NUCLEAR-LOCALIZATION AND TRANSFORMING ACTIVITY OF HUMAN PAPILLOMAVIRUS TYPE-16 E7-BETA-GALACTOSIDASE FUSION PROTEIN - CHARACTERIZATION OF THE NUCLEAR-LOCALIZATION SEQUENCE

A MAb 9F6 was capable of staining HPV16 E7 in a human cervical carcino ma line, CaSki, and rat 3Y1 cells stably expressing HPV16 E7 gene. Con trary to the current understanding of 67 as a nuclear protein, the sit e of staining was clearly cytoplasmic. The subcellular localization of E7 was further studied by using the P-galactosidase (P-gal) receptor method. A fusion protein composed of 67 and P-gal was stably expressed in rat 3Y1 cells. The P-gal activity in these cells was detected most ly in the nucleus, even though 9F6 still stained the cytoplasm of thes e cells. The fusion protein was also found to be oncogenic since trans fected 3Y1 cells acquired transformed phenotypes such as increased sat uration density and anchorage-independent growth. These results indica te that biologically active E7 exists mostly in the nucleus, but nucle ar E7 is masked from 9F6. A series of deletion mutants of E7 further d emonstrated that the amino acid sequence from 16 to 41 was enough to t ransport P-gal into the nucleus. A mutation either at amino acid 24 or 26 which is known to disrupt the binding of 67 to RB, the retinoblast oma gene product, did not strongly affect the nuclear localization of the fusion protein, suggesting that the nuclear transportation of E7 i s mostly independent of RB binding. (C) 1994 Academic Press, Inc.