APOBEC3-independent interferon-induced viral clearance in hepatitis B virus transgenic mice

Interferon (IFN) has been part of the standard treatment of chronic hepatitis B infection for more than 2 decades, yet the mechanism of action of this antiviral remains poorly understood. It was recently observed that members of the human APOBEC family of cytidine deaminases endowed with anti-hepatitis B virus (HBV) activity are upregulated by type I and II IFNs. However, we demonstrated that, in tissue culture, these cellular enzymes are not essential effectors of the anti-HBV action of these cytokines. Here, we show that murine APOBEC3 (muA3) can also block HBV replication. While expressed at low levels in the mouse liver at baseline, muA3 is upregulated upon IFN induction. However, in HBV-transgenic muA3 knockout mice, IFN induction blocked HBV DNA production as efficiently as in control HBV-transgenic muA3-competent animals. We conclude that APOBEC3 is not an essential mediator of the IFN-mediated inhibition of HBV in vivo.

Polynucleotide cytidine deaminases, innate immunity and hepatitis B infection

Stéphanie Jost

More than 350 millions individuals worldwide suffer from chronic hepatitis B virus (HBV) infection, a condition that evolves towards liver insufficiency and hepatocellular carcinoma in approximately 15 to 40 percent of cases. Yet the majority of de novo HBV infections, except for those occurring during the perinatal period, are cleared during the acute phase. This largely appears to result from non-cytopathic host defense mechanisms involving cytokines and notably interferons (IFNs). A key role for IFNs in the biology of HBV infection has been further illustrated by studies in animal models of the disease. Besides, IFN-α is the standard treatment for chronic hepatitis B. Although type I and II IFNs are the most important immune-regulators of HBV replication, the downstream effectors mediating the inhibitory action of IFNs on HBV replication have not yet been identified. In a first place we showed that HBV replication is blocked by human APOBEC3G and APOBEC3F, two closely related editing enzymes belonging to the family of polynucleotide cytidine deaminases, whose catalytic activity results in the conversion of cytidine residues to uridine residues. Both proteins were initially recognized for their ability to inhibit retroviruses, a property which mainly depends on their editing activity, and as the targets of the virion infectivity factor (Vif) protein of human immunodeficiency virus (HIV), which induces their degradation. The family of polynucleotide cytidine deaminases also comprises Activation-Induced cytidine Deaminase (AID), APOBEC1, APOBEC2, and the products of the APOBEC3A through APOBEC3H gene cluster. AID is essential for class switch recombination and somatic hypermutation of the immunoglobulin locus in pre-B lymphocytes, thus for the generation of antibody diversity, and APOBEC1 edits the apolipoprotein B mRNA in the guts, thereby regulating cholesterol metabolism. By comparison, little is known about the physiological functions of the other family members. However, apart from APOBEC2 and APOBEC3A, sequence analysis of the genes encoding these enzymes suggested that they possibly evolved as a host defense against pathogens. This hypothesis was confirmed for most of APOBEC3 proteins, which were independently found to restrict the replication of various exogenous and endogenous retroelements, as well as the adeno-associated virus. For this reason, we next explored the range of cytidine deaminases active on HBV and found that besides APOBEC3G and APOBEC3F, APOBEC3B, AID and APOBEC1 are also potent inhibitors of HBV. In parallel, while examining in details the mechanisms underlying the antiviral activity of APOBEC3G against HBV, we found that this cytidine deaminase blocks HBV infection not by lethal editing of nascent reverse transcripts, as for retroviruses, but rather by impairing the accumulation of pregenomic RNA (pgRNA)-containing viral capsids, the subviral structures in which HBV reverse transcription takes place before virions containing a partially double-stranded DNA genome are released. Remarkably, this is similar to the decreased steady-state levels of pgRNA-containing capsids which are observed upon exposure of HBV infected cells to IFNs. It suggested that IFNs and cytidine deaminases might affect HBV replication through the same pathway. The first step to unveil the in vivo relevance of antiviral cytidine deaminases for the control of HBV replication consisted in our demonstration that type I and II IFN treatment of human hepatocytes induces the production of APOBEC3G, and to a smaller extent, that of APOBEC3F and APOBEC3B, but not that of AID and APOBEC1. These observations led us to ask whether the induction of antiviral cytidine deaminases by cytokines contributes to the IFN-induced clearance of HBV, as IFN-α and IFN-γ are both found in high amounts in the liver of acutely infected individuals. Importantly, we revealed that blocking APOBEC3G, APOBEC3F and APOBEC3B in hepatic cell lines does not abrogate the inhibitory effect of IFNs on HBV, indicating that antiviral cytidine deaminases are not the main mediators of the action of IFNs on this pathogen. Nevertheless, while HBV replication was not impaired in hepatic cells when these enzymes were expressed at their baseline levels, they could edit the HBV genome in these conditions, suggesting that cytidine deaminases might participate in the emergence of new HBV strains, notably those that have been recognized as being of clinical importance. Finally, the results of this work indicate that a therapeutic strategy based on the expression of antiviral cytidine deaminases in the liver could be envisioned to cure HBV chronic infections.

