Recombinant Rotaviruses Rescued by Reverse Genetics Reveal the Role of NSP5 Hyperphosphorylation in the Assembly of Viral Factories

FIG 1. (A) Schematic representation of mutations in gs11 of the corresponding rRV strains. Sequences mutated or deleted in NSP5 are indicated. (B) Profiles of viral dsRNAs of the different rRV strains grown in MA-NSP5 cells. gs11 is indicated by a red asterisk.

FIG 2. Characterization of rRV-NSP5/KO. (A) Western blot analysis of MA104 cell extracts infected with rRV-wt and rRV-NSP5/KO strains (MOI, 1 FFU/cell). (B) Confocal immunofluorescence microscopy of MA104, MA-NSP2-mCherry, and MA-NSP5-EGFP cells infected with rRV-wt or rRV-NSP5/KO (MOI, 1 FFU/cell), using antibodies for NSP5, NSP2, VP2, and VP6, as indicated. Scale bar, 15 μm. (C) A comparison of the replication kinetics of rRV-NSP5/KO in MA-NSP5 and MA-NSP5-EGFP cells. Data are expressed as the means ± standard deviations (n = 3); **, P < 0.01 (Student's t test). (D) Electrophoretic migration pattern of dsRNAs extracted from the rRV-NSP5/KO strain grown in MA-NSP5 or MA104 cells. Genome segments 1 to 11 are indicated on the left. (E) Replication kinetics of rRV-NSP5/KO in MA-NSP5 and MA104 cells. Data are expressed as the means ± standard deviations (n = 3); **, P < 0.01 (Student's t test).

FIG 3. Characterization of rRV-NSP5/S67A. (A) Schematic representation of rRV-NSP5/S67A gs11 and sequence of NSP5-wt and NSP5/S67A mutant (highlighted). (B) Electrophoretic pattern of dsRNA genome segments of rRV-wt and rRV-NSP5/S67A strains grown in MA-NSP5 cells (left panel) and in MA104 wt cells (right panel). (C) Replication kinetics of rRV-wt and rRV-NSP5/S67A in MA104 cells. Data are expressed as the means ± standard deviations (n = 3); ***, P < 0.001 (Student's t test). (D) Western blot analysis of extracts of U2OS, Caco-2, and MA104 cells infected with rRV-wt or rRV-NSP5/S67A strains. (E) Western blot analysis of NSP5 phosphorylation pattern in MA104 cells infected with rRV-wt or rRV-NSP5/S67A (MOI, 1 FFU/cell) at 5 and 10 hpi. (F) Western blot analysis of λ-Ppase and alkaline Ppase treatment of lysates of MA104 cells infected with rRV-wt or rRV-NSP5/S67A. Protein bands corresponding to NSP5 are shown.

FIG 4. Characterization of rRV-NSP5/ΔT and rRV-NSP5/Δ176-180. (A) Western blot analysis of NSP5 and NSP5 mutants from MA104 cells infected with rRV-wt, rRV-NSP5/ΔT, or rRV-NSP5/Δ176-180 at 5 hpi. (B) Electrophoretic dsRNA migration pattern of rRV-NSP5/ΔT and rRV-NSP5/Δ176-180 grown in MA-NSP5 and MA104 cells.

FIG 5. Viroplasm morphology in MA104 cells infected with rRV-NSP5 wt and mutants. (A) Representative confocal immunofluorescence micrographs of MA104 cells infected with rRV-wt and rRV-NSP5/S67A (MOI, 15 FFU/cell) at 5 hpi (upper panels) and 10 hpi (lower panels). Cells were stained with anti-NSP5 and DAPI. (B) Quantitative analysis of viroplasm size (μm2) of MA104 cells infected with rRV-wt and rRV-NSP5/S67A at 5 hpi and 10 hpi. ***, P < 0.001; **, P < 0.01. (C) Confocal immunofluorescence micrographs of MA-NSP5-EGFP and MA-NSP2-mCherry cells infected with rRV-NSP5/S67A (MOI, 15 FFU/cell). (D) Confocal immunofluorescence micrographs of rRV-wt- and rRV-NSP5/S67A-infected MA104 cells (MOI, 15 FFU/cell) stained at 10 hpi with the indicated antibodies. (E and F) Confocal immunofluorescence of MA104 cells infected with rRV-NSP5/Δ176-180 (E) or rRV-NSP5/ΔT (F) and stained with the indicated antibodies and DAPI. Scale bar, 15 μm.

FIG 6. Viral RNA detection in rRV-NSP5/S67A-infected cells. (A) Representative confocal immunofluorescence micrographs of MA104 (upper panel), MA-NSP2-mCherry (middle panel), and MA-NSP5 (lower panel) cells infected with rRV-wt or rRV-NSP5/S67A strains and stained with anti-NSP5 (red in MA104 and MA-NSP5 cells) and 5-EU (green). (B) Single-molecule RNA fluorescence in situ hybridization (smFISH) of MA-NSP5-EGFP cells infected with rRV-wt, rRV-NSP5/S67A, or rRV-NSP5/ΔT strains. Viroplasms detected with NSP5-EGFP (green) and viral RNA (the probe specific for gs6 was Cy3 conjugated; red). Colocalizing viroplasms and RNAs are indicated by white arrows. Scale bar, 15 μm. (C) Quantitative analysis of NSP5-EGFP-positive structures (viroplasms) for RNA (gs6) in MA-NSP5-EGFP cells infected with rRV-wt or rRV-NSP5/S67A. ***, P < 0.001.

