Coimmunoprecipitation assay.

For the coimmunoprecipitation assay of MA-104 cells infected with RVA-NMTL, the cells were seeded in 75-cm² bottles (catalog number 430720; Corning) and then infected with RVA-NMTL at an MOI of 0.1 for 18 h. The control cells were not infected. The cells were then lysed with 1 ml of Pierce immunoprecipitation (IP) lysis buffer (catalog number 87788; Thermo Scientific) containing phenylmethylsulfonyl fluoride (catalog number ST506; Beyotime) and a protease inhibitor (catalog number 4693132001; Roche) for 30 min at 4°C and centrifuged at 12,000 × g for 5 min at 4°C. An aliquot (80 μl) of the supernatant was boiled with 20 μl of 5× SDS-PAGE protein loading buffer. To avoid nonspecific reactions, the remaining supernatant was shaken with protein A/G magnetic beads (catalog number UI287210A; Thermo) and negative mouse IgG (catalog number A7028; Beyotime) for 2 h at 4°C. The beads were removed, and the supernatant was shaken overnight with a mouse anti-DIC monoclonal antibody (catalog number ab23905; Abcam) at 4°C. Protein A/G magnetic beads were added, and the samples were shaken again at 4°C for 4 h. The beads were washed five times with Pierce IP lysis buffer and boiled in 5× SDS-PAGE protein loading buffer. All the samples were analyzed by Western blotting, as described above. For the coimmunoprecipitation assay in HEK-293T cells, the cells were transfected with plasmids pCMV-Myc-DIC and pCAGGS-HA-NSP2 for 48 h, as described above. The control group of cells was cotransfected with pCMV-Myc-DIC and pCAGGS-HA, pCMV-Myc-DIC and pCAGGS-HA-NSP5, pCMV-Myc and pCAGGS-HA-NSP2, or pCMV-Myc and pCAGGS-HA-NSP5. The remaining procedures were the same as those described above for the MA-104 cells but with a mouse anti-Myc monoclonal antibody (catalog number M4439; Sigma) instead of the mouse anti-DIC monoclonal antibody.