Fig. 4

Nsp3.1 interacts with N through its N-terminal domain. A Schematic diagram of PI values of various Nsp3.1 regions generated using Expasy ProtParam tool. Note that a strong acidic region (aa 1–235) is located in the N-terminus of Nsp3.1. HEK293T cells were transfected with the indicated plasmid combinations. The cells were subjected to co-immunoprecipitation with Flag agarose (B) and immunostaining with Flag and HA antibodies (D) and (E). C The intensities of HA or Flag stained bands of each sample were quantified using LI-COR Image Studio software, and ratios of the intensities of HA/Flag bands were calculated. Note that compared with Nsp3.1 (aa 1–749 of Nsp3), aa 1–235 interacted with N in a stronger manner. Similarly, the interaction between N and Δaa 1–102 (G) or Δaa 1–235 (I) of Nsp3.1 was examined using co-immunoprecipitation with HA antibody and immunostaining with Flag and HA antibodies (F). Their interactions were quantified by calculating the ratios of intensities of Flag/HA bands (H). Note that deletion of aa 1–102 impaired the capacity of Nsp3 to interact with N. Data represent one of 3 independent experiments with similar results; error bar represent mean ± s.e.m; *P < 0.05, **P < 0.01, ***P < 0.001; two-tailed unpaired Student’s t-test