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