Functional interaction of a novel cellular protein with the papillomavirus E2 transactivation domain.
The transactivation domain (AD) of bovine papillomavirus type 1 E2 stimulates gene expression and DNA replication. To identify cellular proteins that interact with this 215-amino-acid domain, we used a transactivation-defective mutant as bait in the yeast two-hybrid screen. In vitro and in vivo results demonstrate that the cDNA of one plasmid isolated in this screen encodes a 37-kDa nuclear protein that specifically binds to an 82-amino-acid segment within the E2 AD. Mutants with point mutations within this E2 domain were isolated based on their inability to interact with AMF-1 and were found to be unable to stimulate transcription. These mutants also exhibited defects in viral DNA replication yet retained binding to the viral E1 replication initiator protein. Overexpression of AMF-1 stimulated transactivation by both wild-type E2 and a LexA fusion to the E2 AD, indicating that AMF-1 is a positive effector of the AD of E2. We conclude that interaction with AMF-1 is necessary for the transcriptional activation function of the E2 AD in mammalian cells.
Breiding
D E
DE
Department of Dermatology, New England Medical Center and Tufts University School of Medicine, Boston, Massachusetts 02111, USA.
Sverdrup
F
F
Grossel
M J
MJ
Moscufo
N
N
Boonchai
W
W
Androphy
E J
EJ
D000818:Animals; D001665:Binding Sites; D052688:Bovine papillomavirus 1 / Q000235:genetics / Q000378:metabolism*; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002417:Cattle; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D017354:Point Mutation; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*; D015533:Transcriptional Activation; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
D002352:Carrier Proteins; D004268:DNA-Binding Proteins; C051550:E2 protein, Bovine papillomavirus; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; D015534:Trans-Activators; D014764:Viral Proteins