Replication and transcription of human papillomavirus type 58 genome in Saccharomyces cerevisiae

Abstract

Background: To establish a convenient system for the study of human papillomavirus (HPV), we inserted a Saccharomyces cerevisiae selectable marker, Ura, into HPV58 genome and transformed it into yeast.

Results: HPV58 genome could replicate extrachromosomally in yeast, with transcription of its early and late genes. However, with mutation of the viral E2 gene, HPV58 genome lost its mitotic stability, and the transcription levels of E6 and E7 genes were upregulated.

Conclusions: E2 protein could participate in viral genome maintenance, replication and transcription regulation. This yeast model could be used for the study of certain aspects of HPV life cycle.

Figure 1

Replication of HPV 58 genome in S. cerevisiae. A and B: Episomal replication of HPV58 genome in S. cerevisiae. Yeast DNA underwent Southern blot with an L1 specific mRNA probe. A, (DNA isolated from yeast harboring HPV58-Ura) Lane 1: yeast DNA without enzyme digestion; lane 2-5: yeast DNA digested with Dpn I, Xho I, Bgl II and Hpa I. B, Lane 1: DNA isolated from HPV58-Ura-E2mt transformed yeast and treated with Dpn I; lanes2-6 (yeast DNA isolated from HPV58-Ura-E2mt/pDBLeu-E2 transformed yeast), lane 2: DNA without enzyme digestion; lanes 3-6: yeast DNA digested with Dpn I, Xho I, Bgl II and Hpa I separately. Arrows show the positions of open-circle (OC), linear (L) and supercoiled (SC) forms of episomal HPV58-Ura or HPV58-Ura-E2mt. C. qRT-PCR to compare the E2 transcription levels in yeast cells harboring HPV58-Ura or pDBLeu-E2. D. Expression of E2 protein in yeast. Yeast protein was prepared from yeast harboring pDBLeu-E2 (lane 1), pDBLeu (lane 2), and untransformed yeast (lane 3).

Figure 2

DNA stability assay of HPV58 genome. Yeast containing different recombinant HPV58 genomes were grown under non-selective conditions for 10 cell generations. After the incubation period, the cell cultures were serially diluted from 1 to 10^-5. Equal volumes of each of the dilutions were spotted onto selective (+) and non-selective (-) media. The percentage of DNA loss per cell generation was calculated by subtracting the percentage DNA retained after 10 generations from that at 0 generation and divided by the total number of generations. Values on the right are the means of three independent experiments. NA: non-available.

Figure 3

Transcription of HPV58 genes in S. cerevisiae. A. RT-PCR analysis of transcriptional expression of HPV58 early and late genes in the HPV58-Ura transformed yeast. E1 (lanes 1, 2), E2 (lanes 3, 4), E6 (lanes 5, 6), E7 (lanes 7, 8), L1 (lanes 9, 10) and L2 (lanes 11, 12) from the cDNA samples. Total RNA was isolated from HPV58-Ura transformed yeast and digested with DNase I. The DNase I treated RNA was amplified to ensure the complete digestion of contaminating viral DNA (lanes 2, 4, 6, 8, 10 and 12). DNase I completely digested RNA were then analysized by RT-PCR (lanes 1, 3, 5, 7, 9 and 11). B. Relative replication levels of HPV58 genomes in HPV58-Ura, HPV58-Ura-E2mt and HPV58-Ura-E2mt/pDBLeu-E2 transformed yeast. 18S rDNA was used as internal control. The DNA relative replication levels of HPV58-Ura-E2mt (0.03) and HPV58-Ura-E2mt/pDBLeu-E2 (0.54) are relative to that of HPV58-Ura, which was set to 1.0. Standard deviations are indicated by error bars. C. Relative transcription levels of E6 and E7 genes for HPV58-Ura, HPV58-Ura-E2mt and HPV58-Ura-E2mt/pDBLeu-E2. DNase I treated RNA was analysized by qRT-PCR with 18S rRNA as internal control. As for HPV58 genomes have different replication efficiency in the presence or absence of E2 protein (Figure 3B), the transcription levels of E6 and E7 genes were then standardized to the relative DNA replication assay. The E6 and E7 genes transcriptional levels of HPV58-Ura were set to 1.0. The transcription levels of E6 are 8.62 for HPV58-Ura-E2mt and 1.02 for HPV58-Ura-E2mt/pDBLeu-E2. The E7 transcription levels are 1.96 for HPV58-Ura-E2mt and 0.74 for HPV58-Ura-E2mt/pDB Leu-E2 relative to that of HPV58-Ura, which was set to 1.0. Standard deviations are indicated by error bars.