title: Conditionally activated E7 proteins of high-risk and low-risk human papillomaviruses induce S phase in postmitotic, differentiated human keratinocytes.
abstract: The productive program of human papillomaviruses (HPVs) in epithelia is tightly linked to squamous differentiation. The E7 proteins of high-risk HPV genotypes efficiently inactivate the pRB family of proteins that control the cell cycle, triggering S phase in suprabasal keratinocytes. This ability has until now not been demonstrated for the low-risk HPV-6 or HPV-11 E7 proteins. An inducible system in which HPV-16 E7 is fused to the ligand binding domain of the human estrogen receptor (ER) was described by Smith-McCune et al. (K. Smith-McCune, D. Kalman, C. Robbins, S. Shivakumar, L. Yuschenkoff, and J. M. Bishop, Proc. Natl. Acad. Sci. USA 96:6999-7004, 1999). In the absence of hormone, E7ER is cytoplasmic, and upon addition of 17beta-estradiol, it translocates to the nucleus. Using organotypic epithelial raft cultures developed from primary human keratinocytes, we show that 16E7ER promotes either S-phase reentry or p21cip1 accumulation in differentiated keratinocytes in a stochastic manner as early as 6 h postinduction with 17beta-estradiol. A vector expressing the ER moiety alone had no effect. These observations prove unequivocally that the E7 protein drives S-phase reentry in postmitotic, differentiated keratinocytes rather than preventing S-phase exit while the cells ascend through the epithelium. HPV-11 E7ER and, much less efficiently, HPV-6 E7ER also promoted S-phase reentry by differentiated cells upon exposure to 17beta-estradiol. S-phase induction required the consensus pRB binding motif. We propose that the elevated nuclear levels of the low-risk HPV E7 protein afforded by the inducible system account for the positive results. These observations are entirely consistent with the fact that low-risk HPV genotypes replicate in the differentiated strata in patient specimens, as do the high-risk HPVs.
authors.affiliation: Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, AL 35294-0005, USA.
authors.forename: N Sanjib
authors.initials: NS
authors.lastname: Banerjee
authors.forename: Nicholas J
authors.initials: NJ
authors.lastname: Genovese
authors.forename: Francisco
authors.initials: F
authors.lastname: Noya
authors.forename: Wei-Ming
authors.initials: WM
authors.lastname: Chien
authors.forename: Thomas R
authors.initials: TR
authors.lastname: Broker
authors.forename: Louise T
authors.initials: LT
authors.lastname: Chow
mesh_terms: D021581:Active Transport, Cell Nucleus / Q000187:drug effects / Q000235:genetics; D020816:Amino Acid Motifs / Q000235:genetics; D002454:Cell Differentiation / Q000187:drug effects / Q000235:genetics; D002465:Cell Movement / Q000187:drug effects / Q000235:genetics; D002467:Cell Nucleus / Q000378:metabolism; D002472:Cell Transformation, Viral / Q000187:drug effects / Q000235:genetics; D002478:Cells, Cultured; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D004848:Epithelium / Q000378:metabolism / Q000473:pathology / Q000821:virology; D004958:Estradiol / Q000494:pharmacology; D005838:Genotype; D052162:Human papillomavirus 16 / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D015603:Keratinocytes / Q000378:metabolism* / Q000473:pathology / Q000821:virology; D008297:Male; D009856:Oncogene Proteins, Viral / Q000235:genetics / Q000378:metabolism*; D030361:Papillomavirus Infections / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D011485:Protein Binding / Q000187:drug effects / Q000235:genetics; D011960:Receptors, Estrogen / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012307:Risk Factors; D016196:S Phase / Q000235:genetics; D013997:Time Factors; D014779:Virus Replication / Q000187:drug effects / Q000235:genetics
chemical_list: D050759:Cyclin-Dependent Kinase Inhibitor p21; D009856:Oncogene Proteins, Viral; D011960:Receptors, Estrogen; D011993:Recombinant Fusion Proteins; D004958:Estradiol