Identification of the Cellular Proteins and Pathways Engaged by the Bovine Papillomavirus Type 1 E6 and E7 Proteins

Bovine papillomavirus type 1 (BPV1) induces fibropapillomas in cattle, and has long served as a useful model virus to study the molecular biology of the papillomaviruses. The cellular transforming activity of BPV1 maps to its E5, E6 and E7 genes. While the cellular transformation function of BPV1 E5 is well elucidated, the biological functions of the BPV1 E6 and E7 oncoproteins are still largely unknown. To further our understanding of the cellular functions of BPV1 proteins, I performed an unbiased mass spectrometry-based proteomic analysis to identify their cellular interacting partners. I subsequently focused on characterizing the interactions of the BPV1 E6 and E7 proteins with some of their cellular interactors. I discovered Mastermind-like 1 (MAML1) and the other components of the Notch transcription complex as novel cellular interacting partners of BPV1 E6. A parallel proteomic screen performed in the laboratory using the HPV E6 proteins as baits identified MAML1 and the Notch transcription complex as interactors of the E6 proteins from beta-genus HPVs, but not those from the alpha-genus HPVs. Further investigation revealed that the beta-genus HPV E6 proteins repress the basal expression and transcriptional activation of endogenous Notch target genes in keratinocytes. For the BPV1 E7 protein, I confirmed a previously reported interaction with UBR4, and showed that this interaction is dependent on the UBR box of UBR4 and the N- terminal of E7. Since little is known about the biological function of UBR4, I performed a proteomic screen to identify its interactors. I identified the E2 ubiquitin-conjugating enzyme UBE2A as an interactor of UBR4, and showed that UBR4 is able to discharge ubiquitin from UBE2A in an in vitro discharge assay. Together with the UBR4 auto-ubiquitylation observed in an in vitro ubiquitylation assay, these results suggest that UBR4 can function as an E3 ubiquitin ligase. In summary, these studies lay the groundwork for further systems-level studies of the biological functions of papillomavirus proteins, identify the Notch signaling pathway as a novel target of cutaneous papillomaviruses such as BPV1 and beta-genus HPVs, and provide evidence that the N-recognin UBR4 can act as an E3 ubiquitin ligase.