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 7
Dengue virus NS3 protein also interacts with ADAR1.
(A) Pairwise Yeast diploids co-expressing dengue virus type 2 NS3 helicase fused to Gal4 DNA-binding domain and ADAR1 fused to Gal4 transactivation domain were plated onto a selective medium lacking histidine to determine interaction-dependent transactivation of the HIS3 reporter gene. Negative controls are vectors without insert (pPC97 and pPC86 for bait and prey respectively). (B) HEK293T cells were co-transfected with 3×Flag-tagged ADAR1 and GST-tagged full-length NS3 of dengue virus (GST-DV-NS3) or its helicase domain (GST-DV-NS3 helicase) or GST-tagged full-length NS1 of influenza virus (GST-FLUAV-NS1) as a positive control. Proteins bound to glutathione sepharose beads were analyzed by western blot using antibodies against GST or 3×Flag. (C) ADAR1 expression in Huh-7 cells upon interferon treatment or dengue virus infection. Huh-7 cells were incubated with 0, 100 or 1000 IU/ml of interferon- (IFN)-α2b or infected with dengue virus and analyzed 24 h later for expression of ADAR1, NS3 and GAPDH. (D) Impact of ADAR1 silencing on dengue virus replication in Huh-7. Data are expressed as the percentage of virus titer obtained with control siRNA-transfected cells. A siRNA targeting dengue virus NS1-coding region (siDV-NS1) was used as positive control for the silencing. (E) ADAR editing activity in Huh-7 cells infected with dengue virus. (F) Dengue virus NS3 contribution to ADAR1 editing activity. The NS1 effector domain of influenza virus, full-length NS3 of dengue virus or its helicase domain were co-expressed with ADAR1 and the editing reporter in HEK293T cells. Luminescence reflecting Firefly and Renilla luciferase activities was measured 48 h post-transfection. The influenza virus NS1 effector domain does not interact with ADAR1 and was used as negative control. Data are normalized to the values obtained with the NS1 effector domain. RLU, relative light unit.