Rotavirus Spike Protein VP4 Mediates Viroplasm Assembly by Association to Actin Filaments

Figures

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  FIG 1. Generation of VP4-BAP tagged recombinant rotavirus. (a) Schematic representation of BAP tag inserted into the lectin domain loops of the VP8* subunit of VP4 from RV simian strain SA11 (GenBank: X14204.1). The lysine (K, red) indicates the biotinylation site of BirA ligase. Four different VP4 proteins tagged with BAP (VP4-BAP) were built between amino acid regions T96-R101 (blue), E109-S114 (orange), N132-Q137 (pink), and K145-G150 (green). VP4 trimer ribbon structure is presented for the visualization of VP8* (yellow) and VP5CT (body and stalk, red) fragments. An inset in VP8* indicates the different positions in the hydrophobic loops of VP8* where the BAP tags were inserted and colored in blue, orange, pink, and green. (b) Immunoblot retardation assay of cell lysates transiently expressing wtVP4 and VP4-BAP, tagged at blue, orange, pink, and green positions, respectively. Untreated (-) and streptavidin-treated (+) samples are indicated. The membrane was incubated with anti-VP4 to detect unbound VP4-BAP and VP4-BAP bound to streptavidin (VP4-BAP•StAv). α-tubulin was used as a loading control. (c) dsRNA electropherotype of the genome segments of rRV/wt and rRV/VP4-BAP. The red…

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  FIG 2. RV protein localization upon rRV/VP4-BAP infection. (a) rRV/wt (top panel) and rRV/VP4-BAP (bottom panel) infected MA-cytBirA cells were untreated (–biotin) or treated with biotin (+biotin). Cells were fixed with paraformaldehyde at 6 hpi and stained for the detection of viroplasms (anti-NSP5, green) and biotinylated proteins (streptavidin-Alexa 555, red). Nuclei were stained with DAPI (blue). (b) Immunostaining images of rRV/VP4-BAP-infected MA-cytBirA cells in the presence of biotin. At 6 hpi, paraformaldehyde-fixed cells were stained for the detection of VP4-BAP (StAv, red) in viroplasms (anti-NSP5, green) (upper row) or mature RV particles (anti-VP7 clone 159, green) (bottom row). (c) Immunofluorescence images of cells infected with rRV/wt (upper row) or rRV/VP4-BAP (lower row) comparing the localization of VP4 and VP4-BAP (anti-VP4, red), respectively. The viroplasms were detected with anti-NSP5 (green). Nuclei were stained with DAPI. In all of the figures, the dashed white boxes correspond to the image insets of the right columns. The yellow arrows point to the VP4-BAP or VP4 signal. The scale bar is 10 μm.

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  FIG 3. Replication fitness of rRV/VP4-BAP is delayed. (a) Virus replication fitness curve from 0 to 48 hpi of rRV/wt and rRV/VP4-BAP. The curve represents the mean of three independent experiments. Welch’s t test; **, P < 0.01; ***, P < 0.001. (b) Plot for the quantification of viroplasm per cells at 6 hpi. Data correspond to the mean ± SEM, n > 60 cells. Welch’s t test; ****, P < 0.0001. Quantification of cells with viroplasms of MA104 cells infected with rRV/wt and rRV/VP4-BAP at (c) same MOI (25 VFU/cell) or (d) the same numbers of virus particles. Samples were counted at 6 hpi (n > 120 cells). Welch’s t test; ***, P < 0.001; ****, P < 0.0001.

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  FIG 4. Virus attachment and internalization of RV/wt and rRV/VP4-BAP are comparable. (a) Virus attachment to MA104 cell surface with different amounts of purified rRV/wt (open gray dots) and rRV/VP4-BAP (full green dots). Samples were detected using a guinea pig anti-RV. The result corresponds to the mean of nine normalized independent experiments. Data analysis corresponds to nonlinear regression, P = 0.2987, n = 9. Virus attachment assay to MA104 cell surface with different amounts of unbiotinylated (–biotin, full green dots) and biotinylated (+biotin, green open dots) purified rRV/VP4-BAP detected with (b) guinea pig anti-RV followed by secondary antibody conjugated to HRP. Data analysis corresponds to nonlinear regression, P = 0.6151, n = 6. (c) Same as above, but samples were detected with streptavidin-HRP. Data analysis corresponds to nonlinear regression, P < 0.0001, n = 6. (d) Internalization of purified virions into MA104 cells at 0 min (upper panel) and 2 min (lower panel). Purified virions of rRV/wt and biotinylated rRV/VP4-BAP were previously labeled with StAv-Alexa 555 (red). At the indicated time postinfection, cells were fixed and immunostained for VP7 trimers (MAb…

