RESUMO
The conserved region 3 (CR3) of the E7 protein of human papillomaviruses contains two CXXC motifs involved in zinc binding and in the homodimerization of the molecule. Studies have suggested that the intact CXXC motifs in the CR3 of HPV16 and HPV18 E7 are required for the in vitro transforming activity of these proteins. CR3 also contains a low affinity pRb binding site and is involved in the disruption of the E2F/Rb1 complex. E7 is structurally and functionally related to Adenovirus E1A protein, which also has two CXXC motifs in CR3. However, the Ad E1A transforming activity appears to be independent of the presence of such domains. In fact, this viral protein exists in vivo as two different forms of 289 and 243 amino acids. The shorter Ad E1A form (Ad E1A243), where both CXXC motifs are deleted by internal splicing, retains its in vitro transforming activity. We have investigated if the HPV16 E7 CR3 can be functionally replaced by the Ad E1A243 CR3, which lacks both CXXC motifs. A chimeric protein (E7/E1A243) containing the CR1 and CR2 of HPV16 E7 fused to the CR3 of Ad E1A 243 was constructed. The E7/E1A243 while not able to homodimerize in the S. cerevisiae two-hybrid system retains several of the properties of the parental proteins, HPV16 E7 and Ad E1A. It associates with the 'pocket' proteins, induces growth in soft agar of NIH3T3 cells and immortalizes rat embryo fibroblasts. These data suggest that the CXXC motifs in CR3 of E7 do not play a direct role in the transforming properties of this viral protein but probably are important for maintaining the correct protein configuration.
Assuntos
Transformação Celular Viral/fisiologia , Proteínas Oncogênicas Virais/metabolismo , Zinco/metabolismo , Células 3T3/metabolismo , Proteínas E1A de Adenovirus/metabolismo , Proteínas E1A de Adenovirus/fisiologia , Sequência de Aminoácidos , Animais , Sítios de Ligação , Sequência Conservada , Dimerização , Camundongos , Proteínas Oncogênicas Virais/fisiologia , Papillomaviridae , Proteínas E7 de Papillomavirus , Proteína do Retinoblastoma/metabolismo , Proteína do Retinoblastoma/fisiologia , Dedos de ZincoRESUMO
The canarypox (CP) and fowlpox (FP) viruses, which are unable to replicate productively in non-avian species, have been utilized as live vectors carrying the HIV-1SF2 env gene with the putative immunosuppressive (IS) region complete (CPIS+ and FPIS+) or deleted (CPIS- and FPIS-). To determine if these avipox-env recombinants could be utilized to elicit a specific immune response against HIV-1, six groups of rabbits were immunized with CPIS+, CPIS-, FPIS+, FPIS- constructs or their non-engineered wild-type CPwt or FPwt counterparts. After a primary inoculation and successive boosters, env-specific humoral and cell-mediated immunity were demonstrated by ELISA, immunoblots and lymphoproliferation assays. Antibody titres and neutralization activities were higher in CP- than FP-inoculated rabbits, the CPIS+ always showing a similar immunogenic capacity to CPIS-. Evidence is also presented indicating that rabbit sera possess group-specific antibodies, which were, however, unable to cross-neutralize divergent HIV-1 strains. Although the protective capacity against HIV-1 experimental infection has not yet been determined in these animals, our results suggest that these recombinants might represent promising and safer candidate vaccines against HIV-1.
Assuntos
Avipoxvirus/genética , Genes env/genética , Anticorpos Anti-HIV/biossíntese , HIV-1/genética , Vacinas contra a AIDS/imunologia , Animais , Especificidade de Anticorpos , Western Blotting , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Regulação Viral da Expressão Gênica/genética , Engenharia Genética , HIV-1/imunologia , Humanos , Soros Imunes/imunologia , Imunidade Celular , Ativação Linfocitária , Coelhos , Linfócitos T/citologia , Fatores de Tempo , Vacinação , Vacinas Sintéticas/imunologia , Replicação Viral/genéticaRESUMO
The human papillomavirus type 16 (HPV-16) E7 and adenovirus (Ad) E1A oncoproteins share a common pathway of transformation. They disrupt the cell cycle G1 phase-specific protein complex containing the E2F transcription factor and the regulatory protein Rb1, the retinoblastoma tumour suppressor gene product. In the G1 and S phases of the cell cycle, E7 and E1A bind two other cellular complexes containing the Rb1-related protein p107 and E2F. Ad E1A disrupts both complexes and releases active E2F. In contrast, HPV-16 E7, although it efficiently binds both E2F-p107 complexes, causes dissociation of the G1 phase complex only. Using chimeric proteins of HPV-16 E7 and Ad E1A we were able to demonstrate that the ability of E1A to disrupt both G1 and S phase E2F-p107 complexes is not due to the higher concentration of Ad E1A in the cell, but is an intrinsic property of the Ad E1A transforming region. These data suggest that E1A and E7 may function in cellular transformation in similar, but not identical ways.