Identification of the E9/E2C cDNA and functional characterization of the gene product reveal a new repressor of transcription and replication in cottontail rabbit papillomavirus

Abstract

Cottontail rabbit papillomavirus (CRPV) genomes mutated in the trans-activation domain of the E2 protein, which stimulates both viral DNA replication and transcription, are severely impaired in their ability to induce tumors in New Zealand White rabbits. A number of papillomaviruses encode, in addition to full-length E2, a shortened E2 protein or an E2 protein fused to a short stretch of amino acids derived from the small E8 open reading frame that counteract the activities of E2. We identified and cloned the novel cDNA E9/E2C of CRPV from papillomas of New Zealand White and cottontail rabbits and characterized the functions of the encoded gene product. E9/E2C was shown to be a bona fide repressor of minimal viral promoters, with the E9 domain being essential for this activity, and to repress E1/E2-dependent replication of a CRPV origin construct. In addition, E9/E2C counteracted the transactivation effect of the full-length E2 on minimal promoters containing several E2 binding sites. To investigate the role of E9/E2C in tumorigenesis, we constructed two CRPV genomes mutated in E9/E2C. One, designated CRPV-E9atgmut-pLAII, contained a mutation in the unique start codon in the E9 open reading frame, and the second E9/E2C mutant was constructed by the introduction of a stop codon close to the splice donor site at nucleotide 3714 that additionally prevented the correct splicing of the transcript. When we infected New Zealand White rabbits with these constructs, we surprisingly noted no differences in tumor induction efficiency, viral genome copy number, and viral transcription in comparison to wild-type CRPV.

FIG. 1.

CRPV genome organization and structure of the E9^E2C transcript. The linearized genome of CRPV with the various ORFs (E1 to E9, L1, and L2), the late viral promoter (P_L), and the early viral promoters (P1, P2, and P3) are shown at the top. Below, structure of the E9^E2C transcript with the splice donor (SD) site at nucleotide 1751 linked to the splice acceptor (SA) site at nucleotide 3714, giving rise to a fusion protein consisting of 10 amino acids derived from ORF E9 (black box) linked to the hinge and C terminus of E2 (grey box).

FIG. 2.

E9^E2C represses the basal activity of different promoters in epithelial cells. Reporter plasmids CRPV-P_LP1-pGL3, 6×E2BS-luc, and pC18-SP1-luc, each containing several E2 binding sites (black boxes) upstream of the viral promoters P_L and P1 of CRPV, the simian virus 40 (SV40) early promoter, or the minimal adenoviral promoter (INR) that drive the expression of the luciferase gene (luc) were cotransfected in SCC13 or CRL6502 cells with either 10 ng of CRPV-E9^E2C-pSG5 or 10 ng of the empty backbone expression vector pSG5. Luciferase activities are given relative to the activity in pSG5-transfected cells, which was set to 1. Standard deviations are indicated by error bars. Each measurement presents the average of 6 to 10 independent experiments.

FIG. 3.

E9 domain is responsible for repression of minimal viral promoters. CRL6502 (A and C) or RK1-16E7/ras (B) cells were transfected with 200 ng of the reporter plasmid pC18SP1-luc (A and B) or 200 ng of the reporter plasmid CRPV-P_LP1-pGL3 (C) and 10 ng of the empty expression vector pSG5 or expression vectors for CRPV-E2, wild-type CRPV-E9^E2C, or the E9 deletion mutant CRPV-E9K2A(d3-10)^E2C. Luciferase activities are given relative to the activity in pSG5-transfected cells, which was set to 1. Standard deviations are indicated by error bars. Note logarithmic scale on y axis (A and B).

FIG. 4.

E9^E2C counteracts the transactivation mediated by full-length E2. SCC13 cells were cotransfected with 200 ng of the reporter plasmid pC18-SP1-luc (see Fig. 2 legend), 10 ng of CRPV-E2-pSG5, and an increasing amount of CRPV-E9^E2C-pSG5 or CRPV-E9K2A(d3-10)^E2C. Luciferase activities are shown relative to that of cells not transfected with E9^E2C-pSG5, which was set to 1. Standard deviations are indicated by error bars.

FIG. 5.

E9^E2C represses the E1- and E2-dependent replication of the CRPV origin. Autoradiography of a transient DNA replication assay. SCC13 cells were transfected with 500 ng of the CRPV origin plasmid CRPV-pGL3-NCR and with the expression vectors CRPV-E1-pSG5 (E1) and CRPV-E2-pSG5 (E2) and increasing amounts (in nanograms) of CRPV-E9^E2C-pSG5 (E9^E2C). Low-molecular-weight DNA was extracted, digested with DpnI and HpaI, and analyzed by Southern blot hybridization. Lane M, 100 and 10 pg of CRPV-pGL3-NCR plasmid linearized by HpaI. Quantitative analysis of the amount of replicated DNA is shown below the autoradiograph. The replication efficiencies are expressed relative to the replication level in cells not transfected with E9^E2C-pSG5, which was set to 100%.

FIG. 6.

Construction of E9^E2C mutants in the context of the CRPV genome. The nucleotide sequence of the CRPV E9 gene is shown below the genome of CRPV. Positions of the ATG codon within the ORF E9 and the splice donor (SD) consensus site at nucleotide 1751 are indicated. Mutated nucleotides leading to the disruption of the ATG codon (CRPV-E9atgmut-pLAII) or to the introduction of a stop codon in E9 (CRPV-E9stop-pLAII) are indicated by arrows.

FIG. 7.

Southern blot analysis of DNA isolated from papillomas induced by wild-type CRPV-pLAII (lanes 5, 6, 9, and 10), CRPV-E9atgmut-pLAII (lanes 7 and 8), or CRPV-E9stop-pLAII (lanes 3 and 4). Biopsy DNA (10 μg) was either digested with XhoI (lanes 3, 5, 7, and 9), which does not cut within CRPV-pLAII, or with EcoRI (lanes 4, 6, 8, and 10), which cuts twice within CRPV-pLAII. As marker fragments, we used 20 pg (lane 2) and 200 pg (lane 1) of CRPV-pLAII plasmid DNA digested with EcoRI.

FIG. 8.

Detection of CRPV-E9^E2C transcript by reverse transcription-PCR. A total of 500 ng of RNA isolated from papillomas induced by wild-type CRPV-pLAII (WT), CRPV-E9atgmut-pLAII (E9atgmut), or CRPV-E9stop-pLAII (E9stop) was used in a reverse transcription-PCR with primers P1708 (upstream of SD₁₇₅₁) and P3883 (downstream of SA₃₇₁₄) and Ready-To-Go reverse transcription-PCR beads. The correct size of the cDNA fragment derived from the spliced E9^E2C transcript is shown on a 2% agarose gel. The sizes of molecular size markers are given on the right (in base pairs).

FIG. 9.

Northern blot analysis of polyadenylated RNA isolated from papillomas induced by wild-type CRPV-pLAII (wild type) or CRPV-E9atgmut-pLAII (E9atgmut) or from normal rabbit skin (normal skin). A total of 600 ng of RNA per lane was hybridized to ³²P-labeled CRPV DNA as the probe. The sizes of molecular size markers are given on the left (in kilobases).