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  FIG 5. Viroplasm morphology and behavior for rRV/wt and rRV/VP4-BAP. (a) Representative high-resolution electron micrograph of viroplasms from rRV/wt (upper row) and rRV/VP4-BAP (lower row) infected MA104 cells at 6 hpi (left panel) and 12 hpi (right panel). Scale bars are 200 nm and 500 nm, as indicated. (b) Fluorescence images of FRAP measurement of single viroplasm of cells infected with rRV/VP4-BAP (top) and rRV/wt (bottom) at prebleach, postbleach, and recovery time conditions. Each inset indicates the bleached viroplasm of the images at the right. Nuclei were stained with Hoechst 33342. The scale bar is 10 μm. (c) FRAP recovery curve of NSP2-mCherry in single viroplasm from rRV/VP4-BAP (green) and rRV/wt (gray) infected MA/NSP2-mCherry cells at 5 hpi (n = 27 and 25, respectively). Plots indicating recovery half-time (d) and diffusion (e) means of NSP2-mCherry in single viroplasms of rRV/VP4-BAP and rRV/wt after photobleaching.

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  FIG 6. Transfection of rRV/VP4-BAP DLPs is inefficient in forming viroplasms. (a) Size plot of TLPs and DLPs purified from rRV/wt and rRV/VP4-BAP. The median value is indicated, n > 30 particles, ordinary one-way ANOVA; *, P < 0.05; ****, P < 0.0001. (b) In-cell Western blot of MA104 cells transfected for 4 h with identical numbers of rRV/wt and rRV/VP4-BAP DLPs. RV infection was detected by staining with anti-NSP5 (IDye800, red). The loading control corresponds to cell tag 700 (green). The merge of the two channels is shown at the bottom. (c) Plot showing normalized amounts of NSP5 in rRV/wt and rRV/VP4-BAP DLPs-transfected MA104 cells. Each experiment was done in triplicate. (d) Micrograph of rRV/wt and rRV/VP4-BAP DLPs-transfected MA104 cells immunostained to detect viroplasms with anti-NSP5 (green). Nuclei were stained with DAPI (blue). Open dashed white box shows enlarged viroplasm inset at the right. White arrowheads point to early-stage viroplasm structures. The scale bar is 10 μm. (e) Quantification of the cells showing viroplasms upon transfection with DLPs of rRV/wt and rRV/VP4-BAP. The data correspond to the mean ± SD, Welch's t test; ****, P > 0.0001.

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  FIG 7. VP4 has a role in viroplasm formation. (a) Schematic representation for the characterization of LLPS condensates in viroplasms of RV-infected cells. At 5 hpi, RV-infected MA/NSP2-mCherry cells were treated with 1,6-HD for 6 min. The drug was washed out, and samples were fixed and imaged for viroplasm quantification at 0, 2, 15, and 30 min postrecovery. (b) Representative images of MA/NSP2-mCherry cells infected at 5 hpi with rRV/wt (upper row) or rRV/VP4-BAP (lower row) and treated with 3.5% of 1,6-HD. Cells were washed and monitored for viroplasm formation at 0, 2, 15, and 30 min postrecovery. White arrows point to cells with recovered viroplasms. The scale bar is 10 μm. Plots indicating the ratio of cells with viroplasms (c) and viroplasm counts per cell (d) normalized at initial conditions (5 hpi). Data represent the mean ± SEM of three independent experiments. Student's t test; **, P < 0.001. (e) Immunoblot of cellular extracts prepared at 6 hpi from noninfected or SA11-infected MA104 or MA/NSP2-mCherry cells silenced with siVP4 or control siRNA (scr). The membrane was stained with anti-VP4, anti-NSP5, and anti-GAPDH (loading control). (f) Representative images of…

