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Supporting Information

Additional Supporting Information may be found in the online version of this article:

Figure S1: Clathrin HC, but not AP1 or AP2, colocalizes with µNS in MRV factories. Cells were infected with T1L ISVPs and were fixed for indirect IF microscopy at 0, 3, 7 or 12 h p.i. µNS-specific serum antibodies (α-µNS) and either MAb X22 (A) or MAb 10A (B) were used in IF. Merged images are shown at right (α-µNS, green; α-CHC or α-AP1/2, red). Scale bars, 20 µm.

Figure S2: AP2(σ2)-GFP is recruited to MRV factories late in infection. BSC-1 cells stably expressing AP2(σ2)-GFP were mock infected or infected with either T1L, rT3D-µNS(WT) or rT3D-µNS(L711A) MRV for 42 h. µNS-specific serum antibodies (α-µNS) and Alexa 647-conjugated secondary antibody were used in IF. Representative overlayed images of factories (blue) and AP2(σ2)-GFP (green) are shown. Scale bars, 10 µm.

Figure S3: µNS(471-721) recruits clathrin to factory-matrix structures. µNS (471-721) protein was expressed in CV-1 cells by plasmid-based transfection. At 16 h p.t., cellular localizations of µNS as well as clathrin were visualized by indirect IF microscopy using a combination of µNS-specific serum antibodies (α-µNS) and MAb X22 (α-HC). Scale bar, 10 µm.

Figure S4: Clathrin recruitment by µNS is not essential for virus growth in cultured cells. A) L929 cells were infected with two different preparations each of either purified rT3D-µNS(WT) (closed bars) or rT3D-µNS(L711A) (open bars) at 0.0001 PFU/cell, and infectious titers were determined at indicated times p.i. by plaque assay. Results of a representative experiment are shown. B) CV-1 cells were infected with purified rT3D-µNS(WT) (closed symbols) or rT3D-µNS(L711A) (open symbols) at 0.1 PFU/cell (circles) or 1 PFU/cell (squares), and infectious titers were determined at indicated times p.i. by plaque assay. Yields are expressed relative to paired 0-h samples for each virus. Results of a representative experiment are shown.

Figure S5: Transferrin uptake is not inhibited at 12 h p.i. by MRV. BSC-1 cells stably expressing clathrin LCA fused to tomato fluorescent protein (CLC-tom) were mock infected or infected with either T1L or rT3D-µNS(WT) MRV. At 12 h p.i., fluorescent Tf uptake was performed as described in Materials and Methods, and cells were fixed and immunostained using µNS-specific serum antibodies (α-µNS). Representative confocal images of mock- and MRV-infected cells are shown, with merged images at right (α-µNS, green; CLC-tom, red; Tf, blue). Scale bars, 10 µm.

Video S1: Redistribution of clathrin LCA to MRV factories late in infection. BSC-1 cells stably expressing clathrin LCA tagged with GFP (LG2 cells) were infected with MRV strain T1L and time-lapse video (3 seconds per frame) was recorded at 30 h p.i. Video is shown at the speed of 10 frames per second [actual time passage (hh:min:second) is shown in the lower-left corner].

Video S2: Dynamics of CCP formation in mock-infected cells. BSC-1 cells stably expressing AP2(σ2)-GFP were mock infected 36 h prior to imaging. The movie is a 2-D time series acquired for 300 seconds at 3-second intervals from a single optical section obtained by spinning-disk confocal microscopy and using 100-millisecond exposures.

Video S3: Dynamics of CCP formation in T1L-infected cells. BSC-1 cells stably expressing AP2(σ2)-GFP were infected with MRV strain T1L 36 h prior to imaging. The movie has the same characteristics as Video S2.

Video S4: Dynamics of CCP formation in rT3D-µNS(WT)-infected cells. BSC-1 cells stably expressing AP2(σ2)-GFP were infected with MRV strain rT3D-µNS(WT) 36 h prior to imaging. The movie has the same characteristics as Video S2.

Video S5: Dynamics of CCP formation in rT3D-µNS(L711A)-infected cells. BSC-1 cells stably expressing AP2(σ2)-GFP were infected with MRV strain rT3D-µNS(L711A) 36 h prior to imaging. The movie has the same characteristics as Video S2.

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TRA_1233_sm_Fs5.jpg1011KSupporting info item
TRA_1233_sm_Vs1.mov2623KSupporting info item
TRA_1233_sm_Vs2.mov681KSupporting info item
TRA_1233_sm_Vs3.mov509KSupporting info item
TRA_1233_sm_Vs4.mov444KSupporting info item
TRA_1233_sm_Vs5.mov658KSupporting info item

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