IFNA2 p.Ala120Thr impairs the inhibitory activity of Interferon-α2 against the hepatitis B virus through altering its binding to the receptor

摘要

Abstract Background Our previous study found that a rare genetic mutation IFNA2p.Ala120Thr affects the structure of IFN-α2 and contributes to increased host susceptibility to CHB. However, the way in which the single amino acid residue mutation affects IFN-α2 activity is unclear. The purpose of this research was to investigate the effects and mechanisms of IFNA2p.Ala120Thr on IFN-α2 activity. Methods Plasmid transfection of BL-21 was used to construct both wild type IFNA2 (wt) and p.Ala120Thr IFNA2 (mut) proteins. The HepG2-NTCP model was established using a lentiviral vector (LV003). Anti-HBV activity of wt and mut were tested on HepG2-NTCP infected cells with HBV, through the detection of HBsAg and HBcAg using immunohistochemistry and by detecting HBV DNA with RT PCR. IF and Co-IP were performed in order to investigate the binding of the IFNA2 protein and its receptor. The changes in IFNAR density and signal molecule phosphorylation were measured with western blotting. We used qPCR to further explore anti-HBV protein expression including APOBEC3, MxA, OAS1, and PKR. Results Cell model experiments confirmed that IFNA2p.Ala120Thr impairs anti-HBV activity of IFN-α2. Co-IP tests indicated that the binding of mut-IFNα to IFNR was weaker in the mut-treated group. IFNR density on the cells surface increased after treatment with wt-IFN-α2. Obvious differences in the STAT phosphorylation profiles were seen between the mut-treated and wt-treated groups. The expression of four main kinds of anti-HBV proteins induced by mut was higher in the HepG2-NTCP cells. Thus, IFNA2p.Ala120Thr affects anti-HBV activity of IFN-α2. Conclusion IFNA2p.Ala120Thr impairs the anti-HBV ability of IFN-a2, mainly by reducing its binding to the IFN receptor. Mut IFN-a2 has a very weak binding, barely inducing STAT phosphorylation, and induces the expression of only a low level of related anti-HBV ISG. This is quite different from the effects of wt IFN-a2, implying that modifying the key structural position of IFNa may lead to the modulation of targeted gene expression. Highlights ? We analyse the negative effects of genetic mutation IFNA2 p.Ala120Thr on interferon-α2 activity against hepatitis B virus. ? IFN-a2 carrying this mutation bind loosely to IFNR. ? Wild type IFN-a2 and mutant IFN-a2 have distinct signaling pathway phosphorylation patterns. ? Mutant IFN-a2 induced lower levels of Anti-HBV associated ISG expressions in cell culture.