Full length articleInvolvement of fish signal transducer and activator of transcription 3 (STAT3) in nodavirus infection induced 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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