Classical Swine Fever Virus p7 Protein Interacts with Host Protein CAMLG and Regulates Calcium Permeability at the Endoplasmic Reticulum

Figures

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  Figure 1. Interaction of CSFV p7 protein and host CAMLG detected by yeast two-hybrid system. (A) Yeast strain AH109 was transformed with either GAL4-binding domain (BD) fused to CSFV p7 (BD-p7), or mutated CSFV p7 construct as indicated or human lamin C (BD-LAM), a negative control and GAL4 activation domain (AD) fused to CAMLG (AD-CAMLG) or the negative control pGADT7 as indicated. 2 × 106 yeast cells were spotted onto either SD-Ade/His/Leu/Trp plates (-ALTH), selective media for detecting protein-protein interactions or nonselective SD-Leu/Trp plates (-TL) (B) Schematic representation of CSFV p7 alanine mutants used in the present study. The name of each alanine p7 mutant is shown below the mutated amino acid residues for each particular mutant. All indicated residues were mutated to an alanine. The highlighted p7 mutants indicate alanine substitutions resulting in lack of binding of p7 to CAMLG in the yeast two-hybrid system.

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  Figure 2. (A) Co-localization of p7 and CAMLG in transfected SK6 cells imaged at 1000×. p7-GFP and RFP-CAMLG constructs were co-transfected in SK6 cell monolayers. p7 mutant constructs fused to GFP (7.6-GFP, 7.7-GFP and 7.8-GFP) were co-transfected with RFP-CAMLG in SK6 cells. Preparations were mounted with ProLong Gold antifade reagent containing DAPI to stain the nuclei. In the merged panel, yellow color indicates co-localization of GFP and RFP. (B) Transfection Native and mutated forms of p7 in 293T cells imaged at 630× located in the endoplasmic reticulum. Distribution of p7 and its mutants 7.6, 7.7 and 7.8 by confocal microscopy. Micrographs illustrate individual cells co-expressing GFP constructs and the ER marker mCh-Sec61. The control GFP devoid of membrane anchors labelled the complete cell (top panels), whereas GFP-p7 constructs were excluded from the nucleus and co-localized with mCh-Sec61. (C) Co-localization of mCh-Sec6 and GFP in the samples as calculated with the ImageJ plugin Coloc 2 program (http://imagej.net/Coloc_2). Measurements were carried out in at least 6 cells as those displayed in the previous panel. Bars represent mean values ± SE.

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  Figure 3. Effect of native and mutated forms of p7 in thapsigargin-induced calcium release from the endoplasmic reticulum. (A) Kinetic traces of Ca2+ efflux from the ER into the cytosol in cells transfected with GFP or the GFP fusions as indicated in the panel. (B) Maximal extents of rhod-2/AM intensity change upon thapsigargin addition.

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  Figure 4. In vitro growth kinetics of CSFV mutants 7.6, 7.7 and 7.8 and parental CSFV BICv. (A) SK6 or (B) swine macrophage cell cultures were infected (MOI0.01). Virus yields, were titrated in SK6 cell cultures as described in Material and Methods. Data represent means and standard deviations from two independent experiments. Sensitivity of virus detection: ≥log10 1.8 HAD50/mL.