FIG 7. Electron microscopy of cells infected with rRV-NSP5/S67A. High-definition electron micrographs of MA104 (A) and MA-Δ3 (B) cells infected with rRV-wt (left panels) and rRV-NSP5/S67A (right panels) (MOI, 75 FFU/cell). At 10 hpi, cells were fixed with glutaraldehyde and processed for transmission electron microscopy. V, viroplasm; m, mitochondria; mt, microtubule bundles; Nu, nucleus. The white dashed box indicates the region that is shown magnified in the image to the immediate right. White arrows indicate the endoplasmic reticulum surrounding viroplasms; black arrowheads indicate viral particles with an envelope. Black arrows indicate microtubule bundles. Scale bar, 500 nm.

FIG 8. Phosphorylation of NSP5/S67A. (A) Western blot analysis of extracts of MA104 cells infected with rRV-NSP5/S67A or coinfected with rRV-NSP5/S67A and rRV-NSP5/ΔT. A blue arrowhead indicates NSP5/S67A. The faster-migrating band corresponds to NSP5-ΔT. (B) Western blot analysis of MA104, MA-ΔT, and MA-Δ3 cells infected with rRV-NSP5/S67A. A blue arrowhead indicates NSP5/S67A. (C) Western blot analysis of extracts of MA104 cells coinfected with rRV-NSP5/S67A and rRV-NSP5/Δ176-180. A blue arrowhead indicates NSP5/S67A. (D) λ-Ppase treatment of extracts of MA-Δ3 cells infected with the rRV-NSP5/S67A strain. Filled and open arrowheads indicate dephosphorylated NSP5-S67A and NSP5-Δ3, respectively. (E) Representative confocal immunofluorescence micrographs of MA104, MA-ΔT, and MA-Δ3 cells infected with rRV-NSP5/S67A. Cells were stained with the indicated antibodies. Scale bar, 15 μm. (F) Yield of infectious virus of rRV-NSP5/S67A grown in MA-NSP5, MA104, MA-ΔT, and MA-Δ3 cells at 24 hpi.*, P < 0.05; **, P < 0.01.

FIG 9. Phosphorylation of NSP5/ΔT and NSP5/Δ176-180 and viroplasm formation. Western blots (A and B) and confocal immunofluorescence micrographs (C and D) of MA104, MA-ΔT, and MA-Δ3 cells infected with rRV-NSP5/ΔT (A and C) or with rRV-NSP5/Δ176-180 (B and D). A blue arrowhead indicates NSP5/ΔT and NSP5/Δ176-180, respectively. Open arrowheads indicate NSP5-Δ3 and NSP5-ΔT. Cells were stained with the indicated antibodies and DAPI. Scale bar, 15 μm. (E) Single-step growth of rRV-NSP5/ΔT (left) and rRV-NSP5/Δ176-180 (right) in MA-NSP5, MA104, MA-ΔT, and MA-Δ3 cells, as indicated. The experiment was terminated at 24 hpi. **, P < 0.01; #, viral titer < 300 FFU/ml.

FIG 10. rRV-NSP5/KO in infected cells. Western blot analysis (A) and confocal immunofluorescence microscopy (B) of MA104, MA-ΔT, and MA-Δ3 cells infected with the rRV-NSP5/KO strain. Cells were stained with the indicated antibodies and DAPI. Scale bar, 15 μm. (C) Single-step growth of rRV-NSP5/KO in MA-NSP5, MA104, MA-ΔT, and MA-Δ3 cells, as indicated. The experiment was terminated at 24 hpi. **, P < 0.01; #, viral titer < 300 FFU/ml.

FIG 11. Model of NSP5 hyperphosphorylation and assembly of replication-competent viroplasms. (a) Interaction of nonphosphorylated NSP5 with either NSP2 or VP2 is required to (b) induce conformational changes that make S67 available for CK1α phosphorylation (P). This initial step of the cascade is then (c) sequentially completed by phosphorylation by CK2 of other residues, including serines in domain 4, to generate the NSP5 activator or (c′) a step of interaction with a nonphosphorylated molecule precedes the involvement of CK2. (d) NSP5 interactions to form dimers/multimers are mediated by the carboxy-terminal tails (T). (e) The primed (S67-phosphorylated) activator molecules result in substrate activation and fully hyperphosphorylated NSP5. (f) Assembly of viroplasm scaffolds containing NSP2 serve to (g) recruit the other viroplasmic components, VP1, VP2, VP3, and VP6 to assemble replication-competent viroplasms. P, phosphorylated amino acid. (This image was created using BioRender.)