RESUMO
E6 from high risk human papillomaviruses (HR HPVs) promotes ubiquitination and degradation of p53 tumour suppressor by mediating its binding to ubiquitin ligase E6AP in a ternary complex, contributing to cell transformation in cervical cancer. We have previously shown that Felis catus papillomavirus type -2 (FcaPV-2) E6 is expressed in feline squamous cell carcinoma (SCC) and displays the ability to bind p53 and decrease its protein levels in transfected CRFK cells. However, the mechanism of p53 downregulation has not yet been characterized. Here we show that FcaPV-2 E6 bound to E6AP, which in turn was bound by p53 exclusively in cells expressing the viral oncoprotein (CRFKE6). Furthermore, p53 was highly poly-ubiquitinated and underwent accumulation upon E6AP gene knockdown in CRFKE6. Half-life experiments and proteasome inhibition treatments indicated that down-regulation of p53 protein in CRFKE6 was due to accelerated proteasomal degradation. E6AP/p53 binding was also demonstrated in two feline SCC cell lines expressing FcaPV-2 E6, where p53 protein levels and poly-ubiquitination degree were proportional to E6 mRNA levels. The data obtained in both artificial and spontaneous in vitro models suggest that FcaPV-2 E6 degrades p53 through a molecular mechanism similar to HR HPVs, possibly contributing to the development of feline SCC.
Assuntos
Carcinoma de Células Escamosas/metabolismo , Doenças do Gato/metabolismo , Lambdapapillomavirus/metabolismo , Proteínas Oncogênicas Virais/metabolismo , Infecções por Papillomavirus/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Proteína Supressora de Tumor p53/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/veterinária , Carcinoma de Células Escamosas/virologia , Doenças do Gato/genética , Doenças do Gato/virologia , Gatos , Linhagem Celular , Lambdapapillomavirus/genética , Proteínas Oncogênicas Virais/genética , Infecções por Papillomavirus/genética , Complexo de Endopeptidases do Proteassoma/genética , Proteína Supressora de Tumor p53/genética , Ubiquitina-Proteína Ligases/genéticaRESUMO
Cell-mediated immunity plays a key role in the regression of papillomavirus-induced warts and intra-epithelial lesions but the target antigens that induce this response are not clear. Canine oral papillomavirus (COPV) infection of the oral cavity in dogs is a well-characterized model of mucosal papillomavirus infection that permits analysis of the immune events during the infectious cycle. In this study we show that during the COPV infectious cycle, systemic T cell responses to peptides of several early proteins particularly the E2 protein, as assayed by delayed type hypersensitivity, lymphoproliferation and IFN-gamma ELISPOT, can be detected. The maximal response occurs in a narrow time window that coincides with maximal viral DNA replication and wart regression: thereafter, systemic T cell responses to early proteins decline quite rapidly. Vaccination using particle-mediated immunotherapeutic delivery (PMID) of codon-modified COPV E2 and E1 genes induces strong antigen-specific cell-mediated immune responses in the vaccinated animals. These data show that therapeutic immunization by PMID with codon-modified E2 is completely effective, that to E1 is partially protective, that this correlates with the intensity of antigen-specific cell-mediated immune responses and, further, they emphasize the importance of these responses and the route of immunization in the generation of protective immunity.
