Xinying Zhou

Rapamycin and everolimus facilitate hepatitis e virus replication: Revealing a basal defense mechanism of PI3K-PKB-mTOR pathway

BACKGROUND & AIMS Humans are frequently exposed to hepatitis E virus (HEV). Nevertheless, the disease mainly affects pregnant women and immunocompromised individuals. Organ recipients receiving immunosuppressants, such as rapalogs, to prevent rejection have a high risk for developing chronic hepatitis following HEV infection. Rapalogs constitute potent inhibitors of mTOR including rapamycin and everolimus. As a master kinase, the mechanism-of-action of mTOR is not only associated with the immunosuppressive capacity of rapalogs but is also tightly regulated during pregnancy because of increased nutritional demands. METHODS We thus investigated the role of mTOR in HEV infection by using two state-of-the-art cell culture models: a subgenomic HEV containing luciferase reporter and a full-length HEV infectious cell culture system. RESULTS In both subgenomic and full-length HEV models, HEV infection was aggressively escalated by treatment of rapamycin or everolimus. Inhibition of mTOR was confirmed by Western blot showing the inhibition of its downstream target, S6 phosphorylation. Consistently, stable silencing of mTOR by lentiviral RNAi resulted in a significant increase in intracellular HEV RNA, suggesting an antiviral function of mTOR in HEV infection. By targeting a series of other up- and downstream elements of mTOR signaling, we further revealed an effective basal defense mechanism of the PI3K-PKB-mTOR pathway against HEV, which is through the phosphorylated eIF4E-binding protein 1 (4E-BP1), however independent of autophagy formation. CONCLUSIONS The discovery that PI3K-PKB-mTOR pathway limits HEV infection through 4E-BP1 and acts as a gate-keeper in human HEV target cells bears significant implications in managing immunosuppression in HEV-infected organ transplantation recipients.

Epidemiology and management of chronic hepatitis E infection in solid organ transplantation: a comprehensive literature review

Hepatitis E virus (HEV) infection has emerged as a global public health issue. Although it often causes an acute and self‐limiting infection with low mortality rates in the western world, it bears a high risk of developing chronic hepatitis in immunocompromised patients with substantial mortality rates. Organ transplant recipients who receive immunosuppressive medication to prevent rejection are thought to be the main population at risk for chronic hepatitis E. Therefore, there is an urgent need to properly evaluate the clinical impact of HEV in these patients. This article aims to review the prevalence, infection course, and management of HEV infection after solid organ transplantation by performing a comprehensive literature review. In addition, an in‐depth emphasis of this clinical issue and a discussion of future development are also presented. Copyright

Hepatitis E virus infects neurons and brains.

Hepatitis E virus (HEV), as a hepatotropic virus, is supposed to exclusively infect the liver and only cause hepatitis. However, a broad range of extrahepatic manifestations (in particular, idiopathic neurological disorders) have been recently reported in association with its infection. In this study, we have demonstrated that various human neural cell lines (embryonic stem cell-derived neural lineage cells) induced pluripotent stem cell-derived human neurons and primary mouse neurons are highly susceptible to HEV infection. Treatment with interferon-α or ribavirin, the off-label antiviral drugs for chronic hepatitis E, exerted potent antiviral activities against HEV infection in neural cells. More importantly, in mice and monkey peripherally inoculated with HEV particles, viral RNA and protein were detected in brain tissues. Finally, patients with HEV-associated neurological disorders shed the virus into cerebrospinal fluid, indicating a direct infection of their nervous system. Thus, HEV is neurotropic in vitro, and in mice, monkeys, and possibly humans. These results challenge the dogma of HEV as a pure hepatotropic virus and suggest that HEV infection should be considered in the differential diagnosis of idiopathic neurological disorders.

