Functional analysis of Rousettus aegyptiacus "signal transducer and activator of transcription 1" (STAT1)

Abstract

Bats are now known as the source of several diseases in humans, but few studies regarding immune responses and factors associated with bats have so far been reported. In this study, we focused on STAT1, one of the critical components in interferon (IFN)-signaling and antiviral activity, which is often targeted by viral proteins to reduce antiviral activity and increase viral replication. We found that Rousettus aegyptiacus STAT1 (bat STAT1) is phosphorylatable and translocates to the nucleus when stimulated with human IFN-alpha (hIFN-alpha). Furthermore, phosphorylation of bat STAT1 and inhibition of nuclear translocation was observed in IFN-stimulated cells infected with the HEP-Flury strain of rabies virus, in the same manner as in other mammals. Additionally, quantitative real-time RT-PCR revealed that bat STAT1 mRNA was highly expressed in the liver, while low in muscle and spleen.

Fig. 1

Western blotting of samples from the transfected 293T cells. (A) 293T cells were mock-infected (lanes 1, 2, 5, and 6) or infected (lanes 3, 4, 7, and 8) with RV at an MOI of 1 and transfected with pFlag-STAT1 (lanes 1–4) or empty vector (lanes 5–8). At 24 h post-transfection, cells were mock-treated (lanes 1, 3, 5, and 7) or treated (lanes 2, 4, 6, and 8) with 10 kU/ml of IFN-α for 30 min. The expression of STAT1, phosphorylated STAT1, and Flag were detected by Western blot analysis with specific antibodies. Actin was used as a loading control. (B) The same lysates were immunoprecipitated with Flag antibody. The expression of STAT1, phosphorylated STAT1, and Flag were detected by the immunoblot analysis with specific antibodies. IgG was used as a loading control.

Fig. 2

Confocal imaging of STAT1 in bat cells infected with RV. The IFN-induced nuclear translocation of STAT1 in bat cells is prevented in RV-infected cells. BatK cells were mock-infected (a–f) or infected (g–l) with RV at an MOI of 0.1. At 24 h postinfection, cells were stimulated (d–f and j–l) or unstimulated (a–c and g–i) with 10 kU/ml of IFN-α for 30 min, fixed, and stained with STAT1 antibody as described in Section 2. The arrows point to RV-infected cells and the arrowheads point to uninfected cells. RV was visualized using FITC-labeled RV antibody as described in Section 2s. The scale bars correspond to 20 μm.

Fig. 3

Quantitative real-time RT-PCR analysis of STAT1 gene expression in various tissues. Expression of STAT1 mRNA in various tissues of Rousettus aegyptiacus was analyzed using real-time RT-PCR. Data are averaged from three different bats with standard deviations. The data were normalized to GAPDH gene expression using the 2nd derivative max (ΔΔCt) method.