Role of NS2 specific RNA binding and phosphorylation in liquid–liquid phase separation and virus assembly

Figures

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  Figure 1. Phase separation plot of non-phosphorylated (P-)NS2 and Helix to coil and β sheet transition. NS2 at variable salt and protein concentrations. (A) Protein/NaCl concentrations plotted for optically significant phase separation (OD350nm). A phase boundary (black curve) drawn between positive phase separation score (red circles) and negative score (grey circles). (B) Phase boundary of (P-)NS2 in presence of denatured tRNA. (C) Phase boundary of (P-)NS2 in complex with viral RNA segment (S10). (D) Far-UV spectrum of NS2 apo alone (black circles ●), in the presence of 0.02 μM S10 (grey circles ●), in 0.26 μM S10 (cyan circles ●) and 0.46 μM S10 (blue circles ●). (E) The percentage secondary structure (helix, coil and β strands) estimations of Apo NS2 (black bar) and in presence of increasing concentrations of RNA segment S10. NS2 with 0.02 μM S10 (grey bar), with 0.26 μM S10 (cyan bar) and with 0.46 μM (blue bar).

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  Figure 2. Phosphorylation regulates phase separation of NS2 and CD spectra comparison of phosphorylated wtNS2 and mutants (A) level of NS2 phosphorylation by CK2α kinase as determined by γ-32P signal intensity of NS2. For negative control, NS2 mutant deficit in phosphorylation serine residues (S249A + S259A) is substrate. Numbers above wells are molar concentrations of Kinase CK2α added in enzyme reactions with non-phosphorylated (P-)NS2 or mutant S249A + S259A as substrates. (B) Titration of CK2α concentration against phase separation of NS2 as a function of extent of phosphorylation, OD350nm. (C) Protein/NaCl concentrations plotted for optically significant phase separation of in vitro phosphorylated (P+) NS2. A phase boundary (black curve) drawn across between positive phase separation score (red circles) and negative score (grey circles). (D) SDS-PAGE gel after Coomassie blue staining showing wt and NS2 mutant proteins. M is the molecular mass markers. (E) Comparative far-UV CD spectra of wtNS2 (black circles) and mutants R6A + R7A (red circles), K160A + K162A (blue circles) and K281A + K283A (green circles). (F) Estimation of percent secondary structure contents of wt and mutant NS2…

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  Figure 3. Mobility shift assay of RNA and (P+)NS2 protein complex. The numbers above lane from left to right denotes increasing concentrations of phosphorylated (P+)NS2 protein in micromolar range, μM. (A) The significant shift in viral S10 RNA in complex with wtNS2 on 0.8% agarose gel. Other panels show relative shifts in mutants R6A + R7A (B), K160A + K162A (C) and K281A + K283A (D), of which mutant R6A + R7A showed significant inhibition in mobility shift. (E) The competition binding of viral RNA S10 with wtNS2 or mutants RgA + R7A, K160A + K162A, K281A + K283A (0.8 μM) in molar excess of heat denatured tRNA.

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  Figure 4. Phase separation plot of phosphorylated (P+) NS2 mutants in complex with RNA. Phase boundary (black curve) drawn between positive phase separation score (red circles) and negative score (grey circles) after incubation of the NS2−RNA complexes for 2 h. (A) Protein/NaCl concentrations of (P+) wtNS2 and S10 RNA complex. Phase plot of S10 RNA−protein complexes for mutants R6A + R7A (B), K160A + K162A (C) and K281A + K283A (D).

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  Figure 5. Visualisation of NS2 + S10 RNA condensates under phase contrast microscope. The left-hand side panels are phase separation condensates (visible as droplets) formed by mixing of RNA with non-phosphorylated (P−) NS2 and right-side panels with the phosphorylated (P+) wtNS2 and mutants. (A) Non-phosphorylated (P−)wtNS2. (P−)wtNS2 + denatured tRNA complex (B), (P−)NS2 + S10 RNA (C). Phase separation of S10 RNA + (P+)NS2 complexes for wtNS2 (D), mutant R6A + R7A (E), K160A + K162A (F) and K281A + K283A (G). The white arrow points to small condensates (minute droplets) apparent on close observation. Images are representative of three independent experiments. Scale bars, 40 μm.

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  Figure 6. NS2 forms concentration dependent condensates. The left panel is green channel, middle is bright field and right panel is merged view. (A) purified phosphorylated NS2 and RNA S10 segment complex forms time dependent condensates. At 20 min, ~4.0 mg/ml (100 μM) NS2 (green) shows two populations at 20× magnification, small condensates (small green spots) and those coalescing into large condensates (circular drops). (B) After 40 min of incubation majority of NS2 population coalesced into large condensates. (C) At a higher concentration of RNA, these large condensates further fused to form associated assemblies. Images are representative of three independent experiments. Scale bars, 20 μm. (D) Viral RNA (S10, 1 mg/ml) stained with GelRed® is observed colocalising with NS2 that is stained with Alexa 488 (green fluorescence) in phase separated condensates, visible as large droplets after 40 min of incubation. Panel 1 is bright field (BF) view; 2 is red view; 3, red, BF merged view; 4, green view; 5 is green. Panel 6 is green, red merged view and 7 is green, red, BF merged view. Images are representative of three independent experiments. Scale bars, 5 μm.

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  Figure 7. NS2−RNA complexes are in liquid phases. (A) The quantification of fluorescent intensity recovery after photobleaching from 0 s to 6 min. The green filled-circles represent datasets of intensity counts for the unbleached area A1, while the red filled-circles represent the bleached circle area (A2). The green vertical bars on the X-axis near 9 s denote bleaching doses. (B) The left, middle and right panels are snapshots of NS2−RNA complex droplets in liquid phases at 0 s (no bleaching), 9 s (bleaching) and 6 min (recovery after bleaching). The green fluorescence of NS2 was bleached in the area represented by the red circle (area A2) at 9 s (middle panel) showing final recovery at 6 min (right most panel). Scale bars, 5 μm.

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  Figure 8. NS2−RNA condensates show liquidity in infected cells. (A) The average quantification of VIBs in infected cells with a diameter size of at least 0.5 μm. Un, untreated cells; PEG, PEG 200 treated cells; 1,6HD, 1,6-hexanediol treated cells and 1,6HD R, cells in fresh media with 1,6HD removed. (B) Representative confocal images of fixed cells, corresponding to the quantifications in A, at intervals of 8 min during 1,6HD and PEG treatment and 30 min after removal of 1,6HD. (C) The quantification of fluorescent intensity recovery in infected live cells after photobleaching of VIBs in bleaching area (red dotted circle), from 0 s to 5 min. (D) The top left and right panels show confocal snapshot images of unbleached VIBs (0 second) and just after bleaching (9 s) respectively. The lower panel image shows recovery at around 3 min after photobleaching. Scale bars, 2 μm.

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  Figure 9. Disruption of arginine residues in NS2 affects plaque formation and VIB visualization. (A) BSR and BS8 cell monolayers were transfected with wt and R6A + R7A mutant S8 together with the 9 RNA segments. Images show plaques formed by recovered viruses in BSR and BS8 (BSR cells expressing NS2) complementary cells. (B) NS2 expression at 4 h post-infection of BSR cells with wt virus. (C) Matured VIBs formed of NS2 after 8 h post-infection. (D) NS2 expressions in cells infected with mutant virus (R6A + R7A) at 4 h and 8 h post-infection (E) showing absence of matured VIBs. Images are representative of three independent experiments. Scale bars, 10 μm.