IFN regulatory factor 1 restricts hepatitis E virus replication by activating STAT1 to induce antiviral IFN-stimulated genes

IFN regulatory factor 1 (IRF1) is one of the most important IFN‐stimulated genes (ISGs) in cellular antiviral immunity. Although hepatitis E virus (HEV) is a leading cause of acute hepatitis worldwide, how ISGs counteract HEV infection is largely unknown. This study was conducted to investigate the effect of IRF1 on HEV replication. Multiple cell lines were used in 2 models that harbor HEV. In different HEV cell culture systems, IRF1 effectively inhibited HEV replication. IRF1 did not trigger IFN production, and chromatin immunoprecipitation sequencing data analysis revealed that IRF1 bound to the promoter region of signal transducers and activators of transcription 1 (STAT1). Functional assay confirmed that IRF1 could drive the transcription of STAT1, resulting in elevation of total and phosphorylated STAT1 proteins and further activating the transcription of a panel of downstream antiviral ISGs. By pharmacological inhibitors and RNAi‐mediated gene‐silencing approaches, we revealed that antiviral function of IRF1 is dependent on the JAK‐STAT cascade. Furthermore, induction of ISGs and the anti‐HEV effect of IRF1 overlapped that of IFNα, but was potentiated by ribavirin. We demonstrated that IRF1 effectively inhibits HEV replication through the activation of the JAK‐STAT pathway, and the subsequent transcription of antiviral ISGs, but independent of IFN production.—Xu, L., Zhou, X., Wang, W., Wang, Y., Yin, Y., van der Laan, L. J. W., Sprengers, D., Metselaar, H. J., Peppelenbosch, M. P., Pan, Q. IFN regulatory factor 1 restricts hepatitis E virus replication by activating STAT1 to induce antiviral IFN‐stimulated genes. FASEB J. 30, 3352–3367 (2016). www.fasebj.org

Convergent Transcription of Interferon-stimulated Genes by TNF-α and IFN-α Augments Antiviral Activity against HCV and HEV

IFN-α has been used for decades to treat chronic hepatitis B and C, and as an off-label treatment for some cases of hepatitis E virus (HEV) infection. TNF-α is another important cytokine involved in inflammatory disease, which can interact with interferon signaling. Because interferon-stimulated genes (ISGs) are the ultimate antiviral effectors of the interferon signaling, this study aimed to understand the regulation of ISG transcription and the antiviral activity by IFN-α and TNF-α. In this study, treatment of TNF-α inhibited replication of HCV by 71 ± 2.4% and HEV by 41 ± 4.9%. Interestingly, TNF-α induced the expression of a panel of antiviral ISGs (2-11 fold). Blocking the TNF-α signaling by Humira abrogated ISG induction and its antiviral activity. Chip-seq data analysis and mutagenesis assay further revealed that the NF-κB protein complex, a key downstream element of TNF-α signaling, directly binds to the ISRE motif in the ISG promoters and thereby drives their transcription. This process is independent of interferons and JAK-STAT cascade. Importantly, when combined with IFN-α, TNF-α works cooperatively on ISG induction, explaining their additive antiviral effects. Thus, our study reveals a novel mechanism of convergent transcription of ISGs by TNF-α and IFN-α, which augments their antiviral activity against HCV and HEV.

RIG‐I is a key antiviral interferon‐stimulated gene against hepatitis E virus regardless of interferon production

Interferons (IFNs) are broad antiviral cytokines that exert their function by inducing the transcription of hundreds of IFN‐stimulated genes (ISGs). However, little is known about the antiviral potential of these cellular effectors on hepatitis E virus (HEV) infection, the leading cause of acute hepatitis globally. In this study, we profiled the antiviral potential of a panel of important human ISGs on HEV replication in cell culture models by overexpression of an individual ISG. The mechanism of action of the key anti‐HEV ISG was further studied. We identified retinoic acid–inducible gene I (RIG‐I), melanoma differentiation–associated protein 5, and IFN regulatory factor 1 (IRF1) as the key anti‐HEV ISGs. We found that basal expression of RIG‐I restricts HEV infection. Pharmacological activation of the RIG‐I pathway by its natural ligand 5′‐triphosphate RNA potently inhibits HEV replication. Overexpression of RIG‐I activates the transcription of a wide range of ISGs. RIG‐I also mediates but does not overlap with IFN‐α‐initiated ISG transcription. Although it is classically recognized that RIG‐I exerts antiviral activity through the induction of IFN production by IRF3 and IRF7, we reveal an IFN‐independent antiviral mechanism of RIG‐I in combating HEV infection. We found that activation of RIG‐I stimulates an antiviral response independent of IRF3 and IRF7 and regardless of IFN production. However, it is partially through activation of the Janus kinase (JAK)–signal transducer and activator of transcription (STAT) cascade of IFN signaling. RIG‐I activated two distinct categories of ISGs, one JAK‐STAT‐dependent and the other JAK‐STAT‐independent, which coordinately contribute to the anti‐HEV activity. Conclusion: We identified RIG‐I as an important anti‐HEV ISG that can be pharmacologically activated; activation of RIG‐I stimulates the cellular innate immunity against HEV regardless of IFN production but partially through the JAK‐STAT cascade of IFN signaling. (Hepatology 2017;65:1823‐1839).

