IN-VIVO ANALYSIS OF HPV E7 PROTEIN ASSOCIATION WITH PRB, P107 AND P130

The two-hybrid system was used to detect interactions in vivo between HPV E7 and three 'Rb-like proteins', pRb, p107 and p130. The associati on between pE7 and pRb parallel the oncogenic potential of the specifi c HPV types. In contrast, the interaction between pE7 and p107 or p130 differ. While the HPV 16 E7 protein associates with the 'Rb-like' pro teins strongly, both HPV 18 and 6b E7 proteins bind more weakly. We te sted several HPV 6 E7 mutants carrying single amino acid mutations. Su bstitution of the glycine at position 22 with an aspartate was the onl y mutation capable of increasing the ability of HPV 6 E7 protein to bi nd pRb. However, association with p107 and p130 by the HPV 6 E7 protei n was also increased by mutation of the arginine at position 4 with an aspartate. These data suggest that pRb, p107 and p130 interact with s imilar but non-identical domains of pE7. In addition, we used amphotro pic retroviruses encoding the HPV 18 E6 and the different E7 genes to analyze their immortalizing activity. The wild-type HPV Is and 16 E7 g enes complemented the HPV 18 E6 gene to immortalize human keratinocyte s. In comparison, none of the cells infected with HPV 6 E7, wildtype o r mutant- encoding retroviruses, became immortal. Thus, our data sugge st that HPV 6 E7 lacks a property independent of pRb-association which is required for immortalization of human keratinocytes.