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 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...

Wounding prior to challenge substantially improves infectivity of cottontail rabbit papillomavirus and allows for standardization of infection

“…This characteristic, therefore, allows papillomavirus DNA mutants to be studied in vivo, something that cannot be done in other animal models to date. However, different CRPV DNA challenge methods have been used in different laboratories (Brandsma et al 1991;Jeckel et al 2002;Kreider et al 1995;Lin et al 1993;Nonnenmacher et al 2006;Salmon et al 1997;Salmon et al 2000). Some delivery methods are more efficient than others and these outcomes complicate the interpretation of results obtained in the different laboratories.…”

Influence of HPV16 E2 and its localisation on the expression of matrix metalloproteinase-9

“…The E2 protein of the Cottontail rabbit papilloma virus (CRPV) has previously been demonstrated to play a major role in the development of CRPV dependent skin carcinogenesis (14). This is particularly interesting in light of our recently reported finding that the E2 protein of CRPV (15) has the ability to activate the in vitro expression of matrix metalloproteinase 9 (MMP-9), a protein involved in the degradation and remodelling of the extracellular matrix (ECM).…”

“…4A). We decided not to delete the NES found in HPV16 E2, because the position in the N-terminal domain is within the transactivation domain of the protein with the consecutive risk of false-positive results (14). Using immunofluorescence and the eGFP-tagged E2 mutants, the localisation of the mutant proteins was determined.…”

Cutaneous Papillomavirus E6 Proteins Must Interact with p300 and Block p53-Mediated Apoptosis for Cellular Immortalization and Tumorigenesis

“…Infections of New Zealand White rabbits with CRPV DNA were performed as previously described (10). Tumor numbers and the maximum diameter of each tumor were determined over a period of 5 months.…”

“…A causative relationship between β-HPV infection and nonmelanoma skin cancer development in immunocompetent individuals is suggested by epidemiologic data and by the fact that the associated risk factors (age, UV-mediated local immunosuppression, and transplantation-related immunosuppression) point to an infectious agent, most probably belonging to the β2 group, which includes HPV38 (8,9). An excellent animal model to study papillomavirusinduced skin cancer formation is the New Zealand White rabbit, in which infections with cottontail rabbit papillomavirus (CRPV) causes the development of tumors within 3 to 6 weeks postinfection, progressing within 6 to 12 months into invasive cancer without additional cofactors (10,11). Recently, we reported that the CRPV oncoproteins E6 and E7 are able to immortalize primary rabbit keratinocytes (12), a common feature of all carcinogenic HPV types (13), which is believed to be a key requirement for cancer formation.…”