RESUMO
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.
RESUMO
The COVID-19 pandemic, caused by SARS-CoV-2 coronavirus, is a global health issue with unprecedented challenges for public health. SARS-CoV-2 primarily infects cells of the respiratory tract, via Spike glycoprotein binding angiotensin-converting enzyme (ACE2). Circadian rhythms coordinate an organismâ™s response to its environment and can regulate host susceptibility to virus infection. We demonstrate a circadian regulation of ACE2 in lung epithelial cells and show that silencing BMAL1 or treatment with a synthetic REV-ERB agonist SR9009 reduces ACE2 expression and inhibits SARS-CoV-2 entry. Treating infected cells with SR9009 limits viral replication and secretion of infectious particles, showing that post-entry steps in the viral life cycle are influenced by the circadian system. Transcriptome analysis revealed that Bmal1 silencing induced a wide spectrum of interferon stimulated genes in Calu-3 lung epithelial cells, providing a mechanism for the circadian pathway to dampen SARS-CoV-2 infection. Our study suggests new approaches to understand and improve therapeutic targeting of SARS-CoV-2.
RESUMO
Chronic hepatitis B is one of the world's unconquered diseases with more than 240 million infected subjects at risk of developing liver disease and hepatocellular carcinoma. Hepatitis B virus reverse transcribes pre-genomic RNA to relaxed circular DNA (rcDNA) that comprises the infectious particle. To establish infection of a naïve target cell, the newly imported rcDNA is repaired by host enzymes to generate covalently closed circular DNA (cccDNA), which forms the transcriptional template for viral replication. SAMHD1 is a component of the innate immune system that regulates deoxyribonucleoside triphosphate levels required for host and viral DNA synthesis. Here, we show a positive role for SAMHD1 in regulating cccDNA formation, where KO of SAMHD1 significantly reduces cccDNA levels that was reversed by expressing wild-type but not a mutated SAMHD1 lacking the nuclear localization signal. The limited pool of cccDNA in infected Samhd1 KO cells is transcriptionally active, and we observed a 10-fold increase in newly synthesized rcDNA-containing particles, demonstrating a dual role for SAMHD1 to both facilitate cccDNA genesis and to restrict reverse transcriptase-dependent particle genesis.