Human Papillomavirus 16 Oncoprotein E7 Stimulates UBF1-Mediated rDNA Gene Transcription, Inhibiting a p53-Independent Activity of p14(ARF)

High-risk human papillomavirus oncoproteins E6 and E7 play a major role in HPV-related cancers. One of the main functions of E7 is the degradation of pRb, while E6 promotes the degradation of p53, inactivating the p14(ARF)-p53 pathway. pRb and p14(ARF) can repress ribosomal DNA (rDNA) transcription in part by targeting the Upstream Binding Factor 1 (UBF1), a key factor in the activation of RNA polymerase I machinery. We showed, through ectopic expression and siRNA silencing of p14(ARF) and/or E7, that E7 stimulates UBF1-mediated rDNA gene transcription, partly because of increased levels of phosphorylated UBF1, preventing the inhibitory function of p14(ARF). Unexpectedly, activation of rDNA gene transcription was higher in cells co-expressing p14(ARF) and E7, compared to cells expressing E7 alone. We did not find a difference in P-UBF1 levels that could explain this data. However, p14(ARF) expression induced E7 to accumulate into the nucleolus, where rDNA transcription takes place, providing an opportunity for E7 to interact with nucleolar proteins involved in this process. GST-pull down and co-immunoprecipitation assays showed interactions between p14(ARF), UBF1 and E7, although p14(ARF) and E7 are not able to directly interact. Co-expression of a pRb-binding-deficient mutant (E7C24G) and p14(ARF) resulted in EC24G nucleolar accumulation, but not in a significant higher activation of rDNA transcription, suggesting that the inactivation of pRb is involved in this phenomenon. Thus, p14(ARF) fails to prevent E7-mediated UBF1 phosphorylation, but could facilitate nucleolar pRb inactivation by targeting E7 to the nucleolus. While others have reported that p19(ARF), the mouse homologue of p14(ARF), inhibits some functions of E7, we showed that E7 inhibits a p53-independent function of p14(ARF). These results point to a mutually functional interaction between p14(ARF) and E7 that might partly explain why the sustained p14(ARF) expression observed in most cervical pre-malignant lesions and malignancies may be ineffective.