Liquid-Liquid Phase Separation Underpins the Formation of Replication Factories in Rotaviruses (2021)
Figure 6

Figure 6 | Rotavirus replication factories are RNA-protein condensates sensitive to propylene glycol. (a) RV-infected MA-NSP5-EGFP cells at 6 HPI. NSP5-EGFP-tagged viral factories (green) are dissolved in the presence of 4.7% (v/v) propylene glycol (PG, middle). Viral RNA-protein condensates rapidly reform (<10 min) after replacing the PG-containing cell culture medium (PG removed, right). Seg3 (magenta) and Seg4 (cyan) transcripts are detected by smFISH, and colocalising Seg3 and Seg4 RNA signals (white). Scale bars: 50 µm, zoomed-in regions: 10 µm. (b) Binding of NSP2 to a fluorescently labelled 20-mer ssRNA in the presence of 4.7% propylene glycol (PG), measured by fluorescence anisotropy. Data points represent mean anisotropy values±SD from three measurements (technical replicates, values normalized from 0 to 1). (c-e) Effects of PG treatments on the viral production. PG-containing medium (2% or 4.7% PG), or standard cell culture medium, were applied to RV-infected cells (MOI of 10) 2 HPI (1 hour after virus absorption). (c) Viral titres (PFU/ml) of samples harvested at 11 HPI that were briefly (15 min) treated with 4.7% PG at 4 HPI (15' PG), and those of untreated RV-infected cells harvested at 11 HPI (control). Virus titres are expressed as mean±SD values estimated for four independent repeats, and were compared by a two-tailed Mann-Whitney test (no significant difference, P = 0.0857). (d) Rotavirus titres measured after 10 h of PG treatments. The 2% PG treatment slightly lowered the titre compared to the control group (ns, P = 0.2282), while the 4.7% PG treatment significantly inhibited viral replication (P = 0.0032). Reported viral titres represent mean±SD values estimated for four independent biological replicates. (e) Effects of PG treatments on virus production at different infection points. 4.7% (v/v) PG was applied between 2-7 HPI ('early infection'), and then diluted to 2% between 7-11 HPI, or 4.7% PG was applied between 7-11 HPI ('late infection'), and diluted prior to harvesting the virus. Each group was compared to the control group (10 h treatment with 2% PG). Each group was compared to the control group (10 h treatment with 2% PG). Application of 4.7% PG for 5 h significantly reduced virus replication (P = 0.0016) between 2-7 HPI compared to the control group. PG treatment between 7-12 HPI did not significantly reduce the viral titre compared to the control group (P = 0.1141). Infectious titres represent mean±SD values estimated for four independent biological replicates. Statistical analyses were performed using a Kruskal-Wallis test, followed by uncorrected Dunn's multiple comparisons test (**P < 0.002).

(f, g) 4.7% PG treatment of RV-infected cells results in NSP5 dephosphorylation. (f) Western blot analysis of RV-infected cells harvested at 7 and 12 HPI, treated with 2% or 4.7% PG at different infection time points indicated in the figure. Multiple phosphorylated forms of NSP5 can be seen as higher MW bands. Treatment with 4.7% PG reduces phosphorylation, which is not perturbed in the presence of 2% PG. Treatments administered between 2-7 and 7-12 hpi reduce NSP5 phosphorylation, albeit to a considerably lower degree compared to the 2-12 hpi treatment. (g) Okadaic acid (5 μM) applied to 4.7% PG-treated infected cells blocks NSP5 dephosphorylation restoring its phosphorylation pattern.