Saccharomyces cerevisiae is permissive for replication of bovine papillomavirus type 1

Abstract

We recently demonstrated that Saccharomyces cerevisiae protoplasts can take up bovine papillomavirus type 1 (BPV1) virions and that viral episomal DNA is replicated after uptake. Here we demonstrate that BPV virus-like particles are assembled in infected S. cerevisiae cultures from newly synthesized capsid proteins and also package newly synthesized DNA, including full-length and truncated viral DNA and S. cerevisiae-derived DNA. Virus particles prepared in S. cerevisiae are able to convey packaged DNA to Cos1 cells and to transform C127 cells. Infectivity was blocked by antisera to BPV1 L1 but not antisera to BPV1 E4. We conclude that S. cerevisiae is permissive for the replication of BPV1 virus.

FIG. 1.

Detection of BPV1 L1 protein in S. cerevisiae cells infected with BPV1 virions. S. cerevisiae cells exposed to (A) or not exposed to (B) BPV1 were probed with an L1 specific MAb 20 h after infection. (C) Electron micrograph of BPV1 virus particles purified by density gradient centrifugation from BPV1-infected S. cerevisiae cultures 4 days after infection. The size bar represents 50 nm. (D) Time course analysis of BPV L1 protein in BPV1-yeast cultures without or with cycloheximide treatment (5 μg/ml) by immunoblotting assay. A set of 1 ml of yeast culture (5 × 10⁷cells/ml) was infected with 60 ng of BPV1 virus. At different time points (as shown in Fig. 1D), 100 μl of yeast culture was collected for protein preparation. The collected yeast culture, after pelleted and washed with PBS twice, was resuspended in 50 μl of 1× Laemmli buffer and sonicated for 40 s. Then, 25 μl of protein sample was boiled for 8 min and applied subjected to SDS-PAGE. A total of 100 ng of BPV1 virus was used as positive control (V). Immunoblotting assay for BPV L1 was as described in Materials and Methods section.

FIG. 2.

BPV1 capsid proteins in BPV1-infected S. cerevisiae cultures. (A) BPV1-infected S. cerevisiae cells were homogenized and fractionated on a CsCl density gradient. Fractions were separated by SDS-PAGE and probed for L1 and L2 proteins with specific MAbs. Band of 55 and 77 kDa, corresponding to L1 and L2, respectively, are indicated. (B) PV-infected S. cerevisiae cells were labeled with ³⁵S-labeled methionine and cysteine and fractionated as in panel A. VLPs were immunoprecipitated, and proteins were separated by SDS-PAGE. Arrows indicate L1 and L2 proteins at 55 and 77 kDa, respectively. The numbers represent fractions of CsCl gradients from the bottom (1.45 g/ml) to the top (1.26 g/ml).

FIG. 3.

Characterization of DNA extracted from high (1.35-g/ml)- and low (1.30-g/ml)-density virus-like particles prepared from BPV1-infected S. cerevisiae. (A) Undigested DNase of BPV1 virions (V), dense (D, 1.35 g/ml), and light (L, 1.30 g/ml) VLPs from BPV1-infected S. cerevisiae were electrophoresed on 1% agarose (I) and subjected to Southern blot hybridization with a ³²P-labeled BPV1 genomic probe (II). (B) DNAs of natural virions and dense (1.35 g/ml) VLPs were digested with three enzymes as shown and hybridized with ³²P-labeled BPV1 L1 gene. (C) DNA of light (1.30 g/ml) VLPs was digested with eight restriction enzymes as shown and hybridized with ³²P-labeled BPV1 L1 gene. (D) Predicted hybridization pattern of BPV1 DNA with ³²P-labeled BPV1 L1. (E) Schema showing the restriction sites of eight enzymes for the BPV1 genome and the location of the L1 gene probe.

FIG. 4.

Characterization of DNA extracted from 22 fractions of a CsCl gradient prepared from S. cerevisiae cells infected with BPV1 virions labeled with (A) or without (B and C) [³H]thymidine. (A) Incorporated radioactivity was determined by scintillation counting. The data presented are the average of three independent experiments, and the standard deviations are indicated by error bars. (B) DNA within fractions is characterized by gel electrophoresis (1% agarose). (C) DNA within fractions (see panel B) was blotted and characterized by hybridization with a ³²P-labeled BPV1 genomic probe. The numbers represent CsCl gradient fractions from 1 (1.45 g/ml) to 22 (1.26 g/ml).

FIG. 5.

Delivery of viral DNA by VLPs prepared from BPV1-infected S. cerevisiae protoplast cultures. Cos1 cells were exposed to dense (1.35 g/ml) or light (1.30 g/ml) VLPs. (A) Assay of BPV1 L1 protein in Cos1 cells exposed to dense (1.35 g/ml) VLPs. Cells were exposed as follows: lane 1, VLPs; lane 2, VLPs pretreated with polyclonal L1 antibody (1:1,000); lane 3, VLPs pretreated with monoclonal L1 antibody (1:1,000); lane 4, VLPs pretreated with E4 antibody (1:1,000). (B) Southern hybridization of Hirt DNA from Cos1 cells exposed to VLPs as indicated and probed with BPV1 genomic DNA. Lanes: 1, λ DNA; 2, 1.35 g of VLPs/ml; 3, 1.35 g of VLPs/ml, pretreated with polyclonal L1 antibody (1:1,000); 4, 1.35 g of VLPs/ml, pretreated with monoclonal L1 antibody (1:1,000); 5, 1.35 g of VLPs/ml, pretreated with E4 antibody (1:1,000); 6, 1.30 g of VLPs/ml; 7, 1.30 g of VLPs/ml, pretreated with polyclonal L1 antibody (1:1,000); 8, 1.30 g of VLPs/ml, pretreated with monoclonal L1 antibody (1:1,000); 9, 1.30 g of VLPs/ml, pretreated with E4 antibody (1:1,000). (C) PCR analysis for BPV1 early and late genes of Hirt DNA prepared from Cos1 cells infected with dense VLPs (1.35 g/ml).

FIG. 6.

Focus formation assay. C127 cells were cultured in 12-well plate and exposed to different volumes (vol/vol) of BPV1 virions (V) and light (1.30 g/ml, L) and dense (1.35 g/ml, D) VLPs. A control culture was left without infection (Mock). Cells were stained with 0.5% methylene blue plus 0.25% carbol fuschin for 15 min at 4 weeks after infection.

FIG. 7.

Estimation of VLP production from BPV1-infected S. cerevisiae cells with BPV1 virions as a standard. (A) Coomassie blue staining of SDS-PAGE gel loaded with three concentrations of BPV1 virions (V) and dense (D, 1.35-g/ml) and light (L, 1.30-g/ml) fractions purified from BPV1-infected S. cerevisiae by using a CsCl gradient. (B) Immunoblot of duplicate of panel A with an MAb (MC15) to L1 protein. Densitometry scanning was used to estimate the L1 signal. The signal for 1 μg of BPV1 virions was 7,306 ± 1,846 arbitrary units. Over six experiments, the signal for 1.35 g of VLPs/ml was 5,585 ± 4,130, a value equivalent to 0.72 μg of L1/50 μl, and the signal for 1.30 g of VLPs/ml was 7,651 ± 2,556, a value equivalent to 1.09 μg of L1/50 μl.