Chronic hepatitis B virus (HBV) infection persists due to the lack of therapies that effectively target the HBV covalently closed circular DNA (cccDNA). We used HBV-specific guide RNAs (gRNAs) and ...CRISPR-Cas9 and determined the fate of cccDNA after gene editing. We set up a ribonucleoprotein (RNP) delivery system in HBV-infected HepG2-NTCP cells. HBV parameters after Cas9 editing were analyzed. Southern blot (SB) analysis and DNA/RNA sequencing (DNA/RNA-seq) were performed to determine the consequences of cccDNA editing and transcriptional activity of mutated cccDNA. Treatment of infected cells with HBV-specific gRNAs showed that CRISPR-Cas9 can efficiently affect HBV replication. The appearance of episomal HBV DNA variants after dual gRNA treatment was observed by PCR, SB analysis, and DNA/RNA-seq. These transcriptionally active variants are the products of simultaneous Cas9-induced double-strand breaks in two target sites, followed by repair and religation of both short and long fragments. Following suppression of HBV DNA replicative intermediates by nucleoside analogs, mutations and formation of smaller transcriptionally active HBV variants were still observed, suggesting that established cccDNA is accessible to CRISPR-Cas9 editing. Targeting HBV DNA with CRISPR-Cas9 leads to cleavage followed by appearance of episomal HBV DNA variants. Effects induced by Cas9 were sustainable after RNP degradation/loss of detection, suggesting permanent changes in the HBV genome instead of transient effects due to transcriptional interference.
Hepatitis B virus infection can develop into chronic infection, cirrhosis, and hepatocellular carcinoma. Treatment of chronic hepatitis B requires novel approaches to directly target the viral minichromosome, which is responsible for the persistence of the disease. Designer nuclease approaches represent a promising strategy to treat chronic infectious diseases; however, comprehensive knowledge about the fate of the HBV minichromosome is needed before this potent tool can be used as a potential therapeutic approach. This study provides an in-depth analysis of CRISPR-Cas9 targeting of HBV minichromosome.
Summary The EASL Monothematic Conference on Translational Research in Viral Hepatitis brought together a group of leading scientists and clinicians working on both, basic and clinical aspects of ...viral hepatitis, thereby building bridges from bench to bedside. This report recapitulates the presentations and discussions at the conference held in Lyon, France on November 29–30, 2013. In recent years, great advances have been made in the field of viral hepatitis, particularly in hepatitis C virus (HCV) infection. The identification of IL28B genetic polymorphisms as a major determinant for spontaneous and treatment-induced HCV clearance was a seminal discovery. Currently, hepatologists are at the doorstep of even greater advances, with the advent of a wealth of directly acting antivirals (DAAs) against HCV. Indeed, promising results have accumulated over the last months and few years, showing sustained virological response (SVR) rates of up to 100% with interferon-free DAA combination therapies. Thus, less than 25 years after its identification, HCV infection may soon be curable in the vast majority of patients, highlighting the great success of HCV research over the last decades. However, viral hepatitis and its clinical complications such as liver cirrhosis and hepatocellular carcinoma (HCC) remain major global challenges. New therapeutic strategies to tackle hepatitis B virus (HBV) and hepatitis D virus (HDV) infection are needed, as current therapies have undeniable limitations. Nucleoside/nucleotide analogues (NUC) can efficiently control HBV replication and reduce or even reverse liver damage. However, these drugs have to be given for indefinite periods in most patients to maintain virological and biochemical responses. Although sustained responses off treatment can be achieved by treatment with (pegylated) interferon-α, only about 10–30% of patients effectively resolve chronic hepatitis B. It was the goal of this conference to review the progress made over the last years in chronic viral hepatitis research and to identify key questions that need to be addressed in order to close the gap between basic and clinical research and to develop novel preventive and treatment approaches for this most common cause of liver cirrhosis and HCC.
