FIG. 2.

Assay of mitochondrial proteins and DNA in ASFV-infected cells. (A) Vero cells were mock infected or infected with ASFV, and, at different times postinfection, whole-cell extracts were prepared, fractionated in sodium dodecyl sulfate–10% polyacrylamide gels, and transferred to nylon membranes. The membranes were probed as described previously (19, 37) with a 1:200 dilution of a serum which has been shown to specifically recognize the mitochondrial stress-responsive p74 (1, 9). M, mock-infected cells. The infected cells were collected at 2 (lane 1), 6 (lane 2), 12 (lane 3), 18 (lane 4), and 24 (lane 5) hpi. The histogram at the bottom of the panel illustrates the p74 induction after quantification by densitometric scanning. Each bar represents the mean of six experiments (error bar, standard error of the mean). (B and C) Same as panel A, but with antibodies against cpn 60 (StressGene) and β-F1-ATPase protein (37), respectively. Densitometric scanning of the bands shown in panel B indicated a threefold increase in the cpn 60 contents in the infected cells. Lanes M, mock-infected cells; lanes V, ASFV-infected cells at 16 hpi. (D) Cytochrome c oxidase activity in mock-infected (M) and ASFV-infected (V) cells was assayed in whole-cell extracts at 16 hpi as described previously (37). The activity is expressed in milliunits per milligram of protein. Each bar represents the mean of five experiments (error bar, standard error of the mean). (E) Vero cells were mock infected (M) or infected with ASFV (V) and, at 16 hpi, the total cell DNA was extracted, digested with BamHI, and subjected to electrophoresis in an agarose gel. After transfer to nitrocellulose membranes, the amount of nuclear DNA present in the samples was estimated by hybridization to a ³²P-labeled DNA probe specific for the rat liver β-F1-ATPase gene (11). The bands corresponding to mock- and virus-infected cells were analyzed by densitometry, and the membrane was then stripped and hybridized to a probe corresponding to the gene coding for the 12S mitochondrial rRNA from rat liver (10). After densitometry was performed on the bands obtained, the amount of mitochondrial DNA in the samples was quantified by calculating the ratio of the densitometric value of the mitochondrial DNA to that of the nuclear DNA. The values for these ratios were the following: mock-infected cells, 0.37; and infected cells, 0.32. Hybridization to BamHI-digested rat liver DNA, used as a control (C) is also shown.