The RIG-I-like RNA helicase (RLR)-mediated interferon (IFN) response plays a pivotal role in the hepatic antiviral immunity. The hepatitis A virus (HAV) and the hepatitis C virus (HCV) counter this response by encoding a viral protease that cleaves the mitochondria antiviral signaling protein (MAVS), a common signaling adaptor for RLRs. However, a third hepatotropic RNA virus, the hepatitis E virus (HEV), does not appear to encode a functional protease yet persists in infected cells. We investigated HEV-induced IFN responses in human hepatoma cells and primary human hepatocytes. HEV infection resulted in persistent virus replication despite poor spread. This was companied by a type III IFN response that upregulated multiple IFN-stimulated genes (ISGs), but type I IFNs were barely detected. Blocking type III IFN production or signaling resulted in reduced ISG expression and enhanced HEV replication. Unlike HAV and HCV, HEV did not cleave MAVS; MAVS protein size, mitochondrial localization, and function remained unaltered in HEV-replicating cells. Depletion of MAVS or MDA5, and to a less extent RIG-I, also diminished IFN production and increased HEV replication. Furthermore, persistent activation of the JAK/STAT signaling rendered infected cells refractory to exogenous IFN treatment, and depletion of MAVS or the receptor for type III IFNs restored the IFN responsiveness. Collectively, these results indicate that unlike other hepatotropic RNA viruses, HEV does not target MAVS and its persistence is associated with continuous production of type III IFNs. HEV infection is a common cause for acute viral hepatitis worldwide. Approximately 20 millions of people are infected annually. In immunocompetent hosts the infection is self-limited and mostly asymptomatic, but the virus frequently persists when immunity is compromised leading to increased risk for cirrhosis. Currently there are no FDA-approved diagnostics or treatments for HEV. Understanding how HEV induces and manipulates host innate immune responses will help elucidate the mechanism(s) of HEV persistence and identify potential targets for therapy. Our results show that unlike other hepatotropic RNA viruses, HEV did not cleave MAVS and stimulated a sustained type III IFN response in persistently infected cells. Furthermore, the JAK/STAT pathway was persistently activated in HEV-replicating cells and responded poorly to exogenously added IFNs. This study uncovers a unique interplay between HEV and the host IFN pathway and provides insight into the mechanism of HEV persistence in patients.

Hepatitis E virus (HEV), an enterically transmitted hepatotropic virus, was thought to be non-enveloped for decades. However, recent studies have revealed that the virus circulating in the patient’s blood is completely cloaked in host membranes and resistant to neutralizing antibodies. The discovery of this novel enveloped form of HEV has raised a series of questions about the fundamental biology of HEV and the way this virus, which has been understudied in the past, interacts with its host. Here, we review recent advances towards understanding this phenomenon and discuss its potential impact on various aspects of the HEV life cycle and immunity.

Hepatitis C virus (HCV) is a major cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma in humans. The life cycle of HCV is closely associated with the metabolism of lipids, especially very-low-density lipoprotein (VLDL) in hepatocytes. Hepatocyte nuclear factor 4α (HNF4α), the most abundant transcription factor in the liver, regulates the VLDL secretory pathway. However, the effects of HNF4α on the HCV life cycle are unclear. In this study, we investigated the regulatory effects of HNF4α on HCV assembly and secretion. HCV in HNF4α-deficient hepatocytes showed reduced assembly and secretion but unchanged entry and RNA replication. Bezafibrate, a chemical inhibitor of HNF4α, suppressed HCV assembly and secretion. HNF4α downregulation resulted in rearrangement of cytosolic lipid droplets (LDs), as evidenced by the aggregation of large LDs and distorted cytosolic distribution. Phospholipase A ₂ GXIIB (PLA ₂ GXIIB), an HNF4α-regulated factor involved in VLDL secretion, was found to be crucial in HCV secretion. PLA ₂ GXIIB expression was upregulated in hepatocytes harboring HCV subgenomic replicons or in HCV-infected hepatocytes. This upregulation was transcriptionally controlled in an HNF4α-dependent manner after HCV infection. Furthermore, PLA ₂ GXIIB combined with microsomal triglyceride transfer protein was found to be responsible for the regulation of HNF4α-induced HCV infectivity. These results suggest that HNF4α and its downstream PLA ₂ GXIIB are important factors affecting the late stage of the HCV life cycle and may serve as potential drug targets for the treatment of HCV infection.

