Binding of the human papillomavirus type 16 E7 oncoprotein and the adeno-associated virus Rep78 major regulatory protein in vitro and in yeast and the potential for downstream effects.
OBJECTIVE
Both human papillomavirus (HPV) and adeno-associated virus (AAV) are common anogenital viruses and likely co-infect the epithelium in vivo. However, whereas HPVs are positively associated with cervical cancer, AAV appears to be negatively associated. In tissue culture, AAV-encoded Rep78--which is essential for AAV--inhibits gene expression and oncogenic transformation by HPV-16/18 and bovine papillomavirus type 1. Here we observed whether the HPV-16 E7 oncoprotein might recognize and bind Rep78. Further, upon finding Rep78-E7 binding, we investigated some of the potential downstream effects such an interaction might have. E7 is capable of recognizing a variety of proteins, including RB105, TATA box-binding protein (TBP), TBP-associated factor (TAF)(II)110, E2F, cyclins A and D, and c-jun. Some of these interactions are likely responsible for E7's cancer-promoting activity.


METHODS
Rep78-E7 interaction was investigated in vitro by West(far)-Western and affinity chromatography analysis and in vivo in living yeast by the GAL4 two-hybrid cDNA assay. Mapping of the E7 binding domain within Rep78 was carried out using a series of amino- and carboxy-truncated Rep78 proteins in a West(far)-Western assay. Downstream effects of the interaction were analyzed by competitive affinity chromatography (protein-protein) and competitive electrophoretic mobility shift assay (protein-DNA).


RESULTS
E7 and Rep78 were found to interact both in vitro and in vivo (yeast) in all assays attempted. The E7 binding domain within Rep78 was found to reside within amino acids 121-370. Regarding downstream effects of this interaction, Rep78 was found to mildly inhibit E7-TAF(II)110 and E7-RB105 interaction in vitro but to have little affect on E7-TBP interaction. Finally, it was found that E7 was able to affect Rep78's interaction with AAV's terminal repeat (TR) DNA in vitro, reducing the formation of the largest sized Rep78-TR complexes in a dosage-dependent manner.


CONCLUSIONS
These data suggest that the Rep78-E7 interaction may have repercussions for both viruses. The Rep78-E7 interaction may be a second mechanism, in addition to Rep78 regulation of the p97 promoter, by which AAV inhibits HPV-16 oncogenic transformation. These data also suggest that HPV-16 may affect the AAV life cycle by altering Rep78-TR interaction.
Hermonat
P L
PL
Department of Obstetrics and Gynecology, University of Arkansas for Medical Sciences, Little Rock 72205, USA.
Santin
A D
AD
Zhan
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D000409:Alanine / Q000031:analogs & derivatives / Q000378:metabolism; D001665:Binding Sites; D015153:Blotting, Western; D002846:Chromatography, Affinity; D017931:DNA Primers; D018076:DNA, Complementary / Q000032:analysis; D004279:DNA, Viral / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism* / Q000494:pharmacology; D000229:Dependovirus / Q000737:chemistry / Q000235:genetics; D006801:Humans; D009856:Oncogene Proteins, Viral / Q000235:genetics / Q000378:metabolism*; D027383:Papillomaviridae / Q000737:chemistry* / Q000235:genetics; D050725:Papillomavirus E7 Proteins; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D035381:TATA-Binding Protein Associated Factors; D035181:TATA-Box Binding Protein; D035362:Transcription Factor TFIID; D014157:Transcription Factors / Q000378:metabolism; D014764:Viral Proteins / Q000378:metabolism* / Q000494:pharmacology; D015003:Yeasts / Q000378:metabolism
D017931:DNA Primers; D018076:DNA, Complementary; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D009856:Oncogene Proteins, Viral; D050725:Papillomavirus E7 Proteins; D011993:Recombinant Fusion Proteins; C100838:TAF5 protein, human; D035381:TATA-Binding Protein Associated Factors; D035181:TATA-Box Binding Protein; D035362:Transcription Factor TFIID; D014157:Transcription Factors; D014764:Viral Proteins; C059731:oncogene protein E7, Human papillomavirus type 16; C068734:rep proteins, Adeno-associated virus 2; C086898:N-(2-(mercaptomethyl)-3-phenylbutanoyl)-L-alanine; D000409:Alanine