Upregulated expression of the antioxidant sestrin 2 identified by transcriptomic analysis of Japanese encephalitis virus-infected SH-SY5Y neuroblastoma cells
Japanese encephalitis virus (JEV) exerts a profound burden of viral encephalitis. We have investigated the differentially expressed transcripts in the neuronal transcriptome during JEV infection by RNA sequencing (RNA-Seq) of virus-infected SH-SY5Y human neuroblastoma cells. Gene ontology analysis revealed significant enrichment from two main pathways: endoplasmic reticulum (ER)-nucleus signaling (P value: 5.75E−18; false discovery rate [FDR] 3.11E−15) and the ER unfolded protein response (P value: 7.58E−18; FDR 3.11E−15). qPCR validation showed significant upregulation and differential expression (P < 0.01) of ER stress-signaling transcripts (SESN2, TRIB3, DDIT3, DDIT4, XBP1, and ATF4) at 24 h post-infection for both low (LN) and high (HN) neurovirulence JEV strains. Immunoblot analysis following JEV infection of SH-SY5Y cells showed an increase in levels of SESN2 protein following JEV infection. Similarly, Zika virus (MR766) infection of SH-SY5Y showed a titer-dependent increase in ER stress-signaling transcripts; however, this was absent or diminished for DDIT4 and ATF4, respectively, suggestive of differences in the induction of stress-response transcripts between flaviviruses. Interestingly, SLC7A11 and SLC3A2 mRNA were also both deregulated in JEV-infected SH-SY5Y cells and encode the two constituent subunits of the plasma membrane xCT amino acid antiporter that relieves oxidative stress by export of glutamate and import of cystine. Infection of SH-SY5Y and HEK293T cells by the JEV HN strain Sw/Mie/40/2004 lead to significant upregulation of the SLC7A11 mRNA to levels comparable to DDIT3. Our findings suggest upregulation of antioxidants including SESN2 and, also, the xCT antiporter occurs to counteract the oxidative stress elicited by JEV infection.
KeywordsJapanese encephalitis virus Neuron RNA-Seq Sestrin xCT antiporter
We are extremely grateful to Dr. Tomohiko Takashi from the National Institute of Infectious Diseases, Tokyo for the kind gift of the JEV strains and to Dr. Takashi Kimura (Hokkaido University) for rabbit anti-JEV hyperimmune sera. This study was supported in part by a grant to Dr. Michael Carr from the Japanese Society for the Promotion of Science (JSPS/KAKENHI) of Japan (16K08803). This study was also supported in part by grants to Professor Hirofumi Sawa for Scientific Research on Innovative Areas from the Ministry of Education, Culture, Science, Sports and Technology (MEXT) of Japan (16H06429, 16H06431, 16K21723), grants from the Ministry of Education, Culture, Sports, Science and Technology; the Ministry of Health, Labour and Welfare, Japan (MEXT)/JSPS KAKENHI (16H05805).
All authors contributed to the study design. MC performed the experiments and GG and AM performed the bioinformatic analyses. All authors contributed to data analysis and the writing and editing of the manuscript.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflicts of interest.
Research involving human participants or animals
This article does not contain any study involving either human participants or animals performed by any of the authors.
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