Two distinct regions of the BPV1 E1 replication protein interact with the activation domain of E2

Papillomavirus E1 and E2 proteins co-operation in viral DNA replication is mediated by protein-protein interactions that lead to formation of an E1-E2 complex. To identify the domains involved, portions of the two proteins we re expressed as fusions to the DNA-binding protein LexA or the transactivat ion domain of VP16 and analyzed by the yeast two-hybrid system. The C-termi nal 266 amino acids of BPV1 E1 (E1C266) interacted strongly with E2 in the yeast system and in a mammalian two-hybrid assay. VP16-E1C266 interacted wi th a region encompassing amino acids 1-200 of the transactivation domain of E2 that was fused to LexA. The interaction between E1 full length and E2 w as clearly observed only when E1 was expressed as LexA-E1 chimera. In addit ion, we found that in the LexA context also the N-terminal region encompass ing the first 340 amino acids of E1 (E1N340) interacted with E2 full length . The interactions of E1N340 and E1C266 with E2 were confirmed also by in v itro binding studies. These observations demonstrate that two distinct regi ons of E1 mediate the interaction with E2 in vivo. (C) 1999 Elsevier Scienc e B.V. All rights reserved.