Disparity of basal and therapeutically activated interferon signalling in constraining hepatitis E virus infection

Hepatitis E virus (HEV) represents one of the foremost causes of acute hepatitis globally. Although there is no proven medication for hepatitis E, pegylated interferon‐α (IFN‐α) has been used as off‐label drug for treating HEV. However, the efficacy and molecular mechanisms of how IFN signalling interacts with HEV remain undefined. As IFN‐α has been approved for treating chronic hepatitis C (HCV) for decades and the role of interferon signalling has been well studied in HCV infection, this study aimed to comprehensively investigate virus–host interactions in HEV infection with focusing on the IFN signalling, in comparison with HCV infection. A comprehensive screen of human cytokines and chemokines revealed that IFN‐α was the sole humoral factor inhibiting HEV replication. IFN‐α treatment exerted a rapid and potent antiviral activity against HCV, whereas it had moderate and delayed anti‐HEV effects in vitro and in patients. Surprisingly, blocking the basal IFN pathway by inhibiting JAK1 to phosphorylate STAT1 has resulted in drastic facilitation of HEV, but not HCV infection. Gene silencing of the key components of JAK‐STAT cascade of the IFN signalling, including JAK1, STAT1 and interferon regulatory factor 9 (IRF9), stimulated HEV infection. In conclusion, compared to HCV, HEV is less sensitive to IFN treatment. In contrast, the basal IFN cascade could effectively restrict HEV infection. This bears significant implications in management of HEV patients and future therapeutic development.

Unphosphorylated ISGF3 drives constitutive expression of interferon-stimulated genes to protect against viral infections

A tripartite transcription factor complex mediates an interferon-independent antiviral response. An interferon-independent antiviral defense Virally infected cells produce type I interferons (IFNs), which stimulate the phosphorylation and activation of the STAT1 and STAT2 transcription factors. When combined with the transcriptional regulator IRF9, phosphorylated STAT1 and STAT2 form the ISGF3 complex, which drives the expression of IFN-stimulated genes (ISGs) that are important for antiviral immunity. Wang et al. report the formation of an alternative form of ISGF3, U-ISGF3, which drove the expression of ISGs and protected cells from viral infection in the absence of detection of detectable IFN or IFN signaling. In addition to IRF9, U-ISGF3 contained unphosphorylated STAT1 and STAT2. Together, these data suggest that U-ISGF3 drives constitutive expression of ISGs as part of an IFN-independent, antiviral immune response. Interferon (IFN)–stimulated genes (ISGs) are antiviral effectors that are induced by IFNs through the formation of a tripartite transcription factor ISGF3, which is composed of IRF9 and phosphorylated forms of STAT1 and STAT2. However, we found that IFN-independent ISG expression was detectable in immortalized cell lines, primary intestinal and liver organoids, and liver tissues. The constitutive expression of ISGs was mediated by the unphosphorylated ISGF3 (U-ISGF3) complex, consisting of IRF9 together with unphosphorylated STAT1 and STAT2. Under homeostatic conditions, STAT1, STAT2, and IRF9 were found in the nucleus. Analysis of a chromatin immunoprecipitation sequencing data set revealed that STAT1 specifically bound to the promoters of ISGs even in the absence of IFNs. Knockdown of STAT1, STAT2, or IRF9 by RNA interference led to the decreased expression of various ISGs in Huh7.5 human liver cells, which was confirmed in mouse embryonic fibroblasts (MEFs) from STAT1−/−, STAT2−/−, or IRF9−/− mice. Furthermore, decreased ISG expression was accompanied by increased replication of hepatitis C virus and hepatitis E virus. Conversely, simultaneous overexpression of all ISGF3 components, but not any single factor, induced the expression of ISGs and inhibited viral replication; however, no phosphorylated STAT1 and STAT2 were detected. A phosphorylation-deficient STAT1 mutant was comparable to the wild-type protein in mediating the IFN-independent expression of ISGs and antiviral activity, suggesting that ISGF3 works in a phosphorylation-independent manner. These data suggest that the U-ISGF3 complex is both necessary and sufficient for constitutive ISG expression and antiviral immunity under homeostatic conditions.

