Fig. 4: Effect of G12(S) on the intracellular NP distribution, and in vitro NP-vRNA interaction.

a–d Subcellular localization of OC43 NP and EIGIC-53 or ER (KDEL) proteins. A549 cells infected with OC43 (MOI = 1) were cultured for 48 h. The cells were treated with 2 μM G12(S) for the next 24 h of the culture, and fixed. Immunostaining was performed for OC43 NP with KEDL proteins (a and b) or ERGIC53 (c and d). See Supplementary Fig. 5e for the specificities of the antibody used for OC43 NP, ERGIC-53, and ER protein (anti-KDEL antibody). e Experimental overview of LLPS formation and G12(S) treatment on the LLPS. Droplets of SARS-CoV-2 NP were formed by LLPS in the presence of 1 µM FITC-32 base of the 3’-untranslated region of SARS-CoV-2 RNA and 40 µM SARS-CoV-2 NP overnight. f Effect of TAMRA-G12(S) on droplets of SARS-CoV-2 NP formed by LLPS in the presence of 1 µM FITC-leveled vRNA. Fluorescence and differential interference contrast (DIC), TAMRA (magenta) and FITC (Green) and merged (Merge) images are shown. TAMRA-G12(S) (final 1, 3, and 6 µM) were added and observed at 25 °C using fluorescence and DIC microscopy at the indicated time. See Supplementary Fig. 6 for the time-dependent image data (10 min, 20 min, 30 min, 1 h, 2 h, 3 h, 4 h, 5 h, 6 h and 7 h). g The supernatant (soluble fraction) was collected from the wells forming LLPS 1 h after addition of TAMRA-G12(S) (final 0, 3, and 6 µM), and the adsorbed fraction was collected by suspending the wells in LLPS buffer containing 0.1%SDS. These samples were separated in 7.5 M urea/10 mM KCl containing 15% PAGE. Fluorescent images were taken with a ChemiDoc Touch MP (BioRad) using filter sets for Alexa546 (left) and fluorescein (right). Tamura and FITC fluorescence were observed using the Aelxa546 mode. FITC fluorescence was observed in fluorescein mode (center). An overlay image is shown (right). The positions of ssRNA marker lengths are indicated.