Molecular Mechanism of Inhibition of the PKR-RNA Interaction by the Influenza a Virus NS1 Protein - A Three Colour Based Flim-Fret Approach in Living Cells

Abstract

Influenza A Virus (IAV) protein NS1 is the major antagonist of the host cellular innate immune response. NS1 has a N-terminal RNA binding domain and a C-terminal effector domain for interaction with immune effector proteins, like RIG-I or PKR. Activation of the double stranded (ds)RNA-dependent protein kinase (PKR) by binding dsRNA induces a global block in protein translation. Hence, inhibition of PKR activation is an important function of NS1.Here, we study PKR inhibition in a cell-based system with living cells. Therefore we use IAV NS1 protein tagged with a YFP or Turquoise fluorophore in combination with GFP-tagged PKR. For PKR activation we used dsRNA labelled with Rhodamine (Rhd). This combination of fluorophores allows us to detect interaction of all three components at the same time using fluorescence lifetime imaging with Forster resonance energy transfer (FLIM-FRET). The method allows us to detect an interaction in cell culture based systems with laser microscopy.We show that co-transfection of PKR-GFP, dsRNA-Rhd and NS1-Turquoise leads to a reduction of the FRET efficiency between PKR and dsRNA. That indicates an increase of distance between PKR and dsRNA due to NS1. Furthermore, we could show an enhanced interaction between PKR and NS1 by adding dsRNA leading to the hypothesis that dsRNA is necessary for the interaction between NS1 and PKR. In addition, reduction of FRET suggests that NS1 prevents binding of PKR to dsRNA.Our study identifies for the first time in a cell-based FLIM-FRET system the changes in interaction by analysing different combinations of PKR, NS1 and dsRNA. In upcoming experiments we will analyse NS1 Mutants with our system to check changes in FRET reduction of NS1 and PKR.