HDV RNA replication is associated with HBV repression and interferon-stimulated genes induction in super-infected hepatocytes

Highlights

• A model of super-infection with HDV on HBV-infected hepatocytes was established.

• HDV infection induces a strong IFN response in these immune-competent hepatocytes.

• In this model, HDV infection is associated with HBV inhibition, thus access to recapitulating in vivo viral interference.

• This super infection model is also suitable for the evaluation of novel drugs/antivirals, including immune-modulators.

Abstract

Hepatitis D virus (HDV) super-infection of Hepatitis B virus (HBV)-infected patients is the most aggressive form of viral hepatitis. HDV infection is not susceptible to direct anti-HBV drugs, and only suboptimal antiviral responses are obtained with interferon (IFN)-alpha-based therapy. To get insights on HDV replication and interplay with HBV in physiologically relevant hepatocytes, differentiated HepaRG (dHepaRG) cells, previously infected or not with HBV, were infected with HDV, and viral markers were extensively analyzed. Innate and IFN responses to HDV were monitored by measuring pro-inflammatory and interferon-stimulated gene (ISG) expression. Both mono- and super-infected dHepaRG cells supported a strong HDV intracellular replication, which was accompanied by a strong secretion of infectious HDV virions only in the super-infection setting and despite the low number of co-infected cells. Upon HDV super-infection, HBV replication markers including HBeAg, total HBV-DNA and pregenomic RNA were significantly decreased, confirming the interference of HDV on HBV. Yet, no decrease of circular covalently closed HBV DNA (cccDNA) and HBsAg levels was evidenced. At the peak of HDV-RNA accumulation and onset of interference on HBV replication, a strong type-I IFN response was observed, with interferon stimulated genes, RSAD2 (Viperin) and IFI78 (MxA) being highly induced. We established a cellular model to characterize in more detail the direct interference of HBV and HDV, and the indirect interplay between the two viruses via innate immune responses. This model will be instrumental to assess molecular and immunological mechanisms of this viral interference.

Introduction

Chronic hepatitis delta (CHD) affects 15–20 million people worldwide (5–10% of the hepatitis B virus (HBV)-infected patients) (Hughes et al., 2011). It is considered to be the most aggressive form of chronic viral hepatitis, with an accelerated progression towards fibrosis and cirrhosis and an increased risk of liver disease decompensation, hepatocellular carcinoma and premature death (Fattovich et al., 2000). Pegylated-alpha interferon (Peg-αIFN) remains the sole therapeutic option for these patients, leading to a low virological response rate (<30%) at 24 weeks post-treatment and high rate (>50%) of late relapse (Heidrich et al., 2014). The overall long-term sustained virologic response (SVR) rate is therefore very low in the clinical trial setting, and is even lower in the “real-life” clinical management (Yurdaydin, 2012). HBV-reverse transcriptase inhibitors have no effect on hepatitis D virus (HDV) replication. The pipeline of investigational drugs against HDV infection remains limited due to the fact that i) HDV does not encode enzymatic activities and uses cell DNA-dependent RNA polymerases (particularly RNA pol II) for its replication, ii) there are remaining gaps in the knowledge of the viral life-cycle, and iii) no appropriate in vitro model of this satellite co- or super-infection exists to screen antiviral drugs. Amongst few others, Myrcludex (a viral entry inhibitors) and farnesyl transferase (i.e. Lonafarnib) inhibitors are in early clinical trial evaluation (Koh et al., 2015, Bogomolov et al., 2016).

HDV is a subviral agent satellite of HBV, and its genome, the smallest known among mammalian viruses, has similarities to plant viroids. To ensure propagation, HDV relies on HBV, as HDV ribonucleoproteins are surrounded by HBV envelope-embedded glycoproteins. Furthermore, HDV entry into human hepatocytes is mediated through the large HBV envelope protein (L-HBsAg) interaction with the recently discovered cell surface HBV receptor, i.e. the human sodium taurocholate cotransporting polypeptide (hNTCP) (Yan et al., 2012).

HDV genome is a single-stranded circular RNA of ∼1680 bp, with high intra-molecular base pairing, allowing a rod-like structure folding. Its complementary ‘antigenomic’ strand encompasses the SHD gene that codes a single protein, the small, 24 kDa, HD protein (S-HDAg), which is essential for HDV RNA replication. At a later phase of the HDV replication cycle, SHD stop codon editing, catalyzed by Adenosine Deaminase acting on RNA-1 (ADAR-1), leads to the synthesis of a 19–20 amino-acid (aa) carboxy-terminal extended isoform of HDAg; this large, 27 kDa, protein (L-HDAg), thwarts HDV RNA replication and, in its farnesylated form, is involved in particle assembly (Lai, 2005, Taylor, 2012).

Both clinical and experimental data support the existence of viral interference between HDV and HBV. In the clinical setting, most patients infected with both HBV and HDV feature a pattern of HDV dominance, with a significant decrease in HBV-DNA viral load, when compared to mono-infected patients (Schaper et al., 2010, Krogsgaard et al., 1987, Genesca et al., 1987). Moreover, studies on liver biopsies from chronically HDV-infected patients have shown a decreased level of HBV replicative intermediates in the liver (Pollicino et al., 2011). Finally, this negative interference has been confirmed in vivo, in super-infection conditions, using HBV-infected chimpanzees, woodchuck hepatitis virus (WHV)-infected woodchucks, and more recently HBV-infected humanized mice (Rizzetto et al., 1980, Lütgehetmann et al., 2012, Negro et al., 1989, Sureau et al., 1989).

To understand the molecular basis of HDV interference on HBV, relevant infection-based in vitro models are essential. Viral interference has been observed in Huh7 cells by transfection of DNA vectors expressing HBV and HDV (or either HDAg isoforms) (Wu et al., 1991). Direct inhibition of HBV enhancer-1 and activation of MxA gene, an interferon-stimulated gene (ISG) known to suppress HBV replication, have been documented in the same cell line (Williams et al., 2009). However, transfection models with cDNAs expressing HDV genome have limitations and protein overexpression may lead to inaccurate assumptions. To explore HBV/HDV interference, the access to a cell culture model featuring both cccDNA formation and a competent innate immunity would be instrumental. Until recently, the knowledge on innate immune response related to HDV infection remained scarce. After in vitro studies suggesting a modulation of the IFN response (McNair et al., 1994, Pugnale et al., 2009), recent data from mouse models (both the humanized uPA-SCID and the hNTCP transgenic mice) revealed a strong induction of the intra-hepatocyte ISG expression (Giersch et al., 2015, He et al., 2015). Further knowledge on the interactions between HDV and the innate immune system could be invaluable to get insights on the interplay between HDV and its helper, as well as to identify novel therapeutic strategies.

The aim of this study was to establish a novel cellular model of HDV super-infection, and characterize HBV/HDV interactions via direct viral interference mechanisms or through hepatocyte innate immune response to infection. This model could furthermore allow an evaluation of novel drugs on HDV replication. Using the differentiated HepaRG (dHepaRG) cells, which are immune-competent (Luangsay et al., 2015a), we confirmed an efficient suppression of HBV replication (i.e. inhibition of intracellular HBV RNA and DNA accumulation, as well as HBeAg secretion), with no detectable effect on cccDNA nor HBsAg expression, and showed that HDV infection is associated with induction of ISGs, but not with induction of NF-kappaB regulated genes. Finally, we demonstrate the usefulness of this model with respect to antiviral discovery, by studying the antiviral activity of interferon alpha, specific anti-HBV and investigational specific anti-HDV drugs.