Tg1.4HBV-s-rec mice, a crossbred hepatitis B virus-transgenic model, develop mild hepatitis.

Hepatitis B virus (HBV)-transgenic mice exhibit competent innate immunity and are therefore an ideal model for considering intrinsic or cell-based mechanisms in HBV pathophysiology. A highly replicative model that has been little used, let alone characterized, is the Tg1.4HBV-s-rec strain derived from cross breeding of HBV-transgenic mouse models that either accumulate (Alb/HBs, Tg[Alb1-HBV]Bri44) or lack (Tg1.4HBV-s-mut) the hepatitis B surface antigen (HBsAg). Tg1.4HBV-s-rec hepatocytes secreted HBsAg, Hepatitis B extracellular antigen (HBeAg) and produced HBV virions. Transmission electron microscopy visualised viral particles (Tg1.4HBV-s-rec), nuclear capsid formations (Tg1.4HBV-s-mut and Tg1.4HBV-s-rec) and endoplasmic reticulum malformations (Alb/HBs). Viral replication in Tg1.4HBV-s-rec and Tg1.4HBV-s-mut differed in HBsAg expression and interestingly in the distribution of HBV core antigen (HBcAg) and HBV × protein. While in Tg1.4HBV-s-mut hepatocytes, the HBcAg was located in the cytoplasm, in Tg1.4HBV-s-rec hepatocytes, the HBcAg appeared in the nuclei, suggesting a more productive replication. Finally, Tg1.4HBV-s-rec mice showed symptoms of mild hepatitis, with reduced liver function and elevated serum transaminases, which appeared to be related to natural killer T cell activation. In conclusion, the study of Alb/HBs, Tg1.4HBV-s-mut and their F1 progeny provides a powerful tool to elucidate HBV pathophysiology, especially in the early HBeAg-positive phases of chronic infection and chronic hepatitis.

Targeted mutations in IFNα2 improve its antiviral activity against various viruses.

During viral infections, type I interferons (IFN) are induced and play a key role in counteracting initial viral spread. Twelve different human IFNα subtypes exist that bind the same receptor; however, they elicit unique host responses and display distinct potencies of antiviral activities. Our previous studies on human immunodeficiency virus (HIV) and hepatitis B virus (HBV) demonstrated that the clinically used IFNα2 is not the most effective one among the IFNα subtypes. By sequence modeling, we identified a region in helix B with mainly conserved residues at the outside facing IFNAR1, but variable residues at the inside facing the core of IFNα, potentially representing a putative tunable anchor to tune pleiotropic IFN responses. Using site-directed mutagenesis, various mutations were introduced into the IFNα2b backbone targeting sites which are important for binding to IFNAR1 and IFNAR2, the putative tunable anchor, or outside these three regions. Selected mutations were based on sequence differences to high antiviral subtypes IFNα6 and IFNα14. Treatment assays against HBV and HIV identified several critical residues for the antiviral activity of IFNα mainly in the IFNAR1 binding region. Combined mutations of the IFNα2 IFNAR1/2 binding sites or the IFNAR1 binding region plus the putative tunable anchor by those of IFNα14 further augmented activation of different downstream signaling cascades providing a molecular correlate for the enhanced antiviral activity. We describe here important functional residues within IFNα subtype molecules, which enabled us to design novel and innovative drugs that may have the potential to be used in clinical trials against a variety of different viral infections.IMPORTANCEThe potency of interferon (IFN)α to restrict viruses was already discovered in 1957. However, until today, only IFNα2 out of the 12 distinct human IFNα subtypes has been therapeutically used against chronic viral infections. There is convincing evidence that other IFNα subtypes are far more efficient than IFNα2 against many viruses. In order to identify critical antiviral residues within the IFNα subtype sequence, we designed hybrid molecules based on the IFNα2 backbone with individual sequence motifs from the more potent subtypes IFNα6 and IFNα14. In different antiviral assays with HIV or HBV, residues binding to IFNAR1 as well as combinations of residues in the IFNAR1 binding region, the putative tunable anchor, and residues outside these regions were identified to be crucial for the antiviral activity of IFNα. Thus, we designed artificial IFNα molecules, based on the clinically approved IFNα2 backbone, but with highly improved antiviral activity against several viruses.

SRSF1 acts as an IFN-I-regulated cellular dependency factor decisively affecting HIV-1 post-integration steps.

Efficient HIV-1 replication depends on balanced levels of host cell components including cellular splicing factors as the family of serine/arginine-rich splicing factors (SRSF, 1-10). Type I interferons (IFN-I) play a crucial role in the innate immunity against HIV-1 by inducing the expression of IFN-stimulated genes (ISGs) including potent host restriction factors. The less well known IFN-repressed genes (IRepGs) might additionally affect viral replication by downregulating host dependency factors that are essential for the viral life cycle; however, so far, the knowledge about IRepGs involved in HIV-1 infection is very limited. In this work, we could demonstrate that HIV-1 infection and the associated ISG induction correlated with low SRSF1 levels in intestinal lamina propria mononuclear cells (LPMCs) and peripheral blood mononuclear cells (PBMCs) during acute and chronic HIV-1 infection. In HIV-1-susceptible cell lines as well as primary monocyte-derived macrophages (MDMs), expression levels of SRSF1 were transiently repressed upon treatment with specific IFNα subtypes in vitro. Mechanically, 4sU labeling of newly transcribed mRNAs revealed that IFN-mediated SRSF1 repression is regulated on early RNA level. SRSF1 knockdown led to an increase in total viral RNA levels, but the relative proportion of the HIV-1 viral infectivity factor (Vif) coding transcripts, which is essential to counteract APOBEC3G-mediated host restriction, was significantly reduced. In the presence of high APOBEC3G levels, however, increased LTR activity upon SRSF1 knockdown facilitated the overall replication, despite decreased vif mRNA levels. In contrast, SRSF1 overexpression significantly impaired HIV-1 post-integration steps including LTR transcription, alternative splice site usage, and virus particle production. Since balanced SRSF1 levels are crucial for efficient viral replication, our data highlight the so far undescribed role of SRSF1 acting as an IFN-modulated cellular dependency factor decisively regulating HIV-1 post-integration steps.

