Grass carp reovirus NS26 interacts with cellular lipopolysaccharide-induced tumor necrosis factor-alpha factor, LITAF

The nonstructural protein NS26 of grass carp reovirus (GCRV) is encoded by the 11th genomic dsRNA segment, homolog of which is not found in orthoreoviruses. The role of NS26 in GCRV pathogenesis is still unclear. Previously, grass carp LITAF/SIMPLE protein was identified as a putative binding partner for NS26 in a yeast two-hybrid screen. Here, we further characterized the association between NS26 and LITAF using in vivo and in vitro protein interaction assays. Soluble GST-NS26 and His₆-LITAF were expressed and purified from E. coli; recombinant NS26 tagged with myc and LITAF tagged with GFP were expressed in Ctenopharyngon idellus kidney cells (CIK) by transient transfection experiments. A GST pulldown assay demonstrated that GST-tagged NS26 efficiently bound to His₆-LITAF. Co-immunoprecipitation assays demonstrated that GCRV NS26 reciprocally precipitated endogenous LITAF in CIK cells. Double-immunofluorescent analyses revealed myc-NS26 colocalized with GFP-LITAF in CIK cells. Taken together, the current in vitro and in vivo data demonstrated the interaction between cellular LITAF and GCRV NS26.