A dual role for SAMHD1 in regulating HBV cccDNA and RT-dependent particle genesis

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

Characterization of the envelope glycoproteins of the viral variants involved in the HCV transmission

Les variants viraux impliqués dans la transmission du VHC ont rarement été étudiés en raison des difficultés rencontrées pour recruter des patients au stade de la primo-infection. Pour mener cette étude, nous avons analysé le goulot d’étranglement génétique subit par les quasi-espèces virales au cours de 3 accidents d’exposition au sang impliquant des représentants du personnel soignant contaminés par piqûre d’aiguille. En utilisant la technique d’amplification de génomes uniques nous avons obtenu les gènes codant les glycoprotéines d’enveloppe virales E1E2 des variants viraux présents dans ces quasi-espèces. Ces gènes ont été séquencés et soumis à une analyse phylogénétique. Nous avons ensuite pu étudier les propriétés phénotypiques des glycoprotéines d’enveloppe dérivées de variants qui apparaissent au stade très précoce de l’infection. Pendant cette période, le VHC pourrait être plus vulnérable à l’élimination par des vaccins préventifs ou par des immunothérapies.Little is known about the transmitted variants responsible for the spread of HCV infection, principally because of the difficulties to recruit patients early enough in infection. To address this issue, we proposed to track the genetic bottleneck event in HCV quasispecies, leading to productive clinical infection in three health care workers accidentally contaminated through needlestick accidents. By using a single genome amplification (SGA) approach we identified genes coding the viral envelope glycoprotein E1E2 which composed these quasispecies. The E1E2 sequences were then directly sequenced and subjected to a phylogenetic analysis. By cloning these full-length E1E2 sequences, we investigated the phenotypic properties of the envelope glycoproteins potentially involved in selective HCV transmission and early stage of infection, a period during which the virus might be most vulnerable to elimination by preventive vaccines or immunotherapies

Hypoxic gene expression in chronic hepatitis B virus infected patients is not observed in state-of-art in vitro and mouse infection models

Hepatitis B virus (HBV) is the leading cause of hepatocellular carcinoma (HCC) worldwide. The prolyl hydroxylase domain (PHD)-hypoxia inducible factor (HIF) pathway is a key mammalian oxygen sensing pathway and is frequently perturbed by pathological states including infection and inflammation. We discovered a significant upregulation of hypoxia regulated gene transcripts in patients with chronic hepatitis B (CHB) in the absence of liver cirrhosis. We used state-of-the-art in vitro and in vivo HBV infection models to evaluate a role for HBV infection and the viral regulatory protein HBx to drive HIF-signalling. HBx had no significant impact on HIF expression or associated transcriptional activity under normoxic or hypoxic conditions. Furthermore, we found no evidence of hypoxia gene expression in HBV de novo infection, HBV infected human liver chimeric mice or transgenic mice with integrated HBV genome. Collectively, our data show clear evidence of hypoxia gene induction in CHB that is not recapitulated in existing models for acute HBV infection, suggesting a role for inflammatory mediators in promoting hypoxia gene expression

Genetic, clinical and radiographic signs in knee osteoarthritis susceptibility

Introduction. Osteoarthritis (OA) is considered to be a multifactorial and polygenic disease and diagnosis is mainly clinical and radiological. Correlation between radiographic data and clinical status has been reported. However, very few studies, especially in Caucasian people, describe the association between the Kellgren and Lawrence OA grading scale (KL) and genetic alterations to better understand OA etiopathogenesis and susceptibility. In order to update the knee OA grading, in this study we assessed the associations between KL grade, clinical features such as American Knee Society Score (AKSS), age, and polymorphisms in the principal osteoarthritis susceptibility (OS) genes in Sicilian individuals. Methods. In 66 Sicilian individuals affected by primary knee OA, the clinical and radiographic evaluation was performed using 2 sub-scores of AKSS (knee score (KS) and function score (FS)) and KL. The patients were also classified according to age. Online Mendelian Inheritance in Man (OMIM) and Database of Single Nucleotide Polymorphisms (dbSNP) Short Genetic Variations databases were used to select gene regions containing the following polymorphisms to analyze: FRZB rs288326 and rs7775, MATN3 rs77245812, ASPN D14 repeats, PTHR2 rs76758470, GDF5 rs143383 and DVWA rs11718863. Patient genotypes were obtained using Sanger DNA sequencing analysis. Results. In our cohort of patients a statistical association between the variables analyzed was reported in all associations tested (KL versus KS, FS and age). We observed that a mild to severe OA radiographic grade is related to severe clinical conditions and loss of articular function and that the severity of symptoms increases with age. Concerning the genotyping analysis, our results revealed a significant statistical association between KL grading and GDF5 rs143383 and DVWA rs11718863 genetic alterations. The latter was also associated with a more severe radiographic grade, displaying its predictive role as OA marker progression. Statistically significant association between clinical, radiographic and genetic signs observed, suggests extending the actual grading of knee OA based mainly on X-ray features. Conclusions. This work represents a multidisciplinary and translational medicine approach to study OA where clinical, radiological, and OS5 and OS6 SNPs evaluation could contribute to better define grading and progression of OA and to the development of new therapies

Endothelial function of conduit arteries in patients with ulcerative colitis and metabolic syndrome.

