Showing papers by "Massimo Tommasino published in 1998"

Human Papillomavirus 16 E6 Variants Are More Prevalent in Invasive Cervical Carcinoma than the Prototype

Abstract: High-risk human papillomavirus (HPV) is a known risk factor in the etiology of cervical intraepithelial neoplasia (CIN) I-III and invasive cervical carcinoma (ICC). The most severe preinvasive lesion is CIN III, and it is still not entirely understood why some cases progress to invasion, whereas others do not. Our hypothesis that this could be predicted by intratype variation of the immortalizing and transforming early proteins E6 and E7 was tested. Because HPV16 is frequently detected in cervical neoplastic lesions, 25 CIN III and 17 ICC cases from Swedish women, all positive for this genotype, were selected to investigate the E6 and E7 genes for mutations. PCR-amplified products were sequenced by the fluorescent dideoxy termination method. ICC harbored almost exclusively HPV16 E6 variants (94%) and rarely harbored the prototype (6%), whereas CIN III demonstrated a more uniform distribution of variants (56%) and prototype (44%; P = 0.013). All variants contained variations that were identified in areas likely to be important for protein-protein interaction with p53 or in areas of immunological significance. The most frequent E6 variation was seen at residue 83. This polymorphism was detected alone or in combination with others in 88% of ICC and 44% of CIN III cases. E7 variations were extremely rare and were only detected together with E6 variations in 4% of CIN III and in 6% of ICC cases, suggesting that the HPV16 E7 but not the HPV16 E6 oncoprotein is highly conserved in vivo. This indicates that HPV16 E6 variants, specifically those containing the substitution at residue 83, may be more oncogenic than the prototype and thus carry a higher risk for the development of invasive cervical disease. This may be due to subtle differences in the type of transformation produced or to evasion of host immune defenses. These results might have implications for future in vitro studies, diagnostics, treatment, and vaccine design.

The CXXC Zn binding motifs of the human papillomavirus type 16 E7 oncoprotein are not required for its in vitro transforming activity in rodent cells

Abstract: 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.

E7 protein of human papillomaviruses and its interaction with cellular pathways

Abstract: Human papillomaviruses (HPVs) are small, double-stranded DNA viruses that infect epithelial cells. Their genomes, though quite different in DNA sequences, can be divided into three different regions: (a) a non-coding region (about 1000 bp), which contains several cis and trans elements that control viral replication and transcription; (b) the region that encodes the six early genes, El, E2, E4, E5, E6, E7; and (c) the region that encodes the two late genes, L1, L2 (Fig. 1). To date, 77 HPV genotypes have been isolated (E.M. de Villiers, personal communication; de Villiers, 1994), each independent isolate having less than 50% cross-hybridisation under stringent conditions. As DNA sequences can be easily determined and are more precise than the results of cross-hybridisations, a new definition of genotypes has been agreed upon to which two types share <90% nucleotides in their L1, E6 and E7 sequences (de Villiers, 1994). HPVs can be divided into several subsets on the basis of their epithelial tropism. Involved anatomic regions are the skin, the oral cavity and the anogenital tract. The best characterised HPVs are those that infect the anogenital mucosa. Altogether, 26 such have been found.