In stark contrast, cells co-expressing 3CD and 3A failed to support induction of PI4P biosynthesis (compare 3CD to 3CD+3A in Fig 7A and 7B). cells were identified by changes to pattern of PI4P expression (red). The nucleus was stained with DAPI (blue). (B) Experiment performed as above immunostained for the (R)-Sulforaphane following: (i) Arf1, (ii) GBF1, or (iii) PI4KB (green). Infected cells were identified by 3D/3CD expression (red). The nucleus was stained with DAPI (blue). (C) Experiment performed as above immunostained for the following: (i) Giantin or (ii) Calnexin (green). Infected cells were identified by 3D/3CD expression (red). The nucleus was stained with DAPI (blue).(PDF) ppat.1007086.s002.pdf (1.0M) GUID:?DB4AA7CA-E3EE-4846-8630-7A6A43B77259 S3 Fig: Additional 3CD variants with substitutions in the 3C domain exhibit defects to PI4P induction caused by a block at a step post-Arf1 activation. (A) The indicated 3CD derivatives were expressed individually in (R)-Sulforaphane HeLa cells and immunostained for the presence of PI4P (red) and 3CD (green). The nucleus was stained with DAPI (blue). Neither 3CD derivative altered the level of PI4P or its localization. (B) Quantification of PI4P intensity per cell (n = 20) was performed as described in the legend to Fig 1C. The averages of the normalized values were: 1.06 0.02 (SEM) in mock-transfected cells; 1.09 0.06 (SEM) in 3CR13LD-transfected cells; 0.95 0.05 (SEM) in 3CR84LD -transfected cells. The level of PI4P induction observed in 3CD mutant-transfected cells was not significant when compared to mock-transfected cells based on a Students t-test. (C) Activation of (R)-Sulforaphane Arf1 by the 3CD derivatives was determined as described in the legend to Fig 3A. Both derivatives induced activation of Arf1. (D) The magnitude of Arf1 activation was determined as described in the legend Rabbit Polyclonal to NCAML1 to Fig 3B. The averages of the values for the quotients (n = 3) were: 0.43 0.03 (SEM) in mock-transfected cells; 1.23 0.18 (SEM) in 3CR13LD-transfected cells; 1.50 0.15 (SEM) in 3CR84LD-transfected cells. For mock vs 3CR13LD, mock vs 3CR84LD and 3CR13LD vs 3CR84LD, a Students t-test yielded P values of 0.0112, 0.0024 and 0.3169, respectively. (E) Complementation of 3CR13LD- or 3CR84LD-expressing subgenomic replicons by 3CD-GAA. HeLa cells were transfected with replicon RNA indicated in the key. GAA refers to a replicon (R)-Sulforaphane expressing a catalytically inactive 3D-encoded polymerase; the corresponding 3CD-GAA should function normally (R)-Sulforaphane in PI4P induction. Luciferase activity was measured every hour post-transfection as indicated.(PDF) ppat.1007086.s003.pdf (401K) GUID:?0DA45B96-D22A-497B-97D4-C8B5CDD4B898 S4 Fig: Specificity of the anti-PIP2 antibody. PIP2 is located at the plasma membrane and can only be detected in na?ve HeLa cells at 0C . We confirm the activity and specificity of the antibody used in this study by immunostaining for PIP2 (red) at 0C. The nucleus was stained with DAPI (blue).(PDF) ppat.1007086.s004.pdf (220K) GUID:?F8DED74C-B55C-4A45-B680-0969DD9DAC92 S5 Fig: Induction of PIP2 and PC biosynthesis by 3CD is not sensitive to actinomycin D and does require induction of PI4P biosynthesis. Effect of actinomycin D (AMD) on 3CD-mediated induction of PIP2 (A) and PC (B). HeLa cells were maintained in the absence or presence of AMD (5 g/mL) prior to transfection with 3CD mRNA. Cells were immunostained for PIP2 or PC (red) and 3CD (green); the nucleus was stained with DAPI (blue). The presence of AMD did not interfere with induction of PIP2 or PC. (C) The indicated 3CD derivatives were expressed.
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