Figure S1.
Construction and analyses of recombinant MHV-68 viruses. (A) A schematic diagram of mCherry-ORF52 BAC and mEosEM-ORF52 BAC constructs. (B) Left panel: WT BAC DNA and mCherry-ORF52 BAC DNA were isolated from E. coli strain SHG68 (GS1783). The DNAs were digested with PteI. The arrow indicates the position of a 7.8-kb PteI fragment resulting from mCherry insertion. Right panel: WT BAC DNA and mEosEM-ORF52 BAC DNA were isolated from E. coli strain SHG68 (GS1783). The DNAs were digested with KpnI. The arrow indicates the position of a 7.7-kb KpnI fragment which disappeared as a result of mEosEM insertion. (C) 293T cells were individually transfected with mCherry-ORF52 BAC or mEosEM-ORF52 BAC and images were captured by a fluorescence microscope. Bright-field images of the same area are shown on the bottom. Bar: 200 μm. (D) Multi-step growth curves of the recombinant viruses. BHK cells were infected with the indicated viruses at an MOI = 0.05 and cultured for 4 d. Viral titers were examined at the indicated time points. Values shown are means ± SD (n = 3). (E and F) Quantification of cytoplasmic ORF52 puncta in COS-7 cells infected by WT virus or mCherry-ORF52 virus. COS-7 cells were infected with WT virus or mCherry-ORF52 virus at a MOI of 3 and fixed at 18 hpi. Number of ORF52 puncta per cell (E) and mean surface areas of puncta (F) were measured using the microscopy image analysis software, Imaris. Error bar represents SD. Statistical analyses were performed using a two-tailed non-parametric t test. Data were collected from at least three independent experiments. (E) WT, n = 6; mCherry, n = 7. (F) WT, n = 6; mCherry, n = 5. (G) Analysis of co-localization of ORF52 with TGN. A plasmid expressing GFP-TGN46 was transfected into COS-7 cells. At 12 h post transfection, COS-7 cells were infected with mCherry-ORF52 virus. Cells were fixed at 24 hpi and observed with a confocal microscope (FV1200). Shown are the merged images of GFP-TGN signals (green) and mCherry-ORF52 signals (red) without the bright-field image (Merge 1) or with the bright-field image (Merge 2). Nu, nucleus. Bar: 5 μm. (H) Fluorescent images of COS-7 cells infected with mCherry-ORF52 virus at an MOI = 3 and treated with 6% propylene glycol (PG) at 24 hpi. Bar: 5 μm. Source data are available for this figure: SourceData FS1.

Construction and analyses of recombinant MHV-68 viruses. (A) A schematic diagram of mCherry-ORF52 BAC and mEosEM-ORF52 BAC constructs. (B) Left panel: WT BAC DNA and mCherry-ORF52 BAC DNA were isolated from E. coli strain SHG68 (GS1783). The DNAs were digested with PteI. The arrow indicates the position of a 7.8-kb PteI fragment resulting from mCherry insertion. Right panel: WT BAC DNA and mEosEM-ORF52 BAC DNA were isolated from E. coli strain SHG68 (GS1783). The DNAs were digested with KpnI. The arrow indicates the position of a 7.7-kb KpnI fragment which disappeared as a result of mEosEM insertion. (C) 293T cells were individually transfected with mCherry-ORF52 BAC or mEosEM-ORF52 BAC and images were captured by a fluorescence microscope. Bright-field images of the same area are shown on the bottom. Bar: 200 μm. (D) Multi-step growth curves of the recombinant viruses. BHK cells were infected with the indicated viruses at an MOI = 0.05 and cultured for 4 d. Viral titers were examined at the indicated time points. Values shown are means ± SD (n = 3). (E and F) Quantification of cytoplasmic ORF52 puncta in COS-7 cells infected by WT virus or mCherry-ORF52 virus. COS-7 cells were infected with WT virus or mCherry-ORF52 virus at a MOI of 3 and fixed at 18 hpi. Number of ORF52 puncta per cell (E) and mean surface areas of puncta (F) were measured using the microscopy image analysis software, Imaris. Error bar represents SD. Statistical analyses were performed using a two-tailed non-parametric t test. Data were collected from at least three independent experiments. (E) WT, n = 6; mCherry, n = 7. (F) WT, n = 6; mCherry, n = 5. (G) Analysis of co-localization of ORF52 with TGN. A plasmid expressing GFP-TGN46 was transfected into COS-7 cells. At 12 h post transfection, COS-7 cells were infected with mCherry-ORF52 virus. Cells were fixed at 24 hpi and observed with a confocal microscope (FV1200). Shown are the merged images of GFP-TGN signals (green) and mCherry-ORF52 signals (red) without the bright-field image (Merge 1) or with the bright-field image (Merge 2). Nu, nucleus. Bar: 5 μm. (H) Fluorescent images of COS-7 cells infected with mCherry-ORF52 virus at an MOI = 3 and treated with 6% propylene glycol (PG) at 24 hpi. Bar: 5 μm. Source data are available for this figure: SourceData FS1.

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