Fig. 6

Inhibition of Nsp3-N interaction in trans impaired viral replication. A HEK293T cells were transfected with pVP16-Nsp3.1 expressing AD-Nsp3.1, pM-N expressing BD-N, nuclear-localized Nsp3.1 (NLS-Nsp3.1), and nuclear-localized N (NLS-N). 36 h post-transfection, the cells were subjected to the Dual-Glo Luciferase Assay. Note that in a dose-dependent manner, NLS-N or NLS-Nsp3.1 inhibited the expression of luciferase, the reporter gene in pG5-Luc, promoted by the interaction of AD-Nsp3.1 and BD-N. B HEK293T cells were transfected with Nsp3.1-HA, aa 1–235 of Nsp3.1-HA, Flag-N, LPC vector, and EGFP as an indicator for transfection effection. 36 h post-transfection, the cells were subjected to the co-immunoprecipitation assay and WB analysis. 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 in a dose-dependent manner, aa 1–235 of Nsp3.1 competed with Nsp3.1 for the interaction with N. HEK293T cells were transfected with Rep-Luci, RL, and various truncated Nsp3.1 proteins (D–F). 48 h post-transfection, the cells were subjected to the Dual-Glo Luciferase Assay. Note that all truncated Nsp3.1 proteins except Δ1-235 of Nsp3.1 (F) inhibited the replication of replicon of SARS-CoV-2, and the aa 1–235 of Nsp3.1 inhibited the replication of replicon in a dose-dependent manner (E). 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, ****P < 0.0001; two-tailed unpaired Student’s t-test or one-way ANOVA.