Impact of Borna Disease Virus Infection on the Transcriptome of Differentiated Neuronal Cells and Its Modulation by Antiviral Treatment

Abstract

Borna disease virus (BoDV-1) is a highly neurotropic RNA virus that causes neurobehavioral disturbances such as abnormal social activities and memory impairment. Although impairments in the neural circuits caused by BoDV-1 infection induce these disturbances, the molecular basis remains unclear. Furthermore, it is unknown whether anti-BoDV-1 treatments can attenuate BoDV-1-mediated transcriptomic changes in neuronal cells. In this study, we investigated the effects of BoDV-1 infection on neuronal differentiation and the transcriptome of differentiated neuronal cells using persistently BoDV-1-infected cells. Although BoDV-1 infection did not have a detectable effect on intracellular neuronal differentiation processes, differentiated neuronal cells exhibited transcriptomic changes in differentiation-related genes. Some of these transcriptomic changes, such as the decrease in the expression of apoptosis-related genes, were recovered by anti-BoDV-1 treatment, while alterations in the expression of other genes remained after treatment. We further demonstrated that a decrease in cell viability induced by differentiation processes in BoDV-1-infected cells can be relieved with anti-BoDV-1 treatment. This study provides fundamental information regarding transcriptomic changes after BoDV-1 infection and the treatment in neuronal cells.

Keywords: Borna disease virus; antiviral; differentiation; gene expression; neuronal cells.

Figure 1

Neuronal differentiation of BoDV-1-infected SH-SY5Y cells. (A) Differentiation protocol of SH-SY5Y cells used in this study. SH-SY5Y cells were differentiated with 10 µM retinoic acid (RA) for 2 days. (B–D) Neuronal differentiation of BoDV-1-infected and uninfected SH-SY5Y cells. Neuronal differentiation was evaluated with the cell morphological changes (B), the total length of neurites in a cell (C), and the NCAM2 gene expression (D). (E) BoDV-1 infection in differentiated (RA) and undifferentiated (DMSO) SH-SY5Y/BoDV cells. Bars, 30 µm. We calculated the expression of each gene using the ∆∆Ct method. The expression of each gene was normalized with that of the GAPDH gene in each condition. Values are expressed as the mean percentage + standard error of the mean (SEM) of at least three independent experiments. N.S, non-significant; ***, p < 0.005; ****, p < 0.001.

Figure 2

Differentiation-related genes affected by BoDV-1 infection. (A,B) Scatter plots of RNA-seq analysis. Extracted genes are indicated in red. (A) Comparison between undifferentiated (X-axis) and differentiated (Y-axis) SH-SY5Y cells. (B) Comparison between differentiated SH-SY5Y/BoDV (X-axis) and differentiated SH-SY5Y cells (Y-axis). (C) Venn diagram showing the number of differentiation-related (left circle) and BoDV-1-impaired (right circle) genes identified by RNA-seq. The overlapped genes are indicated. (D) Heatmap showing the expression levels of BoDV-1-impaired, differentiation-related genes.

Figure 3

Suppression of BoDV-1 infection by TD-Borna treatment. (A) Effects of TD-Borna treatment on BoDV-1 load in SH-SY5Y/BoDV cells. SH-SY5Y/BoDV cells were treated with DMSO or RA in the presence or absence of TD-Borna for 2 days. The amounts of BoDV-1 gRNAs in SH-SY5Y/BoDV cells were determined by RT-qPCR analyses. (B–D) The amounts of N (B), X/P (C), and M/G/L (D) mRNAs in SH-SY5Y/BoDV cells. The amounts of BoDV-1 mRNAs were determined by RT-qPCR analyses. (E) The amounts of BoDV-1 proteins in SH-SY5Y/BoDV cells. The amounts of BoDV-1 N, BoDV-1 P, and tubulin were determined by western blot analyses using specific antibodies. siCont, the scrambled siRNA-treated control; TD-Borna, the siRNA cocktail against BoDV-1. We calculated the expression of each gene using the ∆∆Ct method. The expression of each gene was normalized with that of the GAPDH gene in each condition. Values are expressed as the mean percentage + SEM of four independent experiments. ***, p < 0.005; ****, p < 0.001.

Figure 4

Effect of TD-Borna treatment on BoDV-1-related transcriptomic changes in differentiated SH-SY5Y cells. SH-SY5Y/BoDV cells were treated with RA in the presence of the scrambled siRNA (siCont) or TD-Borna for 2 days. The mRNA amounts of differentiation-related genes in the indicated cells were determined by RT-qPCR analyses. We calculated the expression of each gene using the ∆∆Ct method. The expression of each gene was normalized with that of the GAPDH gene in each condition. Values are expressed as the mean percentage + SEM of four independent experiments. *, p < 0.05; ***, p < 0.005; ****, p < 0.001.

Figure 5

Effect of TD-Borna treatment on cell viability during differentiation of SH-SY5Y/BoDV cells. SH-SY5Y or SH-SY5Y/BoDV cells were treated with RA in the presence of the scrambled siRNA (siCont) or TD-Borna for 2 days. The cell viability was evaluated by using a CellTiter Glo Luminescent Cell Viability Assay. Values are expressed as the mean percentage + SEM of three independent experiments. ****, p < 0.001.