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  FIG 8. VP4-BAP does not bind to the actin cytoskeleton. (a) Immunostaining of MA104 cells showing NSP5 and NSP2 VLSs coexpressed with GFP (top panel), VP4-GFP (middle panel), and VP4-BAP-GFP (bottom panel). Cells were fixed with paraformaldehyde and immunostained to detect VLSs (anti-NSP5, red). Nuclei were stained with DAPI (blue). The scale bar is 10 μm. (b) Plot indicating the number of VLSs per cell when coexpressed with GFP, VP4-GFP, and VP4-BAP-GFP.The data represent the mean ± SEM, one-way ANOVA; *, P < 0.05; **, P < 0.001. (c) Immunostaining of MA104 cells expressing VP4-GFP and VP4-BAP-GFP after treatment for 1 h before fixation without (–NOC) or with (+NOC) 10 μM nocodazole. Cells were fixed with methanol at 24 hours posttransfection (hpt) and immunostained for GFP detection (anti-GFP, green) and actin cytoskeleton (anti-actin, red). Nuclei were stained with DAPI (blue). The scale bar is 10 μm. (d) Plot for the quantification of VP4-GFP and VP4-BAP-GFP filament lengths associated with actin filaments in cells untreated or treated with nocodazole. The data represent the mean± SD, Welch's t test: ****, P < 0.0001.

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  FIG 9. rRV/VP4-BAP impaired association with the actin cytoskeleton. (a) Immunostaining of noninfected and SA11-infected MA104 cells. At 6 hpi, cells were fixed with methanol and immunostained to detect viroplasms (anti-NSP5, green) and actin cytoskeleton (anti-actin, cyan). Nuclei were stained with DAPI (blue). The scale bar is 20 μm. Immunostaining of noninfected and rRV/wt- or rRV/VP4-BAP-infected MA104 cells. At 6 hpi, cells were fixed with methanol and immunostained for detection of (b) VP4 (anti-VP4, green) and actin cytoskeleton (anti-actin, cyan) and (c) VP4 (anti-VP5 clone 4G2, cyan) and myosin motor (anti-myosin IIa, green). Nuclei were stained with DAPI (blue). The scale bar is 20 μm. Open yellow and red arrowheads point to stress fibers in the actin cytoskeleton and VP4 fibers, respectively. (d) Immunofluorescence of rRV/wt and rRV/VP4-BAP-infected MA104 cells treated at 1 hpi with either 0.5 μM jasplakinolide (jasp; middle panel) or 10 μM cytochalasin B (cyt B; lower panel). At 6 hpi, cells were fixed and immunostained to detect viroplasms (anti-NSP5, green). Nuclei were stained with DAPI (blue). The dashed open box corresponds to the inset of the enlarged image at the…

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  FIG 10. A peptide mimicking loop K145-G150 rescues the rRV/VP4-BAP phenotype. (a) Immunofluorescence images at 17 hpt of BHK-T7 cells expressing VP4-GFP or VP4-BAP-GFP, untreated or treated with 25 μM SPL added immediately after transfection. Before methanol fixation, cells were treated for 1 h with 10 μM nocodazole. The cells were immunostained for GFP (anti-GFP, red), and nuclei were stained with DAPI (blue). Scale bar is 10 μm. (b) Plot quantifying the length of VP4 filaments untreated or treated with SPL as described in panel a. Welch ANOVA test; **, P < 0.01; ****, P < 0.0001. (c) Representative immunofluorescence microphotograph of viroplasms of rRV/wt- and rRV/VP4-BAP-infected cells untreated (-SPL) or treated (+SPL) at +1 and +3 hpi with 25 μM SPL, as indicated. Viroplasms were immunostained at 6 hpi with anti-NSP5 (red), and nuclei were stained with DAPI (blue). The dashed white boxes correspond to the enlarged picture in the right columns. Scale bar is 10 μm. Quantification plots for the ratio of cells with viroplasms (d) and the numbers (e) and size (f) of viroplasms per cell of MA104 cells infected with the indicated virus and untreated (UT) or treated with 25 μM SPL at +1…