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Fig. 3 | Retrovirology

Fig. 3

From: ATP generation in a host cell in early-phase infection is increased by upregulation of cytochrome c oxidase activity via the p2 peptide from human immunodeficiency virus type 1 Gag

Fig. 3

Mitochondrial cytochrome c oxidase was activated by the p2 peptide. a MAGIC-5 cells transfected with pcDNA4/EGFP-p2x1 were subjected to western immunoblot analysis for MT-CO1 or actin expression. The expression of the EGFP-p2x1 peptide in MAGIC-5 cells did not affect the expression of both MT-CO1 and actin. A representative result is shown from at least three independent experiments. b MT-CO activity measurements show that the p2 peptide activates the enzymatic activity of MT-CO-positive control or native MT-CO1 in mitochondrial fractions from MAGIC-5 cells. Unit Definition: One unit oxidizes one micromole of ferrocytochrome c per minute at 25 °C, pH 7.0. **p < 0.01 versus control; Repeated measures ANOVA and Dunnett’s post hoc test (n = 10). c Sodium azide completely inhibited p2-peptide-dependent enzymatic activation of MT-CO. The significance of difference (Student’s t test) is indicated as follows: **p < 0.01 (n = 5). d Confocal laser scanning microscopy was used for observation of fluorescence in HeLa cells expressing EGFP, or the EGFP-p2x1, EGFP-p2x9, or EGFP-p2x17 peptide. EGFP fluorescence is shown in the left micrographs, MitoTracker-derived fluorescence is shown in the second column of the micrographs, and an overlay of EGFP and MitoTracker signals (Merge) is shown in the rightmost column of micrographs. The nuclei were made visible with DAPI staining (Third column). EGFP-p2 peptide fusion proteins (EGFP-p2x1 peptide, EGFP-p2x9 peptide, or EGFP-p2x17 peptide) colocalized with the mitochondrial marker MitoTracker. A representative image from three independent experiments is shown. e Mitochondria obtained from MAGIC-5 cells expressing the EGFP-p2x1 peptide were incubated with proteinase K (0.05 μg/ml). The lysate of the mitochondrial protein was subjected to SDS-PAGE and examined by western immunoblot analysis using an anti-Xpress, anto-Bcl2 or anti-MT-CO1 antibody. A representative result is shown from at least three independent experiments

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