EPFL2007

Estimating the net contribution of IL28B variation to spontaneous hepatitis C virus clearance

Julia di Iulio, Jacques Fellay, Amalio Telenti

The identification of associations between IL28B variants and spontaneous clearance of hepatitis C virus (HCV) raises the issues of causality and of the net contribution of host genetics to the trait. To estimate more precisely the net effect of IL28B genetic variation on HCV clearance, we optimized genotyping and compared the host contribution in a multiple and a single source cohort to control for viral and demographic effects. The analysis included individuals with chronic or spontaneously cleared HCV infection from a multiple (n=389), and from a single source cohort (n=71). We performed detailed genotyping in the coding region of IL28B and searched for copy number variation to identify the genetic variant or haplotype carrying the strongest association with viral clearance. This analysis was used to compare the effect of IL28B variation in both cohorts. Haplotypes characterized by carriage of the major alleles at IL28B single nucleotide polymorphisms (SNPs) were highly overrepresented in individuals with spontaneous clearance compared to those with chronic HCV infection (66.1% versus 38.6%, P=6*10(-9) ). The Odds Ratio for clearance was 2.1 (CI(95) 1.6-3.0) and 3.9 (CI(95) 1.5-10.2) in multiple and single source cohort, respectively. Protective haplotypes were in perfect linkage (r(2) =1.0) with a non-synonymous coding variant (rs8103142). Copy number variants were not detected. CONCLUSIONS: We identified IL28B haplotypes highly predictive of spontaneous HCV clearance. The high linkage disequilibrium between IL28B SNPs indicates that association studies need to be complemented by functional experiments to identify single causal variant(s). The point estimate for the genetic effect was higher in the single source cohort, which effectively controls for viral diversity, gender and co-infections and therefore offers a precise estimate of the net host genetic contribution. (HEPATOLOGY 2011.).

2011

Human genomics of acute liver failure due to hepatitis B virus infection: An exome sequencing study in liver transplant recipients

Samira Asgari, Nimisha Chaturvedi, Jacques Fellay, Christian Hammer, Petar Scepanovic

Acute liver failure (ALF) or fulminant hepatitis is a rare, yet severe outcome of infection with hepatitis B virus (HBV) that carries a high mortality rate. The occurrence of a life-threatening condition upon infection with a prevalent virus in individuals without known risk factors is suggestive of pathogen-specific immune dysregulation. In the absence of established differences in HBV virulence, we hypothesized that ALF upon primary infection with HBV could be due to rare deleterious variants in the human genome. To search for such variants, we performed exome sequencing in 21 previously healthy adults who required liver transplantation upon fulminant HBV infection and 172 controls that were positive for anti-HBc and anti-HBs but had no clinical history of jaundice or liver disease. After a series of hypothesis-driven filtering steps, we searched for putatively pathogenic variants that were significantly associated with case-control status. We did not find any causal variant or gene, a result that does not support the hypothesis of a shared monogenic basis for human susceptibility to HBV-related ALF in adults. This study represents a first attempt at deciphering the human genetic contribution to the most severe clinical presentation of acute HBV infection in previously healthy individuals.