The Interactomes of Influenza Virus NS1 and NS2 Proteins Identify New Host Factors and Provide Insights for ADAR1 Playing a Supportive Role in Virus Replication
Figure 6
Enhancement of ADAR1 editing activity by influenza virus NS1.
(A) The RNA editing reporter system is composed of the hepatitis D virus minimal sequence edited by ADAR1 and positioned in-between the Renilla luciferase and the Firefly luciferase coding sequence, respectively. The unedited reporter has a stop codon that is converted into Trp codon upon A to I editing by ADAR. Hence, editing is correlated with Firefly luciferase expression while Renilla luciferase expression is used as an internal control. (B) HEK293T were co-transfected with the editing reporter, ADAR1 and NS1 (full-length, RNA-binding domain or effector domain) or the control protein DLG4. Two days post transfection, luciferase activities were determined by luminescence measurement. Data are expressed as percentage of the luciferase activity detected in cells expressing the NS1 effector domain (relative light unit, RLU). (C) Editing activity in HEK293T expressing or not ADAR1, transfected with the editing reporter and infected with influenza virus H1N1. (D, E) A549 cells were transfected with plasmids encoding for wild type or catalytically inactive ADAR1 (ADAR1 E912A, Text S1). Forty eight hours later, cells were infected with the A/H1N1/New Caledonia/2006 virus strain at a MOI of 0.5. After an additional 48 h incubation period, expression of ADAR1 and viral proteins was assessed in cell lysates by using western blot (D) and neuraminidase activities were measured in supernatants (E). Values are normalized to mock-transfected cells.