Extracellular vesicles (EVs) are nano-sized membrane-limited organelles that are liberated from their producer cells, traverse the intercellular space, and may interact with other cells resulting in the uptake of the EV molecular payload by the recipient cells which may become functionally reprogramed as a result. Previous in vitro studies showed that EVs purified from normal mouse AML12 hepatocytes (“EV^Norm”) attenuate the pro-fibrogenic activities of activated hepatic stellate cells (HSCs), a principal fibrosis-producing cell type in the liver. In a 10-day CCl₄ injury model, liver fibrogenesis, expression of hepatic cellular communication network factor 2 [CCN2, also known as connective tissue growth factor (CTGF)] or alpha smooth muscle actin (αSMA) was dose-dependently blocked during concurrent administration of EV^Norm. Hepatic inflammation and expression of inflammatory cytokines were also reduced by EV^Norm. In a 5-week CCl₄ fibrosis model in mice, interstitial collagen deposition and mRNA and/or protein for collagen 1a1, αSMA or CCN2 were suppressed following administration of EV^Norm over the last 2 weeks. RNA sequencing (RNA-seq) revealed that EV^Norm therapy of mice receiving CCl₄ for 5 weeks resulted in significant differences [false discovery rate (FDR) <0.05] in expression of 233 CCl₄-regulated hepatic genes and these were principally associated with fibrosis, cell cycle, cell division, signal transduction, extracellular matrix (ECM), heat shock, cytochromes, drug detoxification, adaptive immunity, and membrane trafficking. Selected gene candidates from these groups were verified by qRT-PCR as targets of EV^Norm in CCl₄-injured livers. Additionally, EV^Norm administration resulted in reduced activation of p53, a predicted upstream regulator of 40% of the genes for which expression was altered by EV^Norm following CCl₄ liver injury. In vitro, EVs from human HepG2 hepatocytes suppressed fibrogenic gene expression in activated mouse HSC and reversed the reduced viability or proliferation of HepG2 cells or AML12 cells exposed to CCl₄. Similarly, EVs produced by primary human hepatocytes (PHH) protected PHH or human LX2 HSC from CCl₄-mediated changes in cell number or gene expression in vitro. These findings show that EVs from human or mouse hepatocytes regulate toxin-associated gene expression leading to therapeutic outcomes including suppression of fibrogenesis, hepatocyte damage, and/or inflammation.

Hepatitis C virus (HCV) causes not only severe liver problems but also extrahepatic manifestations, such as insulin resistance (IR). Wild-type peroxisome proliferator-activated receptor gamma coactivator 1 alpha (WT-PGC-1α) is essential in hepatic gluconeogenesis and has recently been demonstrated to link HCV infection to hepatic insulin resistance (IR). A recent study has characterized a novel human liver-specific PGC-1α (L-PGC-1α) transcript, which is proposed to reflect human adaption to more complex pathways. However, the effect of HCV infection on L-PGC-1α expression and the mechanism by which HCV modulates WT-PGC-1α/L-PGC-1α remain unclear. In this study, we showed that HCV infection upregulated both WT-PGC-1α and L-PGC-1α, which further promoted HCV production. The upregulation of both PGC-1α isoforms depended on HCV RNA replication. By using promoter-luciferase reporters, kinase inhibitors, and dominant negative mutants, we further observed that the HCV-induced upregulation of WT-PGC-1α was mediated by the phosphorylation of cyclic AMP (cAMP)-responsive element-binding protein (CREB), whereas that of L-PGC-1α was mediated by CREB phosphorylation and forkhead box O1 dephosphorylation. Moreover, HCV infection induced endoplasmic reticulum (ER) stress, and pharmacological induction of ER stress upregulated WT-PGC-1α/L-PGC-1α and phosphorylated CREB. In contrast, pharmacological inhibition of HCV-induced ER stress impaired WT-PGC-1α/L-PGC-1α upregulation along with decreased phosphorylated CREB. The correlation of hepatic mPGC-1α with ER stress was further confirmed in mice. Overall, HCV infection upregulates both WT-PGC-1α and L-PGC-1α through an ER stress-mediated, phosphorylated CREB-dependent pathway, and both PGC-1α isoforms promote HCV production in turn. IMPORTANCE HCV causes not only severe liver problems but also extrahepatic manifestations, such as insulin resistance (IR). As a key regulator in energy metabolism, wild-type PGC-1α (WT-PGC-1α), has recently been demonstrated to link HCV infection to hepatic IR. A recent study has characterized a novel human liver-specific PGC-1α (L-PGC-1α), which reflects human adaption to more complex pathways. However, the effect of HCV infection on L-PGC-1α expression and the mechanism by which HCV regulates WT-PGC-1α/L-PGC-1α remain unclear. In this study, we showed that HCV infection upregulated both WT-PGC-1α and L-PGC-1α, which further promoted HCV production. WT-PGC-1α upregulation was mediated by CREB phosphorylation, whereas L-PGC-1α upregulation was mediated by CREB phosphorylation and FoxO1 dephosphorylation. HCV-induced ER stress mediated WT-PGC-1α/L-PGC-1α upregulation and CREB phosphorylation. Overall, this study provides new insights into the mechanism by which HCV upregulates WT-PGC-1α/L-PGC-1α and highlights the novel intervention of HCV-ER stress-PGC-1α signaling for HCV therapy and HCV-induced IR therapy.