The Anti-interferon Activity of Conserved Viral dUTPase ORF54 is Essential for an Effective MHV-68 Infection
Figure 3
ORF54 induces degradation of IFNAR1.
24 hours post transfection, 293T cells were treated with 1 µg/mL of puromycin to select for transfected cells. 48 hours post treatment, cells overexpressing vector control or FLAG-tagged MHV-68 ORFs were: A) treated with human IFN-α for 15 minutes. Immunoblot for phosphorylation of STAT1 on Tyr 701 was assayed first; the membrane was subsequently stripped twice to demonstrate equal levels of total STAT1 and expression of FLAG-ORFs as controls; B) Upper panels: Lysed to analyze level of IFNAR1. The membrane was subsequently stripped to demonstrate equal expression of the FLAG-ORFs and β-actin as controls, and IGF1β to demonstrate specificity of ORF54 to IFNAR1. Lower panels: The same lysates were also subjected to immunoblot against IFNAR2. The membrane was stripped twice to demonstrate equal expression of FLAG-ORFs and β-actin as controls. C) harvested for total RNA isolation. Human IFNAR1 transcript levels were quantified by RT-PCR, normalized first to actin, and are shown relative to vector control transfected cells. Ctrl. is the vector control, ORF48 is an unrelated MHV-68 ORF used as a negative control.