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Upregulated expression of the antioxidant sestrin 2 identified by transcriptomic analysis of Japanese encephalitis virus-infected SH-SY5Y neuroblastoma cells

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Abstract

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.

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Acknowledgements

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).

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Authors and Affiliations

  1. Global Station for Zoonosis Control, Global Institution for Collaborative Research and Education (GI-CoRE), Hokkaido University, N20, W10, Kita-ku, Sapporo, 001-0020, Japan

    Michael Carr, Axel Martinelli, William W. Hall & Hirofumi Sawa

  2. National Virus Reference Laboratory, School of Medicine, University College Dublin, Belfield, Dublin 4, Ireland

    Michael Carr & William W. Hall

  3. Division of Bioinformatics, Research Center for Zoonosis Control, Hokkaido University, N20, W10, Kita-ku, Sapporo, 001-0020, Japan

    Gabriel Gonzalez & Kimihito Ito

  4. Division of Molecular Pathobiology, Research Center for Zoonosis Control, Hokkaido University, N20, W10, Kita-ku, Sapporo, 001-0020, Japan

    Christida E. Wastika, Yasuko Orba, Michihito Sasaki & Hirofumi Sawa

  5. Global Virus Network, Baltimore, MD, 21201, USA

    William W. Hall & Hirofumi Sawa

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  1. Michael Carr
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Contributions

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.

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Correspondence to Michael Carr.

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11262_2019_1683_MOESM1_ESM.tif

Supplementary material 1 (TIFF 1310 kb) Supplementary material 1 (TIFF 1310 kb) Supplementary Figure 1. Immunofluorescence analysis of JEV-infected SH-SY5Y cells. SH-SY5Y neuroblastoma cells were mock-infected or infected with JEV HN strain at a MOI of 10, 25, 50 and 100 and were then fixed 24 hpi. The number of JEV-infected cells was examined by immunofluorescence using rabbit anti-JEV hyperimmune sera.

11262_2019_1683_MOESM2_ESM.tif

Supplementary material 2 (TIFF 351 kb) Supplementary material 2 (TIFF 351 kb) Supplementary Figure 2A. PCA and t-SNE analysis of the RNA-Seq data derived from the JEV-infected SH-SY5Y cells. Principal components analysis (PCA) plot of “Control” (mock-infected SH-SY5Y cells) versus “Infected” (JEV strain Sw/Mie/40/2004-infected) SH-SY5Y neuroblastoma cells at 24 hpi. Supplementary Figure 2B. T-distributed Stochastic Neighbour Embedding (t-SNE) plot of “Control” (mock-infected SH-SY5Y cells) versus “Infected” (JEV strain Sw/Mie/40/2004-infected) SH-SY5Y neuroblastoma cells at 24 hpi.

11262_2019_1683_MOESM3_ESM.tif

Supplementary material 3 (TIFF 861 kb) Supplementary material 3 (TIFF 861 kb) Supplementary Figure 3. CV and BCV of the of the RNA-Seq data derived from the JEV-infected SH-SY5Y cells. Coefficients of variation per gene (standard deviation / mean) among replicates for “control” (mock-infected) and “infected” (JEV-infected) samples. Horizontal and vertical axes show the coefficient of variation (CV) among control replicates and among infected replicates, respectively. A) CV for 15879 genes with reads in all replicates. B) CV for the 50 most significant DEGs. C) Biological coefficient of variation (BCV) for genes with expression (18078 genes). The common dispersion for the BCV was 0.005.

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Supplementary material 4 (TIFF 547 kb) Supplementary material 4 (TIFF 547 kb) Supplementary Figure 4. Venn diagrams of the significantly up- and downregulated transcripts identified in the JEV-infected SH-SY5Y transcriptome. Differential expression analyses were performed by DESeq2 and EdgeR and the Venn diagrams are based on P values adjusted for false discovery rate.

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Supplementary material 5 (TIFF 10219 kb) Supplementary material 5 (TIFF 10219 kb) Supplementary Figure 5. Immunofluorescence analysis of JEV-infected HEK293T cells. HEK293T cells were mock-infected or infected with JEV LN or HN strains at a MOI of 1, 10 and 25 and were then fixed 24 hpi. The number of JEV-infected cells was examined by immunofluorescence using rabbit anti-JEV hyperimmune sera.

Supplementary material 6 (DOCX 11 kb)

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Carr, M., Gonzalez, G., Martinelli, A. et al. Upregulated expression of the antioxidant sestrin 2 identified by transcriptomic analysis of Japanese encephalitis virus-infected SH-SY5Y neuroblastoma cells. Virus Genes 55, 630–642 (2019). https://doi.org/10.1007/s11262-019-01683-x

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  • DOI: https://doi.org/10.1007/s11262-019-01683-x

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