IFNα subtype-specific susceptibility of HBV in the course of chronic infection.

Chronic hepatitis B virus (HBV) infection continues to be a major health problem worldwide and remains hard to be cured. Therapy with interferon (IFN) α is an important method for the clinical treatment of chronic hepatitis B. IFNα exhibits direct antiviral effects as well as immunomodulatory activities, which can induce sustained antiviral responses in part of the treated chronic hepatitis B patients. Numerous IFNα subtypes with high sequence identity between 76-96% exist which are characterized by diverse, non-redundant biological activities. Our previous studies have demonstrated that the clinically approved IFNα2 is not the most effective subtype for the anti-HBV treatment among all IFNα subtypes. So far very little is known about the IFNα subtype expression pattern during early HBV infection and the IFNα subtype-specific susceptibility during persistent HBV infection as well as its related cellular mechanism. Here we determined the Ifna subtype mRNA expression during acute and chronic HBV infection by using the well-established hydrodynamic injection (HDI) mouse model and we revealed a transient but strong expression of a panel of Ifna subtypes in the spleen of HBV persistent replication mice compared to HDI controls. Immunotherapy with distinct IFNα subtypes controlled chronic HBV infection. IFNα subtype-mediated antiviral response and immune activation were comprehensively analyzed in an AAV-HBV persistent infection murine model and murine IFNα2 was identified as the most effective subtype in suppression of HBV replication. Further analysis of the immune response revealed a strong immunomodulatory activity of murine IFNα2 on splenic and intrahepatic NK and T cell activation during persistent HBV infection. Taken together, our data provide IFNα subtype-specific differences in the antiviral and immunomodulatory effector responses and a strong expression of all IFNα subtypes in the spleen during persistent HBV infection in mice. This knowledge will support the development of novel immunotherapeutic strategies for chronic hepatitis B infection.

A ribavirin-induced ORF2 single-nucleotide variant produces defective hepatitis E virus particles with immune decoy function.

Hepatitis E virus (HEV) is the causative agent of hepatitis E in humans and is the leading cause of enterically transmitted viral hepatitis worldwide. Ribavirin (RBV) is currently the only treatment option for many patients; however, cases of treatment failures or posttreatment relapses have been frequently reported. RBV therapy was shown to be associated with an increase in HEV genome heterogeneity and the emergence of distinct HEV variants. In this study, we analyzed the impact of eight patient-derived open reading frame 2 (ORF2) single-nucleotide variants (SNVs), which occurred under RBV treatment, on the replication cycle and pathogenesis of HEV. The parental HEV strain and seven ORF2 variants showed comparable levels of RNA replication in human hepatoma cells and primary human hepatocytes. However, a P79S ORF2 variant demonstrated reduced RNA copy numbers released in the supernatant and an impairment in the production of infectious particles. Biophysical and biochemical characterization revealed that this SNV caused defective, smaller HEV particles with a loss of infectiousness. Furthermore, the P79S variant displayed an altered subcellular distribution of the ORF2 protein and was able to interfere with antibody-mediated neutralization of HEV in a competition assay. In conclusion, an SNV in the HEV ORF2 could be identified that resulted in altered virus particles that were noninfectious in vitro and in vivo, but could potentially serve as immune decoys. These findings provide insights in understanding the biology of circulating HEV variants and may guide development of personalized antiviral strategies in the future.

Differential interferon-α subtype induced immune signatures are associated with suppression of SARS-CoV-2 infection.

Type I interferons (IFN-I) exert pleiotropic biological effects during viral infections, balancing virus control versus immune-mediated pathologies, and have been successfully employed for the treatment of viral diseases. Humans express 12 IFN-alpha (α) subtypes, which activate downstream signaling cascades and result in distinct patterns of immune responses and differential antiviral responses. Inborn errors in IFN-I immunity and the presence of anti-IFN autoantibodies account for very severe courses of COVID-19; therefore, early administration of IFN-I may be protective against life-threatening disease. Here we comprehensively analyzed the antiviral activity of all IFNα subtypes against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to identify the underlying immune signatures and explore their therapeutic potential. Prophylaxis of primary human airway epithelial cells (hAEC) with different IFNα subtypes during SARS-CoV-2 infection uncovered distinct functional classes with high, intermediate, and low antiviral IFNs. In particular, IFNα5 showed superior antiviral activity against SARS-CoV-2 infection in vitro and in SARS-CoV-2-infected mice in vivo. Dose dependency studies further displayed additive effects upon coadministration with the broad antiviral drug remdesivir in cell culture. Transcriptomic analysis of IFN-treated hAEC revealed different transcriptional signatures, uncovering distinct, intersecting, and prototypical genes of individual IFNα subtypes. Global proteomic analyses systematically assessed the abundance of specific antiviral key effector molecules which are involved in IFN-I signaling pathways, negative regulation of viral processes, and immune effector processes for the potent antiviral IFNα5. Taken together, our data provide a systemic, multimodular definition of antiviral host responses mediated by defined IFN-I. This knowledge will support the development of novel therapeutic approaches against SARS-CoV-2.