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

The conserved region 3 (CR3) of the E7 protein of human papillomavirus es contains two CXXC motifs involved in zinc binding and in the homodi merization of the molecule, Studies have suggested that the intact CXX C motifs in the CR3 of HPV16 and HPV18 E7 are required for the in vitr o transforming activity of these proteins, CR3 also contains a low aff inity pRb binding site and is involved in the disruption of the E2F/Rb 1 complex, E7 is structurally and functionally related to Adenovirus E 1A protein, which also has two CXXC motifs in CR3. However, the Ad E1A transforming activity appears to be independent of the presence of su ch domains, In fact, this viral protein exists in vivo as two differen t forms of 289 and 243 amino acids, The shorter Ad E1A form (Ad E1A243 ), where both CXXC motifs are deleted by internal splicing, retains it s in vitro transforming activity, We have investigated if the HPV16 E7 CR3 can be functionally replaced by the Ad E1A243 CR3, which lacks bo th 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, HP V16 E7 and Ad E1A, It associates with the 'pocket' proteins, induces g rowth in soft agar of NIH3T3 cells and immortalizes rat embryo fibrobl asts, 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 bu t probably are important for maintaining the correct protein configura tion.