Up-regulation of nuclear factor E2 – Related factor 2 upon SVCV infection

doi:10.1016/j.fsi.2014.07.015

Fish & Shellfish Immunology

Volume 40, Issue 1, September 2014, Pages 245-252

https://doi.org/10.1016/j.fsi.2014.07.015 Get rights and content

Highlights

•
The Nrf2 gene of Pimephales promelas was cloned.
•
The expression profile of Nrf2 was investigated during SVCV infection in EPC cells.
•
SVCV infection facilitated the nuclear accumulation of Nrf2 and up-regulated its transcription and translation in EPC cells.

Abstract

Nuclear factor E2 – related factor 2 (Nrf2) is a crucial transcription factor that regulates the basal and inducible expression of many antioxidant response element (ARE)-dependent genes, including heme oxygenase-1 (HO-1) and superoxide dismutase 1 (SOD1). The Nrf2/ARE pathway has been regarded as a critical switch in the initiation of cellular defence systems for surviving oxidative insults and viral infection. In this study, the Nrf2 gene of EPC cells, which is originally derived from Pimephales promelas, was cloned, and an investigation on the interactions between Nrf2 and spring viraemia of carp virus (SVCV) was performed. These results demonstrated that the virus facilitated the nuclear accumulation of Nrf2 and up-regulated its transcriptional and protein profiles in EPC cells. In addition, exogenous activation of Nrf2 conferred EPC cells with a higher cellular total antioxidant capacity via an increase in the expression of HO-1 and SOD1, but did not suppress the replication of SVCV.

Introduction

An increasing number of publications have documented that oxidative stress induced by viral infection is one of the major pathogenic mechanisms(s) and may contribute to inflammatory responses and tissue injury by undermining the balance of intracellular redox-cycling [1], [2]. The imbalance between ROS/RNS production and antioxidant defence system is not only involved in the perturbation/disruption of normal cellular functions but also in the progression of viral amplification [3], [4], [5].

Nuclear factor E2 – related factor 2 (Nrf2), which manipulates versatile cytoprotective genes, is the master gene of cellular defence in the counteraction with oxidative stress [6]. Normally, Nrf2 is anchored to the cytoplasm, via covalent linkage with the actin-binding protein, Keap1 (Kelch-like ECH-associated protein 1). However, once exposed to an array of stimuli, Nrf2 escapes from Keap1's suppression and translocates into the nucleus, thereby initiating cellular stress responses driven by AREs [7].

Downstream effective genes, which are mediated by the Nrf2/ARE signal transduction pathway, play a fundamental role in cellular detoxification, anti-oxidative, ant-inflammatory as well as anti-apoptotic capacities [8], [9], [10]. They may also affect viral infection [11]. Activation or overexpression of Nrf2 has been shown to nullify potentially detrimental chemicals, relieve the ROS-related diseases (Alzheimer's syndrome, Parkinsonism disease) [12], [13], [14] and lower specific virus-induced oxidative injury [15]. Therefore, a novel antiviral strategy, including targeting of the Nrf2-ARE pathway should be tested. However, with regard to fish viruses, few studies have examined in the role of Nrf2 in viral infection.

Spring Viraemia of Carp Virus (SVCV), which is the etiological agent of spring viraemia of carp (SVC), is a member of the genus Vesiculovirus (family Rhabdoviridae) [16], was used to investigate the interactions between Nrf2 and the virus. SVCV is a highly pathogenic virus, and SVC is a widespread disease in the aquaculture of common carp (Cyprinus carpio). Outbreaks of SVC usually cause mass morbidity and mortality [17]. However, the pathogenesis of SVCV remains unclear, even though SVC was listed as a notifiable disease by the OIE (Office International des Epizooties) early in 2000.

In the present study, we demonstrated that the transcription and expression level of Nrf2 was up-regulated in SVCV-infected cells. In addition, sulforaphane (SFN) exogenously induced the nuclear translocation of Nrf2, thereby enhancing the cellular total antioxidant capacity, but did not suppress the replication of SVCV.

Section snippets

Cell, virus and chemicals

Epithelioma Papulosum Cyprini (EPC, ATCC: CRL-2872) cells are SVCV-sensitive and were cultured in Eagle's minimum essential medium (MEM, Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS, Gibco, Thermo Fisher) at 25 °C. SVCV (ATCC: VR-1390) was used for infection and was a kind gift from Professor Yuanan Lu (University of Hawaii at Manoa). The Nrf2-inducer, d,l-sulforaphane (SFN), was purchased from Sigma–Aldrich (S4441) and diluted in a series of gradient concentrations

Molecular characterization of Nrf2

Full-length Nrf2 cDNA derived from EPC cells was generated using RT-PCR and 5′/3′-RACE. According to sequence analysis, the Nrf2 cDNA was 2115 bp long, with 159 bp and 183 bp presented in the 3′/5′ flanking section, respectively. A typical poly(A) tail signal (AATAAA) was also be found in the 3′ non-coding region. The deduced amino acid sequence, encoded by a 1773 bp open reading frame, predicted a protein of 590 amino acid residues with a highly conserved CNC-bZip domain (AA⁴⁴¹–AA⁵⁵²) (Fig. 1

Discussion

As an important transcription factor, Nrf2 plays a pivotal role in maintaining intracellular homoeostasis and in the defence against invasive pathogens by coordinately activating many cytoprotective genes [24]. In this study, we cloned the Nrf2 gene from EPC cells and performed a detailed investigation of the interactions between Nrf2 and SVCV infection for the first time. Our data demonstrated that SVCV infection up-regulated the expression of Nrf2, and exogenous activation of Nrf2 was

Authors' contributions

JY and ZW conceived and designed the study. YY and LL performed most of the experiment, analysed the data and drafted the manuscript. JH and YZ participated in the determination of cellular total antioxidant capacity. JY, XC, LL and XL participated in the coordination of the study and revised the manuscript. All of the authors read and approved the final version of the manuscript.

Acknowledgements

We acknowledge with gratitude Dr Ken McColl (Australian Animal Health Laboratory, Geelong, Victoria) for critically reading the manuscript. This work was jointly supported by the National Natural Science Foundation of China (30700624) and the Fundamental Research Funds for the Central Universities (2662013PY070).

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Full length articleUp-regulation of nuclear factor E2 – Related factor 2 upon SVCV infection

Highlights

Abstract

Introduction

Section snippets

Cell, virus and chemicals

Molecular characterization of Nrf2

Discussion

Authors' contributions

Acknowledgements

Virology

Exp Mol Pathol

Adv Pharmacol

Mutat Res

Med Mal Infect

Free Radic Biol Med

Anal Biochem

Fish Shellfish Immunol

Biochem Pharmacol

Redox Biol

Toxicol Appl Pharmacol

Fish Shellfish Immunol

J Biol Chem

J Biol Chem

Food Chem Toxicol

Free Radic Biol Med

J Mol Biol

J Virol Methods

Interplay between Hepatitis C virus and redox cell signaling

Int J Mol Sci

Redox imbalance and its control in HIV infection

Antioxid Redox Signal

Viral-mediated inhibition of antioxidant enzymes contributes to the pathogenesis of severe respiratory syncytial virus bronchiolitis

Am J Respir Crit Care Med

Molecular mechanisms of the Keap1–Nrf2 pathway in stress response and cancer evolution

Genes Cells

Molecular mechanisms activating the Nrf2–Keap1 pathway of antioxidant gene regulation

Antioxid Redox Signal

Full length article
Up-regulation of nuclear factor E2 – Related factor 2 upon SVCV infection