Elsevier

Journal of Hepatology

Volume 61, Issue 4, October 2014, Pages 746-754
Journal of Hepatology

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

https://doi.org/10.1016/j.jhep.2014.05.026Get rights and content

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.

Introduction

Although hepatitis E virus (HEV) infection is underdiagnosed, it is clear that the virus represents one of the most abundant infectious challenges to humans [1]. In Western countries, HEV infection of healthy individuals almost exclusively remains subclinical and otherwise causes an acute and self-limiting infection in immune-competent individuals with low mortality rates [2]. In contrast, patients with HEV infection in immunocompromised individuals that include organ transplantation recipients [3], HIV patients [4] and cancer patients receiving chemotherapy [5] have a substantially high risk of developing chronic hepatitis. The use of immunosuppressants, such as rapalogs, in organ transplant recipients to prevent rejection is associated with substantial pathology and in particular an increased risk of developing chronic hepatitis with substantial graft loss and mortality rates [6].

However, in undernourished populations in the developing world, fulminant hepatitis and high mortality are described, reaching 25% in the case of pregnant women [7]. In the current (2012–2013) hepatitis E outbreak among refugees in South Sudan, a total of 5080 acute jaundice syndrome cases had been reported from all four Maban County refugee camps, as of January 27, 2013. An acute jaundice syndrome case-fatality rate of 10.4% was observed among pregnant women across all camps [8]. Humans appear to have powerful HEV combating mechanisms, but these apparently require a good nutritional and host defence status for optimal functionality [9]. The nature of these mechanisms has not been characterised, due to the lack of robust HEV cell culture models. The advent of new technology that mimics the HEV infectious process in vitro, in particular the development of in vitro adapted infectious clones and subgenomic HEV reporters, has led to hopes that the mechanisms that control HEV infection in normal physiology can now be identified [10], [11].

Rapalogs comprise, amongst others rapamycin (RAPA, rapamune, sirolimus; originally isolated from Streptomyces hygroscopicus) and everolimus (the 40-O-[2-hydroxyethyl] derivative of rapamycin). This immunosuppressive medication is gaining increasing popularity in the transplantation context, mainly because of its low nephrotoxicity [12]. Their molecular mode of action is well characterised and involves inhibition of the mammalian target of rapamycin (mTOR) pathway. mTOR is a central element within the phosphatidylinositol-3 kinase (PI3K)-protein kinase B (PKB)-mTOR signaling [13] and integrates nutritional information and receptor tyrosine kinase signaling to control cellular growth via a variety of cellular effectors, including activation of p70 S6 kinase and subsequent protein synthesis as well as inhibition of autophagy. Activation of PI3K-PKB-mTOR signaling following viral infection of liver cells has been reported and linked to both viral supportive functions (e.g., prevention of apoptosis in hepatitis C-infected cells) [14], but also to the induction of the production of antiviral interferons [15]. Thus, generally speaking the role of this signaling cascade in combating viral infection of the liver remains unclear, prompting further research.

Given the important and increasing role of rapalog implications in clinical practice and the lack of insight into the mechanisms employed by the body to constrain HEV infection, we investigated the role of the PI3K-PKB-mTOR signaling cascade in HEV infection using state-of-the-art cell culture models. These results show that mTOR inhibition drastically promotes HEV replication in an autophagy-independent fashion but through phosphorylated 4E-BP1 in infected hepatocytes.

Section snippets

Reagents

Stocks of rapamycin (Merck, Schiphol-Rijk, The Netherlands) and everolimus (Sigma-Aldrich, St Louis, MO) were dissolved in dimethyl sulfoxide (DMSO) (Sigma-Aldrich, St Louis, MO) with a final concentration of 2 mM. Stocks of LY294022, an inhibitor of PI3K-PKB (Sigma-Aldrich), BEZ235, a dual inhibitor of PI3K-PKB and mTOR (Selleck Chemicals), FG-4592, an inhibitor of HIF-1α (Selleck Chemicals) and PF-478671, an inhibitor of p70 S6 kinase (Selleck Chemicals) were dissolved in DMSO. All agents were

mTOR inhibition by rapalogs facilitates HEV replication

The 7.2-kb genome of HEV is a single strand positive-sense of RNA containing three overlapping reading frames (ORFs). We employed a model, in which human hepatoma cells (HuH7) were transfected with a 3′ subgenomic construct of the HEV coding sequence, in which the 5′ portion of ORF2 was replaced with the in-frame secreted form of luciferase derived from the marine copepod Gaussia princeps (p6-luc) (Supplementary Fig. 1). Accumulation of luciferase in HuH7 cells thus serves as reporter for HEV

Discussion

Large zoonotic reservoirs of hepatitis E exist in cattle and poultry and it is generally accepted that humans are frequently infected with the virus [7]. Almost invariably, however, the disease remains subclinical [2]. Here we present evidence that the inability of HEV to effectively replicate in humans is linked to constitutive mTOR activation. This novel action of mTOR in directly counteracting viral replication in liver cells themselves rather than acting through the adapted immune system,

Conflict of interest

The authors who have taken part in this study declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.

Financial support

Supported by the Netherlands Organisation for Scientific Research (NOW/ZonMw) for a VENI grant (No. 916-13-032) (to Q. Pan), the Dutch Digestive Foundation (MLDS) for a career development grant (No. CDG 1304) (to Q. Pan), the European Association for the Study of the Liver (EASL) for a Sheila Sherlock Fellowship (to Q. Pan), the Daniel den Hoed Foundation for a Centennial Award grant (to Q. Pan) and the China Scholarship Council for funding PhD fellowships to X. Zhou (No. 201206150075) and Y.

Authors’ contributions

X.Z. performed the experiments, analysed data and wrote a draft of the paper. Y.W. performed some experiments. H.J.M. and H.L.J. discussed the project and edited the manuscript. M.P.P. and Q.P. conceived the ideas, designed and discussed experiments, supervised the project and extensively edited the manuscript.

Acknowledgement

The authors would like to thank Dr. Suzanne U. Emerson (National Institute of Allergy and Infectious Diseases, NIH, USA) for generously providing the plasmids to generate subgenomic and full-length HEV genomic RNA.

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