Cross Talk between Nucleotide Synthesis Pathways with Cellular Immunity in Constraining Hepatitis E Virus Replication

ABSTRACT Viruses are solely dependent on host cells to propagate; therefore, understanding virus-host interaction is important for antiviral drug development. Since de novo nucleotide biosynthesis is essentially required for both host cell metabolism and viral replication, specific catalytic enzymes of these pathways have been explored as potential antiviral targets. In this study, we investigated the role of different enzymatic cascades of nucleotide biosynthesis in hepatitis E virus (HEV) replication. By profiling various pharmacological inhibitors of nucleotide biosynthesis, we found that targeting the early steps of the purine biosynthesis pathway led to the enhancement of HEV replication, whereas targeting the later step resulted in potent antiviral activity via the depletion of purine nucleotide. Furthermore, the inhibition of the pyrimidine pathway resulted in potent anti-HEV activity. Interestingly, all of these inhibitors with anti-HEV activity concurrently triggered the induction of antiviral interferon-stimulated genes (ISGs). Although ISGs are commonly induced by interferons via the JAK-STAT pathway, their induction by nucleotide synthesis inhibitors is completely independent of this classical mechanism. In conclusion, this study revealed an unconventional novel mechanism of cross talk between nucleotide biosynthesis pathways and cellular antiviral immunity in constraining HEV infection. Targeting particular enzymes in nucleotide biosynthesis represents a viable option for antiviral drug development against HEV. HEV is the most common cause of acute viral hepatitis worldwide and is also associated with chronic hepatitis, especially in immunocompromised patients. Although often an acute and self-limiting infection in the general population, HEV can cause severe morbidity and mortality in certain patients, a problem compounded by the lack of FDA-approved anti-HEV medication available. In this study, we have investigated the role of the nucleotide synthesis pathway in HEV infection and its potential for antiviral drug development. We show that targeting the later but not the early steps of the purine synthesis pathway exerts strong anti-HEV activity. In particular, IMP dehydrogenase (IMPDH) is the most important anti-HEV target of this cascade. Importantly, the clinically used IMPDH inhibitors, including mycophenolic acid and ribavirin, have potent anti-HEV activity. Furthermore, targeting the pyrimidine synthesis pathway also exerts potent antiviral activity against HEV. Interestingly, antiviral effects of nucleotide synthesis pathway inhibitors appear to depend on the medication-induced transcription of antiviral interferon-stimulated genes. Thus, this study reveals an unconventional novel mechanism as to how nucleotide synthesis pathway inhibitors can counteract HEV replication.

Requirement of the eukaryotic translation initiation factor 4F complex in hepatitis E virus replication.

Hepatitis E virus (HEV) infection, one of the foremost causes of acute hepatitis, is becoming a health problem of increasing magnitude. As other viruses, HEV exploits elements from host cell biochemistry, but we understand little as to which components of the human hepatocellular machinery are perverted for HEV multiplication. It is, however, known that the eukaryotic translation initiation factors 4F (eIF4F) complex, the key regulator of the mRNA-ribosome recruitment phase of translation initiation, serves as an important component for the translation and replication of many viruses. Here we aim to investigate the role of three subunits of the eIF4F complex: eukaryotic translation initiation factor 4A (eIF4A), eukaryotic translation initiation factor 4G (eIF4G) and eukaryotic translation initiation factor 4E (eIF4E) in HEV replication. We found that efficient replication of HEV requires eIF4A, eIF4G and eIF4E. Consistently, the negative regulatory factors of this complex: programmed cell death 4 (PDCD4) and eIF4E-binding protein 1 (4E-BP1) exert anti-HEV activities, which further illustrates the requirement for eIF4A and eIF4E in supporting HEV replication. Notably, phosphorylation of eIF4E induced by MNK1/2 activation is not involved in HEV replication. Although ribavirin and interferon-α (IFN-α), the most often-used off-label drugs for treating hepatitis E, interact with this complex, their antiviral activities are independent of eIF4E. In contrast, eIF4E silencing provokes enhanced anti-HEV activity of these compounds. Thus, HEV replication requires eIF4F complex and targeting essential elements of this complex provides important clues for the development of novel antiviral therapy against HEV.