Hepatitis B virus X protein inhibits extracellular IFN-α-mediated signal transduction by downregulation of type I IFN receptor

Abstract

We have previously shown that hepatitis B virus (HBV) protein X (HBX), a regulatory protein of HBV, activates Stat1, leading to type I interferon (IFN) production. Type I IFN secreted from HBX-expressing hepatic cells enforces antiviral signals through its binding to the cognate type I IFN receptor. We therefore investigated how cells handle this detrimental situation. Interestingly, compared to Chang cells stably expressing an empty vector (Chang-Vec), Chang cells stably expressing HBX (Chang-HBX) showed lower levels of IFN-α receptor 1 (IFNAR1) protein, a subunit of type I IFN receptor. The levels of IFNAR1 transcripts detected in Chang-HBX cells were lower than the levels in Chang-Vec cells, indicating that HBX regulates IFNAR1 at the transcriptional level. Moreover, we observed that HBX induced the translocation of IFNAR1 to the cytoplasm. Consistent with these observations, HBX also downregulated Tyk2, which is required for the stable expression of IFNAR1 on the cell surface. Eventually, Chang-HBX cells consistently maintained a lower level of IFNAR1 expression and displayed no proper response to IFN-α, while Chang-Vec cells exhibited a proper response to IFN-α treatment. Taken together, we propose that HBX downregulates IFNAR1, leading to the avoidance of extracellular IFN-α signal transduction.

Figure 1

HBX downregulates IFNAR1 at the transcriptional level. (A) Cell lysates of Chang-Vec and Chang-HBX cells were electrophoretically separated on 10% SDS-PAGE. IFNAR1 and IFNγR were detected with the corresponding antibodies. (B) Total-RNA (3 μg) isolated from Chang-Vec and Chang-HBX cells was subjected to RT-PCR. The IFNAR1 sequence was amplified using its specific primers and visualized on a 1.5% agarose gel by ethidium bromide staining. β-actin was used as an internal control.

Figure 2

HBX induces translocation of IFNAR1 into the cytoplasm. (A) Chang-Vec and Chang-HBX cells were fixed with 4% paraformaldehyde and permeabilized with cold acetone. The cells were incubated with anti-IFNAR1 and visualized by confocal microscopy after staining with Alexa Fluor 680-conjugated goat anti-rabbit IgG antibody. (B) Chang cells were transfected with a Flag-tagged IFNAR1 vector with or without a Myc-tagged HBX vector and fixed 2 days after transfection. The cells were incubated with rabbit polyclonal c-Myc antibodies (HBX) and mouse monoclonal anti-Flag antibody and visualized by confocal microscopy after staining with Alexa Fluor 680-conjugated goat anti-rabbit IgG antibody together with Alexa Fluor 514-conjugated goat anti-mouse IgG antibody.

Figure 3

HBX-mediated decrease of Tyk2 causes a reduction in IFNAR1 levels. (A) Cell lysates from Chang-Vec and Chang-HBX cells were electrophoretically separated on 10% SDS-PAGE. Tyk2, Jak1 and Stat1 were detected with the corresponding antibodies. (B) HEK293 T cells were transfected with a Flag-tagged IFNAR1 vector (1 μg) with or without a Tyk2 expression vector (1 μg) using the calcium phosphate precipitation method. Two days post-transfection, cells were harvested and the cell lysates separated on 10% SDS-PAGE followed by immunoblotting with anti-Flag and anti-Tyk2 antibodies. (C) Chang cells were transfected with Tyk2 siRNA (60 nM) and control siRNA (60 nM). Two days post-transfection, cells were harvested and the cell lysates were separated on 10% SDS-PAGE followed by immunoblotting with anti-IFNAR1 and anti-Tyk2 antibodies. (D) Chang cells were transfected with a Myc-tagged HBX vector (2 μg) and endogenous IFNAR1 and Tyk2 were detected by immunoblotting 2 days post-transfection.

Figure 4

Chang-HBX cells display no alterations in IFNAR1 expression during exogenous IFN-α signaling. Chang-Vec and Chang-HBX cells were treated with IFN-α (1,000 U/ml) for 12 h. Cells were harvested and the cell lysates separated on 10% SDS-PAGE, followed by immunoblotting with anti-IFNAR1 antibody.