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Snakehead vesiculovirus (SHVV) causes enormous economic losses in snakehead fish (Ophicephalus striatus) culture. Understanding replication mechanisms of virus is considerable significance in preventing and treating viral disease. In our previous studies, we have reported that glutamine starvation could significant inhibit the replication of SHVV. Furthermore, we also showed that SHVV infection could cause apoptosis of striped snakehead fish cells (SSN-1). However, the underlying mechanisms remain enigmatic. To decipher the relationships among the viral infection, glutamine starvation and apoptosis, SSN-1 cells transcriptomic profilings of SSN-1 cells infected with or without SHVV under glutamine deprived condition were analyzed. RNA-seq was used to identify differentially expressed genes (DEGs). Our data revealed that 1215 up-regulated and 226 down-regulated genes at 24 h post-infection were involved in MAPK, apoptosis, RIG-1-like and toll-like receptors pathways and glutamine metabolism. Subsequently, DEGs of glutamine metabolism and apoptosis pathways were selected to validate the sequencing data by quantitative real-time PCR (qRT-PCR). The expression patterns of both transcriptomic data and qRT-PCR were consistent. We observed that lack of glutamine alone could cause mild cellular apoptosis. However, lack of glutamine together with SHVV infection could synergistically enhance cellular apoptosis. When the cells were cultured in complete medium with glutamine, overexpression of glutaminase (GLS), an essential enzyme for glutamine metabolism, could significantly enhance the SHVV replication. While, SHVV replication was decreased in cells when GLS was knocked down by specific siRNA, indicating that glutamine metabolism was essential for viral replication. Furthermore, the expression level of caspase-3 and Bax was significantly decreased in SHVV infected cells with GLS overexpression. By contrast, they were significantly increased in SHVV infected cells with GLS silence by SiRNA, indicating that SHVV infection activated the Bax and caspase-3 pathways to induce apoptosis independent of glutamine. Our results reveal that SHVV replication and starvation of glutamine could synergistically promote the cellular apoptosis, which will pave a new way for developing strategies against the vial infection. • Transcriptomic data of cells infected by SHVV with glutamine deprivation was analyzed. • Lack of glutamine and SHVV infection enhanced cellular apoptosis synergistically. • SHVV infection activated the Bax and caspase-3 pathways independent of glutamine. [ABSTRACT FROM AUTHOR]

Abstract Autophagy is a degradation cellular process which also plays an important role in virus infection. Glutamine is an essential substrate for the synthesis of glutathione which is the most abundant thiol-containing compound within the cells and plays a key role in the antioxidant defense and intracellular signaling. There is an endogenous cellular glutathione pool which consists of two forms of glutathione, i.e. the reduced form (GSH) and the oxidized form (GSSG). GSH serves as an intracellular antioxidant to maintain cellular redox homeostasis by scavenging free radicals and other reactive oxygen species (ROS) which can lead to autophagy. Under physiological conditions, the concentration of GSSG is only about 1% of total glutathione, while stress condition can result in a transient increase of GSSG. In our previous report, we showed that the replication of snakehead fish vesiculovirus (SHVV) was significant inhibited in SSN-1 cells cultured in the glutamine-starvation medium, however the underlying mechanism remains enigmatic. Here, we revealed that the addition of L-Buthionine-sulfoximine (BSO), a specific inhibitor of the GSH synthesis, could decrease the γ- glutamate-cysteine ligase (GCL) activity and GSH levels, resulting in autophagy and significantly inhibition of the replication of SHVV in SSN-1 cells cultured in the complete medium. On the other hand, the replication of SHVV was rescued and the autophagy was inhibited in the SSN-1 cells cultured in the glutamine-starvation medium supplemented with additional GSH. Furthermore, the inhibition of the synthesis of GSH had not significantly affected the generation of reactive oxygen species (ROS). However, it significantly decreased level of GSH and enhanced the level of GSSG, resulting in the decrease of the value of GSH/GSSG, indicating that it promoted the cellular oxidative stress. Overall, the present study demonstrated that glutamine starvation impaired the replication of SHVV in SSN-1 cells via inducing autophagy associated with the disturbance of the endogenous glutathione pool. Highlights • Glutamine starvation induced autophagy in SSN-1 cells. • Inhibition of the synthesis of glutathione induced autophagy, resulting in the inhibiting of SHVV replication. • Glutamine starvation induced autophagy by disturbing endogenous glutathione pool as well as GSH/GSSG ratio. [ABSTRACT FROM AUTHOR]