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Involvement of fish signal transducer and activator of transcription 3 (STAT3) in nodavirus infection induced cell death

https://doi.org/10.1016/j.fsi.2014.12.031Get rights and content

Highlights

  • STAT3 was activated during RGNNV infection in grouper cells.

  • Inhibition of Ec-STAT3 in vitro did not affect RGNNV replication.

  • RGNNV induced autophagy was significantly increased after inhibition of STAT3.

  • Inhibition of STAT3 significantly decreased RGNNV induced necrotic cell death.

  • Inhibition of STAT3 regulated the expression of host factors induced by RGNNV.

Abstract

The Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signaling pathway is an important signaling pathway activated by interferons in response to virus infection. Fish STAT3 has been demonstrated to be involved in Singapore grouper iridovirus (SGIV) infection and virus induced paraptosis, but its effects on the replication of other fish viruses still remained uncertain. Here, the roles of grouper STAT3 (Ec-STAT3) in red spotted grouper nervous necrosis virus (RGNNV) infection were investigated. The present data showed that the distribution of phosphorylated Ec-STAT3 was altered in RGNNV infected fish cells, and the promoter activity of STAT3 was significantly increased during virus infection, suggesting that STAT3 activation was involved in RGNNV infection. Using STAT3 specific inhibitor, we found that inhibition of Ec-STAT3 in vitro did not affect the transcription and protein synthesis of RGNNV coat protein (CP), however, the severity of RGNNV induced vacuolation and autophagy was significantly increased. Meanwhile, at the late stage of virus infection, RGNNV induced necrotic cell death was significantly decreased after inhibition of Ec-STAT3. Further studies indicated that Ec-STAT3 inhibition significantly increased the transcript level of autophagy related genes, including UNC-51–like kinase 2 (ULK2) and microtubule-associated protein 1 light chain 3-II (LC3-II) induced by RGNNV infection. Moreover, the expression of several pro-inflammatory factors, including TNFα, IL-1β and IL-8 were mediated by Ec-STAT3 during RGNNV infection. Together, our results not only firstly revealed that STAT3 exerted novel roles in response to fish virus infection, but also provided new insights into understanding the roles of STAT3 in different forms of programmed cell death.

Introduction

The Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway is an important and conserved cascade which can transduce a multitude of signals in development and immune response [1], [2]. Among these STATs members, STAT3 activation not only played important roles in cell proliferation and apoptosis in the tumor microenvironment, but also mediated the innate immune response in response to various pathogens [3], [4], [5], [6], [7], [8], [9]. Recent studies revealed that the activation and increased expression of STAT3 exerted crucial roles in virus replication by suppressing the type I IFN-mediated antiviral response or regulating microtubule dynamics [3], [10], [11], [12], [13], [14]. In addition, STAT3 was also involved in regulation of different forms of programmed cell death, including interferon induced apoptosis, TNF-induced necroptosis, starvation-induced autophagy, and virus induced paraptosis [10], [15], [16], [17], [18].

With the rapid development of commercial aquaculture industry, viral nervous necrosis disease has attracted much attention due to the increased frequency of outbreak in both freshwater and marine fishes from different geographical areas [19], [20], [21], [22], [23]. To our knowledge, nervous necrosis virus (NNV) infection has became one of the most important threats to grouper (Epinephelus spp.) which are widely cultured in China and Southeast Asian countries, and caused great economic losses in grouper industry [20], [24]. To date, a number of NNV isolates have been identified from different species of grouper, including brown-spotted grouper Epinephelus malabaricus (Bloch et Schneider), orange spotted grouper Epinephelus coiodes (Hamilton), giant grouper Epinephelus ianceolatus (Bloch), red spotted grouper Epinephelus akaara (Temminck & Schlegel), yellow grouper Epinephelus awoara (Temminck & Schlegel), greasy grouper Epinephelus tauvina (Forsskal) [21], [24], [25], [26], [27], [28]. The critical events in virus entry and virus infection induced cell death were disclosed in recent years [29], [30], [31]. Although different forms of programmed cell death, including apoptosis, autophagy and necrotic cell death were explored during NNV infection [27], [31], [32], [33], the host factors involved in NNV replication and virus induced cell death remained largely unknown [34], [35].

In this study, using an in vitro cell culture infection model for red-spotted grouper nervous necrosis virus (RGNNV) [36], we investigated the roles of orange-spotted grouper STAT3 (Ec-STAT3) during RGNNV replication and RGNNV infection induced cell death. Our results provided new insights into exploring the molecules involved in nodavirus induced cell death, but also contributed greatly to understanding the mechanism of nodavirus pathogenesis.

Section snippets

Cell and virus

The grouper brain (GB) cells were established and maintained in our lab [36]. GB cells were grown in Leibovitz's L15 containing 10% fetal bovine serum (FBS, Gibco) at 25 °C. The RGNNV used in this study was kept in our lab. For RGNNV infection, GB cells were infected with RGNNV at a multiplicity of infection (MOI) of 5 in the following experiment.

Immune fluorescence microscopy

The phosphorylation of Ec-STAT3 and protein synthesis of CP in mock or RGNNV infected cells were examined using immune fluorescence assay as described

RGNNV infection induced the activation of STAT3 in vitro

To determine the activation of Ec-STAT3 during RGNNV infection, we firstly detected the phosphorylation of Ec-STAT3 using immune fluorescence assay. As shown in Fig. 1, the phosphorylated Ec-STAT3 was mainly observed in the nucleus of control cells. Differently, after RGNNV infection, the distribution of phosphorylated Ec-STAT3 was altered, and minority of the red fluorescence were observed in the cytoplasm of the cells.

It has been demonstrated that the STAT3 promoter contains a GAS site and is

Discussion

Activation of STAT3 plays important roles not only in cell proliferation, apoptosis, as well as tumor angiogenesis, invasion, and migration, but also in virus infection [3], [4], [7], [8]. Mammalian viruses, including DNA and RNA virus, could activate STAT3 for efficient infection [3], [49]. Our previous studies demonstrated that fish STAT3 was involved in Singapore grouper iridovirus (SGIV) infection, and inhibition of STAT3 activation decreased virus replication and inhibited virus induced

Acknowledgment

This work was supported by grants from the National Basic Research Program of China (973) (2012CB114402; 2012CB114404), the National Natural Science Foundation of China (31172437, 30930070) and the National High Technology Development Program of China (863) (2014AA093507).

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