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 carcinoma line, CaSki, and rat 3Y1 cells stably expressing HPV16 E7 gene. Contrary to the current understanding of E7 as a nuclear protein, the site of staining was clearly cytoplasmic. The subcellular localization of E7 was further studied by using the beta-galactosidase (beta-gal) receptor method. A fusion protein composed of E7 and beta-gal was stably expressed in rat 3Y1 cells. The beta-gal activity in these cells was detected mostly in the nucleus, even though 9F6 still stained the cytoplasm of these cells. The fusion protein was also found to be oncogenic since transfected 3Y1 cells acquired transformed phenotypes such as increased saturation density and anchorage-independent growth. These results indicate that biologically active E7 exists mostly in the nucleus, but nuclear E7 is masked from 9F6. A series of deletion mutants of E7 further demonstrated that the amino acid sequence from 16 to 41 was enough to transport beta-gal into the nucleus. A mutation either at amino acid 24 or 26 which is known to disrupt the binding of E7 to RB, the retinoblastoma gene product, did not strongly affect the nuclear localization of the fusion protein, suggesting that the nuclear transportation of E7 is mostly independent of RB binding.
Fujikawa
K
K
Shionogi Institute for Medical Science, Osaka, Japan.
Furuse
M
M
Uwabe
K
K
Maki
H
H
Yoshie
O
O
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001483:Base Sequence; D002460:Cell Line; D002467:Cell Nucleus / Q000737:chemistry*; D002471:Cell Transformation, Neoplastic; D006801:Humans; D007150:Immunohistochemistry; D008969:Molecular Sequence Data; D009856:Oncogene Proteins, Viral / Q000032:analysis* / Q000737:chemistry / Q000502:physiology; D027383:Papillomaviridae / Q000378:metabolism; D050725:Papillomavirus E7 Proteins; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000032:analysis*; D013329:Structure-Activity Relationship; D001616:beta-Galactosidase / Q000032:analysis*
D000911:Antibodies, Monoclonal; D009856:Oncogene Proteins, Viral; D050725:Papillomavirus E7 Proteins; D011993:Recombinant Fusion Proteins; C059731:oncogene protein E7, Human papillomavirus type 16; D001616:beta-Galactosidase