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.Papillomaviruses have attracted special attention because persistent infection with a subset of human papillomaviruses (HPV) is a necessary factor for the development of anogenital cancer (4, 43). In contrast, the role of HPV in skin cancer, which is the most common cancer of the Caucasian race, is still undefined, although a very first link was obtained in 1935, when Rous et al. (30) described the development of squamous cell carcinomas in rabbits after experimental infection with cottontail rabbit papillomavirus (CRPV). Skin cancer in humans has been recognized as a common side effect in longterm immunosuppressed patients, who have a 65-fold increased risk compared to immunocompetent individuals. Shortly after the onset of immunosuppressive therapy, HPVinduced warts develop in these individuals, and 20 years after the beginning of immunosuppression nonmelanocytic skin cancer can be found in up to 70% of these patients (6). A similar situation has been described in the rare genetic disorder epidermodysplasia verruciformis, but the mechanisms leading to the development of skin cancer and especially the role of papillomaviruses are not yet fully understood (3,12,38).The only model system for studying tumor induction and progression after infection of the skin with papillomaviruses is the domestic rabbit infected with CRPV, in which local tumors develop within 3 to 6 weeks postinfection and progress within 6 to 1...
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 replicationcompetent 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. A suitable animal model in which to investigate molecular processes involved in tumor induction and progression by papillomaviruses is the New Zealand White rabbit, which develops local papillomas within 3 to 8 weeks after injection of cottontail rabbit papillomavirus (CRPV) DNA into the skin (45). In contrast to the productive virus infection observed in the cottontail rabbit, which is the natural host, infection of New Zealand White rabbits with CRPV causes an abortive infection (45). As many as 80% of the initially benign epithelial tumors progress to carcinomas within the next 6 to 14 months, and these finally metastasize without the need for cofactors (45). Previous studies using deletion or insertion mutations within the genome have shown that all open reading frames (ORF) of CRPV, except for E5 and L2, are required for tumor induction in rabbits (5,6,25,26,46). In contrast, tissue culture experiments have demonstrated that expression of CRPV LE6 and E7 is sufficient to induce anchorage-independent growth of NIH 3T3 cells and to induce tumors in nude mice injected with the respective cell lines (25). When viral transcripts from benign and malignant tumors were compared by a number of different techniques, no major quantitative or qualitative differences that would have been indicative of a prominent role of a single viral gene product could be observed (27,29,48).A master regulator of early gene expression and viral DNA replication is the m...
While high-risk human papillomaviruses (HPVs) have been identified as necessary risk factors for the development of cervical cancer (7), the role of HPV in skin cancer, which is the most common cancer in Caucasians, is still undefined (3, 23). A first link between papillomaviruses and skin cancer was obtained in 1935 from an animal model system, when Rous and Beard (40) described the development of squamous cell carcinomas (SCCs) in rabbits after experimental infection with cottontail rabbit papillomavirus (CRPV). Initial benign warts develop into invasively growing SCCs without any further cocarcinogens within 6 to12 months in the vast majority of the animals (50). A similar situation has been described for humans, where in the rare genetic disorder epidermodysplasia verruciformis, patients develop flat HPV-induced warts which progress in up to 60% of the cases into mostly primary SCCs (36, 37). Skin cancer has also been recognized as a common side effect in long-term-immunosuppressed patients, who have a 65-fold-increased risk for this disease. Shortly after the onset of immunosuppressive therapy, HPV-induced warts develop, and after 20 years of immunosuppression, nonmelanocytic skin cancer can be found in up to 70% of the patients (8). As in the case of epidermodysplasia verruciformis patients, the lesions seem to progress from warts through dysplastic lesions into SCC (4,16,43). Although HPV-associated SCCs have been found in 65% (43) or 81% (4) of the cases, the mechanisms leading to the development of skin cancer and especially the role of papillomaviruses are not yet fully understood.Thus far, the only model system for studying the progression of papillomavirus-induced skin tumors is the domestic rabbit infected with CRPV. Infection of domestic rabbits with CRPV particles or viral DNA leads to the development of local tumors within 3 to 6 weeks postinfection. These papillomas progress within 6 to 12 months in more than 80% of the cases into invasively growing and finally metastasizing carcinomas (45; S. Jeckel, unpublished data). Only in very few individual rabbits do papillomas persist in the benign state, and even fewer regress. This progressive behavior does not depend on any other known cofactor besides the viral infection, which makes CRPV an important model for papillomavirus-associated skin disorders in order to study the role of viral and cellular gene expression during cancer development.Earlier studies using more descriptive methods, such as RNA-RNA in situ hybridization, showed no pronounced qualitative or quantitative changes in viral gene expression during malignant progression (50). The primary target cells localized in the bulge region of hair follicles already revealed rather high expression of E6 and E7 mRNAs (42), which remains a constant feature from papillomas through carcinomas to lung metastases (50, 52). Therefore it seems most likely that alterations
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