The two-hybrid system was used to detect interactions in vivo between HPV E7 and three 'Rb-like proteins', pRb, p107 and p130. The association between pE7 and pRb parallel the oncogenic potential of the specific HPV types. In contrast, the interaction between pE7 and p107 or p130 differ. While the HPV 16 E7 protein associates with the 'Rb-like' proteins strongly, both HPV 18 and 6b E7 proteins bind more weakly. We tested several HPV 6 E7 mutants carrying single amino acid mutations. Substitution of the glycine at position 22 with an aspartate was the only mutation capable of increasing the ability of HPV 6 E7 protein to bind pRb. However, association with p107 and p130 by the HPV 6 E7 protein was also increased by mutation of the arginine at position 4 with an aspartate. These data suggest that pRb, p107 and p130 interact with similar but non-identical domains of pE7. In addition, we used amphotropic retroviruses encoding the HPV 18 E6 and the different E7 genes to analyze their immortalizing activity. The wild-type HPV Is and 16 E7 genes complemented the HPV 18 E6 gene to immortalize human keratinocytes. In comparison, none of the cells infected with HPV 6 E7, wildtype or mutant- encoding retroviruses, became immortal. Thus, our data suggest that HPV 6 E7 lacks a property independent of pRb-association which is required for immortalization of human keratinocytes.

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