Replication of bovine papillomavirus type 1 (BPV-1) DNA in Saccharomyces cerevisiae following infection with BPV-1 virions

Abstract

Saccharomyces cerevisiae protoplasts exposed to bovine papillomavirus type 1 (BPV-1) virions demonstrated uptake of virions on electron microscopy. S. cerevisiae cells looked larger after exposure to BPV-1 virions, and cell wall regeneration was delayed. Southern blot hybridization of Hirt DNA from cells exposed to BPV-1 virions demonstrated BPV-1 DNA, which could be detected over 80 days of culture and at least 13 rounds of division. Two-dimensional gel analysis of Hirt DNA showed replicative intermediates, confirming that the BPV-1 genome was replicating within S. cerevisiae. Nicked circle, linear, and supercoiled BPV-1 DNA species were observed in Hirt DNA preparations from S. cerevisiae cells infected for over 50 days, and restriction digestion showed fragments hybridizing to BPV-1 in accord with the predicted restriction map for circular BPV-1 episomes. These data suggest that BPV-1 can infect S. cerevisiae and that BPV-1 episomes can replicate in the infected S. cerevisiae cells.

FIG. 1.

Morphological observations and Southern blot analysis of BPV-1-infected S. cerevisiae culture. (A) Morphology of S. cerevisiae protoplasts after 2 days in culture without and with BPV-1 virus exposure. (B) Cell wall regeneration of S. cerevisiae protoplasts with and without exposure to BPV-1 virions at various time points in culture as indicated, demonstrated in the lower panels by staining with Calcofluor. Upper panels in each case show light microscopy of the same field. (C) Transmission electron micrograph of a nuclear section of S. cerevisiae exposed to BPV-1 virus. NM indicates the nuclear membrane. Bars, 200 nm. The inset shows an enlargement of a selected part (small square) of the main picture, and bar represents 50 nm. (D) Southern blot analysis of two S. cerevisiae strains, exposed as shown to BPV-1 virus 48 h previously. S. cerevisiae (5 ml; 5 × 10⁷cells/ml) was infected with 0.2 μg of BPV-1 virus and cultured with gentle shaking at 28°C in the dark. At 24 h, 5 ml of fresh medium was added. S. cerevisiae was collected for Hirt DNA preparation at 48 h, and 10 μg of DNA was used for Southern blot analysis. N, nicked circle; L, linear; S, supercoiled DNA.

FIG. 2.

BPV-1 DNA detected by Southern blot in a BPV-infected S. cerevisiae culture over 5 days. S. cerevisiae (15 ml; 5 × 10⁷cells/ml) was infected with 0.9 μg of BPV-1 virus. At 24 h, 10 ml of S. cerevisiae was collected, with 5 ml each being used for Hirt DNA and RNA preparation. Then 10 ml of fresh medium was added for continuing culture. Thereafter, 10 ml of S. cerevisiae was collected for Hirt DNA preparation and 10 ml of fresh medium was added every 24 h until 120 h. A total of 15 ml of medium was added to 0.9 μg of BPV-1 virus and similarly sampled and diluted. Hirt DNA prepared from 5 ml of S. cerevisiae cultures for each time point was resuspended in 200 μl of TE buffer. Then 20 μl of Hirt DNA, after digesting partially with HindIII for about 2 h, was electrophoresed on a 1% agarose gel and blotted onto a nylon membrane. DNA blots were hybridized with whole BPV-1 DNA probe. As controls, 5 ml of medium infected with BPV-1 virus was pelleted for viral DNA extraction at high speed (10,000 rpm) for 30 min. The pellets were resuspended in 400 μl of lysate buffer and incubated with 100 μg of proteinase K at 37°C for 1 h. The DNA was extracted with Tris-buffered phenol and chloroform. The extracted DNA was also resuspended in 200 μl of TE buffer. Then 20 μl of the DNA suspensions was run on an agarose gel and blotted onto nylon membrane for BPV-1 DNA hybridization. The time point and effective culture dilution are shown above each lane. *1 indicates that the Hirt DNA contained the viral DNA from 0.03 μg of BPV-1 virions. The viral DNA dilution at the different time point is shown above each lane.

FIG. 3.

(A) BPV-1 DNA detected by Southern blot in a BPV-infected S. cerevisiae culture over 55 days. S. cerevisiae cells (10 ml; 5 × 10⁷cells/ml) infected with 0.6 μg of BPV-1 was cultured at 28°C in the dark. At 1 day, 5 ml of S. cerevisiae cells was collected for Hirt DNA preparation, with 5 ml of fresh medium added for continuous culture. This was repeated every day until 5 days. At 5 days, 5 ml of S. cerevisiae cells was held at 4°C for 15 days. Then 5 ml of fresh medium was added, and cells were cultured at 28°C. From day 21 to day 25, 5 ml of S. cerevisiae culture was collected for Hirt DNA and 5 ml of fresh medium was added every day. A total of 5 ml of the 25-day S. cerevisiae culture was held at 4°C for 25 days. Fresh medium (5 ml) was added, and cells were cultured at 28°C, diluted, and sampled as previously from 51 days to 55 days. Hirt DNA (20 μl) was resuspended in 200 μl of TE buffer, partially (days 1, 2, 21, and 22) or completely (days 54 and 55) digested with HindIII, electrophoresed on a 1% agarose gel, and blotted onto a nylon membrane. *1 indicates that the Hirt DNA contained the viral DNA from 0.03 μg of BPV-1 virus. Viral DNA dilution at the different time points is shown above each lane. The time point and effective culture dilution are shown above each lane. (B) BPV-1 DNA detected by Southern blot in a BPV-infected S. cerevisiae culture over 75 days. A total of 2 ml of S. cerevisiae cells (5 × 10⁷cells/ml) infected with 0.06 μg of BPV-1 was cultured at 28°C in the dark. Then 2 ml of fresh medium was added once a week for continuous culture. Hirt DNA prepared from S. cerevisiae infected with BPV-1 virus 62 and 75 days postinfection was electrophoresed on a 1% agarose gel without enzyme digestion. All blots were hybridized with BPV-1 DNA using [α-³²P]dCTP at 3,000 Ci/mmol and exposed using Kodak BioMax film at −70°C for 24 h.

FIG. 4.

Two-dimensional gel electrophoresis of Hirt DNA from BPV-1-infected S. cerevisiae cultures. A total of 10 μg of Hirt DNA was electrophoresed as described in the text, blotted onto nylon, and hybridized with a BPV-1 DNA probe. The left panel shows the hybridization results of the first- and second-dimensional blots, and the right panel shows the diagrammed pattern of the replication intermediates.

FIG. 5.

Restriction enzyme analysis of Hirt DNA prepared from BPV-1-infected S. cerevisiae cells in a 5-day culture (A) and a 55-day culture (B). A total of 10 μg of Hirt DNA, after overnight digestion with enzymes as shown, was electrophoresed on a 1% agarose gel (upper panels), blotted onto nylon, and hybridized with the ³²P-labeled BPV-1 L1 gene (lower panels). (C) Schema showing the restriction sites of seven enzymes for the BPV-1 genome and the location of the L1 probe.