Chronic hepatitis B virus (HBV) infection is a major risk factor for hepatocellular carcinoma (HCC) and current treatments for chronic hepatitis B and HCC are suboptimal. Herein, we identified ...cellular serine/threonine Polo‐like‐kinase 1 (PLK1) as a positive effector of HBV replication. The aim of this study was to demonstrate the proviral role of PLK1 in HBV biosynthesis and validate PLK1 inhibition a potential antiviral strategy. To this end, we employed physiologically relevant HBV infection models of primary human hepatocytes (PHHs) and differentiated HepaRG cells in conjunction with pharmacologic PLK1 inhibitors, small interfering RNA (siRNA)‐mediated knockdown, and overexpression of constitutively active PLK1 (PLK1CA). In addition, a humanized liver Fah−/−/Rag2−/−/Il2rg−/− (FRG) mouse model was used to determine the antiviral effect of PLK1 inhibitor BI‐2536 on HBV infection in vivo. Finally, in vitro PLK1 kinase assays and site‐directed mutagenesis were employed to demonstrate that HBV core protein (HBc) is a PLK1 substrate. We demonstrated that HBV infection activated cellular PLK1 in PHHs and differentiated HepaRG cells. PLK1 inhibition by BI‐2536 or siRNA‐mediated knockdown suppressed HBV DNA biosynthesis, whereas overexpression of PLK1CA increased it, suggesting that the PLK1 effects on viral biosynthesis are specific and that PLK1 is a proviral cellular factor. Significantly, BI‐2536 administration to HBV‐infected humanized liver FRG mice strongly inhibited HBV infection, validating PLK1 as an antiviral target in vivo. The proviral action of PLK1 is associated with the biogenesis of the nucleocapsid, as BI‐2536 leads to its decreased intracellular formation/accumulation. In this respect, our studies identified HBc as a PLK1 substrate in vitro, and mapped PLK1 phosphorylation sites on this protein. Conclusion: PLK1 is a proviral host factor that could be envisaged as a target for combined antiviral and antitumoral strategies against HBV infection and HBV‐mediated carcinogenesis. (Hepatology 2017;66:1750–1765)
Plasmacytoid dendritic cells (pDCs) play a key role in detecting pathogens by producing large amounts of type I interferon (IFN) by sensing the presence of viral infections through the Toll-Like ...Receptor (TLR) pathway. TLR9 is a sensor of viral and bacterial DNA motifs and activates the IRF7 transcription factor which leads to type I IFN secretion by pDCs. However, during chronic hepatitis B virus (HBV) infection, pDCs display an impaired ability to secrete IFN-α following ex vivo stimulation with TLR9 ligands. Here we highlight several strategies used by HBV to block IFN-α production through a specific impairment of the TLR9 signaling. Our results show that HBV particle internalisation could inhibit TLR9- but not TLR7-mediated secretion of IFN-α by pDCs. We observed that HBV down-regulated TLR9 transcriptional activity in pDCs and B cells in which TLR9 mRNA and protein levels were reduced. HBV can interfere with TLR9 activity by blocking the MyD88-IRAK4 axis and Sendai virus targeting IRF7 to block IFN-α production. Neutralising CpG motif sequences were identified within HBV DNA genome of genotypes A to H which displayed a suppressive effect on TLR9-immune activation. Moreover, TLR9 mRNA and protein were downregulated in PBMCs from patients with HBV-associated chronic hepatitis and hepatocellular carcinoma. Thus HBV has developed several escape mechanisms to avoid TLR9 activation in both pDCs and B lymphocytes, which may in turn contribute to the establishment and/or persistence of chronic infection.
