Persistent borna disease virus infection confers instability of HSP70 mRNA in glial cells during heat stress

Abstract

Borna disease virus (BDV) is a highly neurotropic RNA virus that causes neurological disorders in many vertebrate species. Although BDV readily establishes lasting persistence, persistently infected cells maintain an apparently normal cell phenotype in terms of morphology, viability, and proliferation. In this study, to understand the regulation of stress responses in BDV infection, we investigated the expression of heat shock proteins (HSPs) in glial cells persistently infected with BDV. Interestingly, we found that BDV persistence did not upregulate HSP70 expression even in cells treated with heat stress. Furthermore, BDV-infected glial cells exhibited rapid rounding and detachment from the culture plate under various stressful conditions. Immunofluorescence analysis demonstrated that heat stress rapidly disrupts the cell cytoskeleton only in persistently infected cells, suggesting a lack of thermotolerance. Intriguingly, we found that although persistently infected glial cells expressed HSP70 mRNA after heat stress, its expression rapidly disappeared during the recovery period. These observations indicated that persistent BDV infection may affect the stability of HSP70 mRNA. Finally, we found that the double-stranded RNA-dependent protein kinase (PKR) is expressed at a constant level in persistently infected cells with or without heat shock. Considering the interrelationship between HSP70 and PKR production, our data suggest that BDV infection disturbs the cellular stress responses to abolish antiviral activities and maintain persistence.

FIG. 1.

HSP expression in persistently BDV-infected glial cells. (A) Morphology and BDV antigen expression in persistently BDV-infected glial cell lines. The C6 (a to d) and OL (e to h) cell lines were grown under normal culture conditions. Subpanels: a, b, e, and f, uninfected; c, d g, and h, persistently infected. The cells were fixed and stained with hematoxylin. BDV antigen was detected by immunofluorescence assay with an anti-P polyclonal antibody. (B) Proliferation of persistently infected glial cells. Cells (2 × 10⁵) were seeded in 60-mm-diameter culture plates, and their proliferation was evaluated every 12 h. (C) Expression of large HSPs in persistently infected cell lines. Cells were lysed, and equal amounts of protein were subjected to Western blot analysis with antibodies against HSP90, HSP70, HSC70, HSP60, and BDV P and N. The GAPDH control shows that each lane contains approximately the same amount of protein. Data from one experiment representative of three independent tests are shown.

FIG. 2.

BDV infection inhibits HSP70 expression induced by heat shock in glial cells. (A) C6 and OL cells were exposed to heat shock at 44°C and allowed to recover at 37°C for different periods. Cells were harvested at the indicated times during the recovery period. Nontreated control cells (NT) were maintained at 37°C and harvested together with heat-stressed cells. After being washed with ice-cold PBS, the cells were lysed and separated by SDS-PAGE (10% acrylamide). Western blot analysis was carried out as described in Materials and Methods. Data from one experiment representative of six independent tests are shown. (B) For quantitative analysis of HSP expression, band intensities were determined with NIH Image software. Values were normalized to GAPDH levels.

FIG. 3.

Suppression of the thermotolerance of persistently infected cell lines. (A) Morphological changes of BDV-infected glial cells. Cells were exposed to heat stress at 44°C for 1 h or to oxidative stress with 10 μM hydrogen peroxide for 0.5 h. In the case of oxidative stress, cells were allowed to recover for 24 h in normal medium at 37°C. For morphological analysis, cells were visualized under a phase-contrast microscope. (B and C) Disruption of the actin cytoskeleton and focal adhesion complexes in persistently BDV-infected C6 cells. C6 cells were exposed to heat stress at 44°C for 1 h. Subpanels: a and b, uninfected; c and d, persistently infected. Stress-treated (b and d) and untreated (a and c) cells were fixed and stained with rhodamine-phalloidin (B) or anti-FAK antibody (C). Arrowheads indicate FAK at the lamellipodia. (D) After heat stress, cells were recovered at 37°C. The floating cells in the culture supernatants were counted at 0 and 1.5 h of recovery. (E) The floating cells were stained with 0.4% trypan blue, and the viable cells were counted. The data are expressed as percentages of the total number of cells counted. For statistical analysis, the data were expressed as the mean plus the standard error. Comparisons of two groups were performed by Student's t test with the Statcel software. Double asterisks indicate statistically significant differences (P < 0.01). NT, nontreated control cells.

FIG. 4.

Effects of BDV infection on HSP70 induction in primary glial cells. (A) Primary glial cells were isolated and infected with BDV as described in Materials and Methods. BDV antigen was detected by immunofluorescence assay with an anti-P polyclonal antibody. Subpanels: a, uninfected; b, BDV infected. (B) Primary glial cells were exposed to heat shock at 44°C and allowed to recover at 37°C for different times. Cells were harvested at the indicated times in the recovery period. Nontreated control cells (NT) were maintained at 37°C and harvested together with heat-stressed cells. After being washed with ice-cold PBS, the cells were lysed and separated by SDS-PAGE (10% acrylamide). Western blot analysis was carried out as described in Materials and Methods. Data from one experiment representative of two independent tests are shown. (C) For quantitative analysis of HSP expression, band intensities were determined with NIH Image software. Values were normalized to GAPDH levels. (D) Morphological changes in BDV-infected primary glial cells. Cells were exposed to heat stress at 44°C for 1 h. For morphological analysis, cells were visualized under a phase-contrast microscope. Subpanels: a and b, uninfected; c and d, persistently infected. Stress-treated (b and d) and untreated (a and c) cells were fixed and stained with hematoxylin.

FIG. 5.

Stability of HSP70 mRNA in BDV-infected glial cells. C6 cells were exposed to heat stress at 44°C for 1 h and allowed to recover at 37°C for the times indicated. (A) Total RNA was extracted, and semiquantitative RT-PCR for HSP70 mRNA was performed as described in Materials and Methods. As a control for the input RNA, the GAPDH transcript in the cells was also amplified. The amplification products were analyzed by 1.5% agarose gel electrophoresis. Gels were stained with ethidium bromide. The images of agarose gels were captured electronically, and the pixels were inverted. Data from one experiment representative of three independent tests are shown. (B) For quantitative analysis, band intensities were determined with NIH Image software. Values were normalized to GAPDH levels. The data are expressed as the mean plus the standard error. (C) C6 cells were exposed to heat stress at 44°C for 1 h and then allowed to recover at 37°C for various times in the presence of actinomycin D. Total RNA was extracted, and levels of HSP70 mRNA in the extract were analyzed by Northern blotting as described in Materials and Methods. The levels of 28S and 18S rRNAs are also shown. NT, nontreated control cells.

FIG. 6.

Induction of autophosphorylated PKR in BDV-infected glial cells during heat stress. (A) After heat shock, uninfected and BDV-infected C6 cells were washed with ice-cold PBS and lysed in lysis buffer. Nontreated control cells (NT) were maintained at 37°C and harvested together with heat-stressed cells. Equivalent amounts of proteins were separated by SDS-PAGE (10% acrylamide). Western blot analysis was carried out to examine relative levels of phosphorylated PKR (P-PKR). Data from one experiment representative of three independent tests are shown. (B) For quantitative analysis, band intensities were determined with NIH Image software. Values were normalized to GAPDH levels. The data are expressed as the mean plus the standard error.