Domains of NSP1 and MAVS responsible for the interaction.

<p><b>A)</b> Diagrammatic representation of mutants of NSP1 and MAVS used in the study. For NSP1 there were 3 mutants; pcD-NSP1-100 which comprised the putative ubiquitin ligase domain, pcD-NSP1-C-395 containing the remaining 395 amino acids of C-terminal and pcD-NSP1ΔIRF3BD comprising the N-terminal 327 amino acids. For MAVS, two constructs were generated, one comprising the CARD and TM domain (pFLAG-CARD-TM) and other vector encoding the intermediate proline rich domain of MAVS (pFLAG-Pro-MAVS). <b>B)</b> To map the domain of NSP1 responsible for this interaction, HEK293 cells were co-transfected with the NSP1 mutants and pFLAG-MAVS. Pull down was performed with anti FLAG Ab which revealed a positive interaction between pcD-NSP1-C-395 and MAVS. Membrane was probed with Anti-FLAG Ab. <b>C)</b> Relative fold change over control was measured for IFN-β and ISG 56 gene transcripts in cells co-expressing full length NSP1-N-100, NSP1-C-395 and NSP1ΔIRF3BD with MAVS. The data shown are means ± the SD (n = 3). * Significantly different in comparison to mock transfected condition. P<0.05.<b>D)</b> To identify the domain of MAVS protein responsible for the interaction, two construct pFLAG-CARD-TM (Mini MAVS) and pFLAG Pro-MAVS were co-transfected with pcD-NSP1 and pulled down with anti-His Ab. CARD-TM domain of MAVS interacts with NSP1. <b>E)</b> Relative fold change in IFN-β gene transcript in cells expressing mini-MAVS in presence or absence of NSP1 with respect to control was measured by real-time PCR. The data shown are means ± the SD (n = 3). * Significantly different in comparison to pcD-NSP1 untransfected condition. P<0.05.