Despite the existence of a preventive vaccine, chronic infection with Hepatitis B virus (HBV) affects more than 250 million people and represents a major global cause of hepatocellular carcinoma ...(HCC) worldwide. Current clinical treatments, in most of cases, do not eliminate viral genome that persists as a DNA episome in the nucleus of hepatocytes and constitutes a stable template for the continuous expression of viral genes. Several studies suggest that, among viral factors, the HBV core protein (HBc), well-known for its structural role in the cytoplasm, could have critical regulatory functions in the nucleus of infected hepatocytes. To elucidate these functions, we performed a proteomic analysis of HBc-interacting host-factors in the nucleus of differentiated HepaRG, a surrogate model of human hepatocytes. The HBc interactome was found to consist primarily of RNA-binding proteins (RBPs), which are involved in various aspects of mRNA metabolism. Among them, we focused our studies on SRSF10, a RBP that was previously shown to regulate alternative splicing (AS) in a phosphorylation-dependent manner and to control stress and DNA damage responses, as well as viral replication. Functional studies combining SRSF10 knockdown and a pharmacological inhibitor of SRSF10 phosphorylation (1C8) showed that SRSF10 behaves as a restriction factor that regulates HBV RNAs levels and that its dephosphorylated form is likely responsible for the anti-viral effect. Surprisingly, neither SRSF10 knock-down nor 1C8 treatment modified the splicing of HBV RNAs but rather modulated the level of nascent HBV RNA. Altogether, our work suggests that in the nucleus of infected cells HBc interacts with multiple RBPs that regulate viral RNA metabolism. Our identification of SRSF10 as a new anti-HBV restriction factor offers new perspectives for the development of new host-targeted antiviral strategies.
The way forward in HCV treatment - finding the right path Manns, Michael P; Foster, Graham R; Rockstroh, Jürgen K ...
Nature reviews. Drug discover/Nature reviews. Drug discovery,
12/2007, Letnik:
6, Številka:
12
Journal Article
Recenzirano
Odprti dostop
Infection with the hepatitis C virus (HCV) represents an important health-care problem worldwide. The prevalence of HCV-related disease is increasing, and no vaccine is yet available. Since the ...identification of HCV as the causative agent of non-A, non-B hepatitis, treatment has progressed rapidly, but morbidity and mortality rates are still predicted to rise. Novel, more efficacious and tolerable therapies are urgently needed, and a greater understanding of the viral life cycle has led to an increase in the number of possible targets for antiviral intervention. Here we review the specific challenges posed by HCV, and recent developments in the design of vaccines and novel antiviral agents.
Chronic infection by the hepatitis C virus (HCV) is a major cause of liver diseases, predisposing to fibrosis and hepatocellular carcinoma. Liver fibrosis is characterized by an overly abundant ...accumulation of components of the hepatic extracellular matrix, such as collagen and elastin, with consequences on the properties of this microenvironment and cancer initiation and growth. This review will provide an update on mechanistic concepts of HCV-related liver fibrosis/cirrhosis and early stages of carcinogenesis, with a dissection of the molecular details of the crosstalk during disease progression between hepatocytes, the extracellular matrix, and hepatic stellate cells.
Background & Aims Long-term viral suppression is a major goal to prevent disease progression in patients with HBV. Aim of this study was to investigate the efficacy and safety of entecavir plus ...tenofovir combination in 57 CHB partial responders or multidrug resistant patients. Methods Investigator-initiated open-label cohort study. Quantitative HBV-DNA measurement and resistance testing (line-probe-assays and direct-sequencing) at baseline and every 3 months. Results Fifty seven patients (37 HBeAg+), median age 45 years, previously treated with a median of three lines of antiviral therapy (range 1–6), 24/57 with advanced liver disease, were included. Median ALT at baseline was 1.0 ULN (range 0.3–22) and HBV-DNA 1.5 × 104 IU/ml (range 500–1 × 1011 IU/ml). Median treatment duration of combination therapy was 21 months. HBV-DNA level dropped 3 logs (median, range 0–8 log; p <0.0001), 51/57 patients became HBV-DNA undetectable, median after 6 months (95% CI, 4.6–7). The probability for HBV DNA suppression was not reduced in patients with adefovir or entecavir resistance or in patients with advanced liver disease. Viral suppression led to decline in ALT (median 0.7 ULN; range 0.2–2.4; p = 0.001). Five patients lost HBeAg (after 15, 18, 20, 21, and 27 months, respectively), one patient showed HBs-seroconversion. Patients with advanced disease did not show clinical decompensation, two patients with cirrhosis and undetectable HBV DNA developed HCCs. No death, newly induced renal impairment or lactic acidosis were reported. Conclusions Rescue therapy with entecavir and tenofovir in CHB patients harboring viral resistance patterns or showing only partial antiviral responses to preceding therapies was efficient, safe, and well tolerated in patients with and without advanced liver disease (249).
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