RNA-dependent RNA polymerase of predominant human norovirus forms liquid-liquid phase condensates as viral replication factories

Fig. 1.. GII.4 RdRp has the required properties to undergo LLPS. (A) Bioinformatics analysis of the primary amino acid sequences of all nonstructural proteins of HuNoV GII.4 to predict LLPS propensity using DeePhase. The dotted black line indicates the threshold LLPS score of proteins to undergo LLPS. (B and C) The disorder prediction of GII.4 RdRp primary amino acid sequence using bioinformatics tools (B) PONDR and (C) IUPred2. The dotted black line in both panels indicates the threshold disorder score and the dashed pink oval shows the predicted disordered N-terminal region. (D and E) The oligomeric state of GII.4 RdRp analyzed using (D) size exclusion chromatography and (E) sedimentation velocity analytical ultracentrifugation. (F) A cartoon representation of the crystal structure of GII.4 RdRp showing the disordered/flexible N-terminal region colored in pink.

Fig. 2.. HuNoV GII.4 RdRp undergoes LLPS in vitro. (A) Differential interference contrast (DIC) and confocal images of phase-separated RdRp (1% Atto 488 labeled and 99% unlabeled). Phase-separated RdRp has liquid-like properties. (B) Effect of 1,6 HD on LLPS of RdRp. (C) FRAP of RdRp in condensate photobleached at the position indicated by the red circle. (D) The graph shows the recovery curve where normalized fluorescence intensity was plotted against time. (E) Time-lapse images of RdRp condensates exhibit the formation of larger condensates by the fusion of smaller condensates over time. The red arrows indicate the site of fusion. (F) Surface wetting and dripping using confocal microscopy. The red arrows indicate the position of surface wetting and dripping.

Fig. 3.. Phase separation of GII.4 RdRp is modulated by PEG-3350 concentration, pH, and salt concentration. Effect of (A) protein and PEG-3350 concentrations, (C) protein concentrations and pH, and (E) protein and salt concentrations on the phase separation of RdRp was assessed by measuring the turbidity of the samples at an optical density of 350 nm. (B, D, and F) A phase diagram of RdRp at (B) protein and PEG-3350 concentrations, (D) protein concentrations and pH, and (F) protein and salt concentrations constructed from the confocal microscopy images. The green boxes indicate LLPS (condensate state), and the white boxes indicate no LLPS (soluble state).

Fig. 4.. Effect of N-terminal deletion on phase separation of GII.4 RdRp. Purified RdRp WT and RdRp-NΔ51 were analyzed by (A) CD spectroscopy, (B and C) enzyme activity, (D) size exclusion chromatography, and (E) confocal microscopy.

Fig. 5.. RdRp forms liquid-like condensates in GII.4-infected HIEs. (A) Confocal microscopy images of GII.4 HuNoV–infected HIEs (TCH 12-580) at different time points after infection showing condensate formation detected by anti-RdRp antibody (green). Uninfected HIEs were used as negative control. White arrows indicate the position of representative condensates. (B) Effect of 1,6 HD on the condensates formed in the GII.4 HuNoV–infected HIEs suggesting their liquid-like nature.

Fig. 6.. GII.4 RdRp condensates are the sites for viral replication. Confocal images of the GII.4 HuNoV–infected HIEs showing (A) colocalization (yellow) of dsRNA (red) and RdRp (green) detected by respective antibodies and (B) colocalization (yellow) of VPg (red) and RdRp (green) detected by respective antibodies. In both panels, white arrows indicate the site of colocalization.

Fig. 7.. Association of RdRp condensates with membrane markers. GII.4 HuNoV–infected HIEs were fixed and dual labeled with markers for RdRp (green) and various cellular membrane markers (red) (A) for the trans-Golgi (GalT), (B) cis-Golgi (GM130), and (C) ER (calnexin).