Human Papillomavirus 16 Oncoprotein E7 Stimulates UBF1-Mediated rDNA Gene Transcription,Inhibiting a p53-Independent Activity of p14ARF

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.