Although ulcerative colitis (UC) is characterized by chronic inflammation and elevated circulating plasma levels of C reactive protein and TNF-alpha, well recognized cardiovascular risk factors, several studies have suggested that patients with UC do not bear an increased risk for cardiovascular diseases. However, recent reports have shown that UC is associated with increased intima-media thickness and impaired endothelial function. On the other hand, endothelial dysfunction is strictly related to premature atherosclerosis and cardiovascular diseases in different populations. In addition, metabolic syndrome (MS) is associated with impairment of endothelial function in the general population, while the impact of MS on endothelial function in UC patients is unknown. Aim of this study was firstly to clarify, in a large group of patients, whether endothelial function of conduit arteries is affected by UC. Secondly, to investigate whether MS in patients with UC has an impact on the endothelial function. Among the patients with UC, twenty-three were also affected by MS, whereas twenty-two patients with MS were present in the control group. FMD was similar in patients with UC and controls (11.8±0.9% and 12.2±0.8, respectively, p=NS). While the presence of MS in the control group was associated with impairment of endothelial function (13.6±0.8% in healthy controls and 10.2±0.6 in patients with MS, p=0.003), patients with MS and UC did not show any worsening of their endothelial function (11.4±1.0% in patients with UC and SM and 12.1±1.3 in patients with UC without MS; p=0.35). NMD was similar in all the groups

コウゾウ シュギ ニ モトズク ヒハン ノ ロンリ : 1970ネンダイ ニオケル アメリカ シャカイカ ブンカ ガクシュウ オ ジレイ トシテ

Hepatitis B virus (HBV) is a unique, tiny, partially double-stranded, reverse-transcribing DNA virus with proteins encoded by multiple overlapping reading frames. The substitution rate is surprisingly high for a DNA virus, but lower than that of other reverse transcribing organisms. More than 260 million people worldwide have chronic HBV infection, which causes 0.8 million deaths a year. Because of the high burden of disease, international health agencies have set the goal of eliminating HBV infection by 2030. Nonetheless, the intriguing HBV genome has not been well characterized. We summarize data on the HBV genome structure and replication cycle, explain and quantify diversity within and among infected individuals, and discuss advances that can be offered by application of next-generation sequencing technology. In-depth HBV genome analyses could increase our understanding of disease pathogenesis and allow us to better predict patient outcomes, optimize treatment, and develop new therapeutics

Inflammatory Gene Expression Associates with Hepatitis B Virus cccDNA- but Not Integrant-Derived Transcripts in HBeAg Negative Disease

Chronic hepatitis B virus (HBV) infection is a global health problem that presents as a spectrum of liver disease, reflecting an interplay between the virus and the host immune system. HBV genomes exist as episomal covalently closed circular DNA (cccDNA) or chromosomal integrants. The relative contribution of these genomes to the viral transcriptome in chronic hepatitis B (CHB) is not well-understood. We developed a qPCR method to estimate the abundance of HBV cccDNA- and integrant-derived viral transcripts and applied this to a cohort of patients diagnosed with CHB in the HBe antigen negative phase of disease. We noted a variable pattern of HBV transcripts from both DNA templates, with preS1/S2 mRNAs predominating and a significant association between increasing age and the expression of integrant-derived mRNAs, but not with inflammatory status. In contrast, cccDNA-derived transcripts were associated with markers of liver inflammation. Analysis of the inflammatory hepatic transcriptome identified 24 genes significantly associated with cccDNA transcriptional activity. Our study uncovers an immune gene signature that associates with HBV cccDNA transcription and increases our understanding of viral persistence

Hypoxia inducible factors regulate hepatitis B virus replication by activating the basal core promoter

PMC8214165BACKGROUND & AIMS: Hypoxia inducible factors (HIFs) are a hallmark of inflammation and are key regulators of hepatic immunity and metabolism, yet their role in HBV replication is poorly defined. HBV replicates in hepatocytes within the liver, a naturally hypoxic organ, however most studies of viral replication are performed under conditions of atmospheric oxygen, where HIFs are inactive. We therefore investigated the role of HIFs in regulating HBV replication. METHODS: Using cell culture, animal models, human tissue and pharmacological agents inhibiting the HIF-prolyl hydroxylases, we investigated the impact of hypoxia on the HBV life cycle. RESULTS: Culturing liver cell-based model systems under low oxygen uncovered a new role for HIFs in binding HBV DNA and activating the basal core promoter, leading to increased pre-genomic RNA and de novo HBV particle secretion. The presence of hypoxia responsive elements among all primate members of the hepadnaviridae highlights an evolutionary conserved role for HIFs in regulating this virus family. CONCLUSIONS: Identifying a role for this conserved oxygen sensor in regulating HBV transcription suggests that this virus has evolved to exploit the HIF signaling pathway to persist in the low oxygen environment of the liver. Our studies show the importance of considering oxygen availability when studying HBV-host interactions and provide innovative routes to better understand and target chronic HBV infection. LAY SUMMARY: Viral replication in host cells is defined by the cellular microenvironment and one key factor is local oxygen tension. Hepatitis B virus (HBV) replicates in the liver, a naturally hypoxic organ. Hypoxia inducible factors (HIFs) are the major sensors of low oxygen; herein, we identify a new role for these factors in regulating HBV replication, revealing new therapeutic targets