Imaging-Based Reporter Systems to Define CVB-Induced Membrane Remodeling in Living Cells

Figures

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  Figure 1. Validation of enterovirus plasmid-based reporters. (a) Left, RepER cassette driven by the CMV promoter. The RepER cassette contains an ER-localized mCherry containing a signal peptide (SP) and KDEL ER retention sequence followed by signal peptidase site fused to a transmembrane domain (TM) followed by the viral 3C protease (3Cpro) recognition sequence and GFP fused to a nuclear localization sequence (NLS). Right, Schematic of the predicted model for visualizing the ER and enterovirus infection. In uninfected cells (left) mCherry and GFP colocalize in the ER. During enterovirus infection (right,) viral 3Cpro cleavage releases the GFP-NLS, which translocates to the nucleus. (b) Immunoblot of GFP, myc, and vinculin from lysates from U2OS cells transfected with RepER and empty (-), CVB 3Cpro-myc, or catalytically inactive CVB 3Cpro-myc (3Cmut) expression plasmids. (c) Immunoblot for GFP, VP1, and GAPDH from lysates from CVB infected (100 PFU/cell) U2OS cells transfected with RepER or RepER containing an AA mutation to the QG in the 3Cpro recognition sequence. The presence of a weak lower RepER band indicates poor host signal peptidase-dependent release of mCherry-KDEL from the TM…

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  Figure 2. Time-lapse imaging of CVB infected cells expressing RepER. (a) Representative time-points of an image series from live-cell imaging of GFP (reporter, green), mCherry (ER, red), and merged panels from mock or CVB infected U2OS cells expressing RepER. Scale bars are 20 μm. (b) Quantification of infection in individual cells, defined as the ratio of nuclear to ER-dense microtubule-organizing center signal intensities, from individual RepER expressing cells either mock (n = 9) or CVB (n = 23) infected. Measurements were taken every 15 min, and data is shown as the average ± SD of the fold change in infection signal compared to 0 hpi. (c) Determination of the time point at which 50% maximum infection signal was observed (HPI50) as determined by nonlinear regression curve analysis for each CVB infected cell. Each data point shown is the HPI50 for an individual cell (n = 23), and the line represents the average ± SD. (d) Quantification of infection (green) and peripheral ER integrity (red) for the mock (dashed lines, n = 9) and CVB (solid lines, n = 23) infected cells shown as the average fold change compared to 0 hpi; the gray dashed line shows fold change of 1. (e) Scatter plot of…

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  Figure 3. Time-lapse imaging of CVB infected cells expressing RepOr. (a) Top, the bicistronic RepOr cassette contains a Golgi localized mEmerald (mEm-Golgi) followed by an IRES-driven RepER, where the GFP has been substituted with BFP. Signal peptide (SP), transmembrane domain (TM), and nuclear localization signal (NLS). Bottom, Schematic of the predicted model for visualizing the Golgi, ER, and enterovirus infection. In uninfected cells (left), mCherry and BFP colocalize in the ER (purple), and mEmerald is localized to the Golgi (green). During enterovirus infection (right), viral 3Cpro cleavage releases the BFP-NLS, which translocates to the nucleus, the mCherry is retained in the ER (red) and the Golgi remains (green). (b) Representative time-points of an image series from live-cell imaging of mEmerald (Golgi, green), BFP (reporter, gray), mCherry (ER, red), and merged panels from U2OS cells expressing RepOr treated with DMSO, brefeldin A, or infected with CVB. Scale bars are 20 μm. (c) Quantification of infection (blue) and Golgi area (green) in mock (dashed lines, n = 16) and CVB (solid lines, n = 16) infected cells shown as the average fold change compared to 0 hpi; the gray dashed…

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  Figure 4. Growth kinetics of CVB in reporter expressing cells. U2OS cells transfected with the indicated expression plasmids were bound with CVB (100 PFU/cell) at 4 °C for 1 h, washed, and infection was allowed to proceed at 37 °C for 24 h. Plaque assays were performed on samples collected at the indicated time points. Data represents the average ± SD titer (plaque-forming units [PFU]/mL) from two independent experiments.

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  Figure 5. ER calcium channel inhibition alters CVB-induced ER remodeling. (a) Representative time points of an image series from live-cell imaging of mEmerald (Golgi, green), BFP (reporter, gray), mCherry (ER, red), and merged panels from U2OS cells expressing RepOr treated with DMSO or 2APB (100 μM) with CVB. Aberrant ER structures are indicated as protein aggregates (*), inflated ER (white arrowhead in ER panels), which exclude cytoplasmic BFP (empty arrowheads in reporter panels). Scale bars are 20 μm. (b) CVB infection kinetics of individual U2OS cells expressing RepOr treated with DMSO (n = 24) or 2APB (100 μM, n = 24). Data is shown as the average (dots) ± SD (dashed lines) infection signal every 20 min from 0 to 8 hpi, presented as fold change compared to 0 hpi. (c) Phenotypes of cells from live-cell imaging of DMSO or 2APB (100 μM) treated U2OS cells expressing RepOr infected with CVB. Data is shown as the percentage of cells showing nuclear translocation of the reporter at 8 hpi (Infection), percentage of cells showing infection containing aberrant ER structures (Aberrant ER), or the percentage of cells showing reporter translocation at 8 hpi that resulted in lysis by 16 hpi…