production. This is reversed in cells, in which m6A METTL3 and METTL14 are overexpressed. The m6A modification of viral RNAs renders RIG-I signaling less effective, and while single nucleotide mutation of m6A consensus motif of viral RNAs, enhances RIG-I sensing activity. Importantly, m6A reader proteins (YTHDF2 and YTHDF3) inhibit RIG-I transduced signaling activated by viral RNAs by occupying m6A modified RNAs and inhibiting RIG-I recognition. Collectively, our results provide new insights into the mechanism of immune evasion via m6A modification of viral RNAs.

Interferon (IFN) stimulates a whole repertoire of cellular genes, collectively referred to as ISGs (Interferon-stimulated genes). ISG20, a 3´-5´exonuclease enzyme, has been previously shown to bind and degrade hepatitis B Virus (HBV) transcripts. Here, we show that the N6-methyladenosine (m 6 A)-modified HBV transcripts are selectively recognized and processed for degradation by ISG20. Moreover, this effect of ISG20 is critically regulated by m 6 A reader protein, YTHDF2 (YTH-domain family 2). Previously, we identified a unique m 6 A site within HBV transcripts and confirmed that methylation at nucleotide A1907 regulates HBV lifecycle. In this report, we now show that the methylation at A1907 is a critical regulator of IFN-α mediated decay of HBV RNA. We observed that the HBV RNAs become less sensitive to ISG20 mediated degradation when methyltransferase enzymes or m 6 A reader protein YTHDF2 are silenced in HBV expressing cells. By using an enzymatically inactive form ISG20 D94G , we further demonstrated that ISG20 forms a complex with m 6 A modified HBV RNA and YTHDF2 protein. Due to terminal redundancy, HBV genomic nucleotide A1907 position is acquired twice by pregenomic RNA (pgRNA) during transcription and therefore the sites of methylation are encoded within 5´and 3´epsilon stem loops. We generated HBV mutants that lack m 6 A site at either one (5´or 3´) or both the termini (5´& 3´). Using these mutants, we demonstrated that m 6 A modified HBV RNAs are subjected to ISG20-mediated decay and propose sequence of events, in which ISG20 binds with YTHDF2 and recognizes m 6 A-modified HBV transcripts to carry out the ribonuclease activity. This is the first study, which identifies a hitherto unknown role of m 6 A modification of RNA in IFN-α induced viral RNA degradation and proposes a new role of YTHDF2 protein as a cofactor required for IFN-α mediated viral RNA degradation.

Two new potent anti-Gram negative compounds, coralmycins A (1) and B (2), were isolated from cultures of the myxobacteria Corallococcus coralloides M23, together with another derivative (3) that was identified as the very recently reported cystobactamid 919-2. Their structures including the relative stereochemistry were elucidated by interpretation of spectroscopic, optical rotation, and CD data. The relative stereochemistry of 3 was revised to "S*R*" by NMR analysis. The antibacterial activity of 1 was most potent against Gram-negative pathogens, including Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumanii, and Klebsiella pneumoniae, with MICs of 0.1-4 μg/mL; these MICs were 4-10 and 40-100 times stronger than the antibacterial activities of 3 and 2, respectively. Thus, these data indicated that the β-methoxyasparagine unit and the hydroxy group of the benzoic acid unit were critical for antibacterial activity.

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 (m6A) addition. HBV also affects m6A 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 m6A 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 m6A 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, m6A‐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 m6A 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 m6A modification of the host PTEN mRNAs.

Purpose Glioblastoma (GBM) inevitably recurs despite surgery, radiation and chemotherapy. A subpopulation of tumor cells, GBM stem cells (GSCs), has been implicated in this recurrence. The chemotherapeutic agent, etoposide is generally reserved for treating recurrent tumors, however its effectiveness is limited due to acute and cumulative toxicities to normal tissues. We investigate a novel combinatorial approach of low dose etoposide with an oncolytic HSV to enhance anti-tumor activity and limit drug toxicity. Experimental design In vitro, human GBM cell lines and GSCs were treated with etoposide alone, oHSV-G47Δ alone or the combination. Cytotoxic interactions were analyzed using the Chou-Talalay method and changes in caspase-dependent apoptosis and cell cycle were determined. In vivo, the most etoposide-resistant human GSC, BT74, was implanted intracranially and treated with either treatment alone or the combination. Analysis included effects on survival, therapy-associated adverse events and histological detection of apoptosis. Results GSCs varied in their sensitivity to etoposide by over 50-fold in vitro, while their sensitivity to G47Δ was similar. Combining G47Δ with low dose etoposide was moderately synergistic in GSCs and GBM cell lines. This combination did not enhance virus replication, but significantly increased apoptosis. In vivo, the combination of a single cycle of low dose etoposide with G47Δ significantly extended survival of mice bearing etoposide-insensitive intracranial human GSC-derived tumors. Conclusions The combination of low dose etoposide with G47Δ increases survival of mice bearing intracranial human GSC-derived tumors without adverse side effects. These results establish this as a promising combination strategy to treat resistant and recurrent GBM.