A transactivator function of cottontail rabbit papillomavirus e2 is essential for tumor induction in rabbits

Abstract

Infection of domestic rabbits with cottontail rabbit papillomavirus (CRPV) causes local papillomas which progress to carcinomas in more than 80% of cases. This animal model system therefore allows the identification of molecular mechanisms required for the induction and progression of epithelial tumors. The viral E2 protein stimulates both viral DNA replication and transcription, and these functions can be genetically separated. We introduced the respective mutations into CRPV E2 and found, in line with published data for other papillomavirus E2 proteins, that mutation of the highly conserved amino acid 37 or 73 resulted in replication-competent but transactivation-deficient E2 proteins, whereas E2 proteins with mutations at residue 39 were replication deficient and transactivation competent. The R37A, I73L, and I73A E2 mutants, showing a loss of transactivation function, and the R37K E2 mutant, which is still transactivation competent, were introduced into the whole genome of CRPV, which was then injected into the skin of rabbits. Strikingly, the ability to induce tumors within 6 weeks was abolished by each of the E2 mutations, in contrast to the tumor induction rate (93%) obtained with wild-type CRPV DNA. Two small papillomas induced by mutant E2 I73A CRPV DNA appeared as late as 12 or 24 weeks postinjection, were significantly smaller, and showed no further extension of growth. These data suggest that functionally conserved amino acids in the transactivation domain of E2 are also required for the induction and growth of epithelial tumors in rabbits infected with CRPV.

FIG. 1.

Linear map of CRPV. Open boxes, ORF. The early promoters P1, P2, and P3 and the late promoter PL are indicated. The structure of the CRPV E2 protein is diagrammed below. Solid rectangle, amino-terminal domain of E2, required for stimulation of DNA replication and transcription. Striped rectangle, carboxy-terminal domain, mediating sequence-specific DNA binding and dimerization of E2 proteins. Mutations resulting in amino acid changes of highly conserved residues (residues 37, 39, and 73) are marked by vertical white lines.

FIG. 2.

(A) Western blot analysis of nuclear extracts from sf1Ep cells transfected with CRPV wt E2-pSG5 or one of the E2 mutants R37K-, R37A-, E39Q-, E39A-, I73L-, and I73A-pSG5. The control lane contains an extract from cells transfected with the parental pSG5 vector. The position of the E2 proteins is indicated by an arrow. A molecular-size marker (in kilodaltons) is shown on the left. (B) Electromobility shift assay of CRPV wt and mutant E2 proteins present in nuclear extracts of transfected sf1Ep cells. Control lanes contained no protein extract (oligo) or an extract from cells transfected with the parental vector (pSG5). The retarded bands corresponding to the E2-DNA complex (a and b) and the unbound ³²P-labeled oligonucleotide containing an E2 binding site (f) are indicated on the right.

FIG. 3.

Transient luciferase expression assays. SCC13 cells were transfected with expression vectors for CRPV wt E2 or the R37K, R37A, E39Q, E39A, I73L, or I73A E2 mutant protein and the E2-responsive reporter plasmid p6xE2BS-luc and were analyzed for luciferase activity. The reporter plasmid, diagrammed below the graph, consists of six E2-binding sites (solid boxes) upstream of the minimal simian virus 40 (SV40) early promoter, which drives the expression of the luciferase gene (luc). The luciferase activity obtained by cotransfection of each E2 mutant expression plasmid is given relative to the activity of wt E2-transfected cells, which was set to 1. Error bars, standard deviations.

FIG. 4.

Autoradiograph of a transient DNA replication assay. SCC-13 cells were transfected with plasmid CRPV-pGL3-NCR either alone (−) or together with an expression vector for CRPV E1 and an expression vector for CRPV wt E2 or the R37K, R37A, E39Q, E39A, I73L, or I73A mutant E2 protein. Low-molecular-weight DNA was extracted, digested with DpnI and HpaI, and analyzed by Southern blot hybridization. The position of DpnI-resistant CRPV-pGL3-NCR DNA is indicated by an arrow. Percentages given refer to the amounts of replicating plasmids of the respective E2 mutants in relation to the wt E2 protein (100%).

FIG. 5.

Photograph of the back of a rabbit 6 weeks after infection with CRPV-pLAII wt DNA (WT), E2-R37K-CRPV DNA (R37K), E2-R37A-CRPV DNA (R37A), or E2-I73L-CRPV DNA (I73L) by use of a gene gun. Papilloma development was visible only at sites injected with wt CRPV-pLAII DNA.