Fig. 8: Rotavirus NSP1 is a viral antagonist of SAMD9.

A HEK293 cells transfected with GFP-ev, or GFP-tagged RV-encoded proteins (VP1-VP4, VP6, VP7, and NSP1-NSP6) for 48 h and cell lysates harvested for SDS-PAGE electrophoresis with indicated antibodies. B MA104 cells infected by indicated recombinant simian RV SA11 (MOI = 3) for 8 h and cell lysates harvested for SDS-PAGE electrophoresis with indicated antibodies. C Schematic representation of N-terminally GFP-tagged WT and truncated RV NSP1 constructs. D Immunoblotting analysis of anti-GFP antibody immunoprecipitates of cell lysates from HEK293 cells transfected with GFP-tagged WT or truncated NSP1s. E Schematic representation of N-terminally 3 × Flag-tagged WT and truncated SAMD9 constructs. F HEK293 cells co-transfected with Flag-tagged SAMD9 mutants and GFP-ev or GFP-NSP1 for 48 h and cell lysates harvested for SDS-PAGE electrophoresis with indicated antibodies. G Immunoblotting analysis of anti-GFP antibody immunoprecipitates of cell lysates from HEK293 cells co-transfected with GFP-tagged NSP1 and Flag-tagged SAMD9 mutants. H HEK293 cells co-transfected with SAMD9-Myc-DDK and GFP-ev or GFP-NSP1 for 36 h, treated with MG132 (10 μM), PYR-41 (50 μM), bortezomib (10 μM), or MLN4924 (10 μM) for another 12 h, and cells lysates harvested for SDS-PAGE electrophoresis with indicated antibodies. I HEK293 cells transfected with control or NEDD8-specific siRNA for 48 h, then co-transfected with SAMD9-Myc-DDK and GFP-ev or GFP-NSP1 for another 36 h, and cell lysates harvested for SDS-PAGE electrophoresis with indicated antibodies. J Relative abundance of SAMD9 and SAMD9L protein levels from indicated species in SAMD9 KO HEK293 co-transfected with indicated SAMD9 or SAMD9L and NSP1s derived from different RV strains. Bv bovine. Darker colors indicate a higher level of SAMD9/9L inhibition by NSP1. For all figures, experiments were repeated at least three times.