A conserved amino acid in the C-terminus of HPV E7 mediates binding to PTPN14 and repression of epithelial differentiation

Abstract

The human papillomavirus (HPV) E7 oncoprotein is a primary driver of HPV-mediated carcinogenesis. The E7 proteins from diverse HPV bind to the host cellular non-receptor protein tyrosine phosphatase type 14 (PTPN14) and direct it for degradation through the activity of the E7-associated host E3 ubiquitin ligase UBR4. Herein we show that a highly conserved arginine residue in the C-terminal domain of diverse HPV E7 mediates interaction with PTPN14. We found that disruption of PTPN14 binding through mutation of the C-terminal arginine did not impact the ability of several high-risk HPV E7 proteins to bind and degrade the retinoblastoma tumor suppressor or activate E2F target gene expression. HPVs infect human keratinocytes and we previously reported that both PTPN14 degradation by HPV16 E7 and PTPN14 CRISPR knockout repress keratinocyte differentiation-related genes. Now we have found that blocking PTPN14 binding through mutation of the conserved C-terminal arginine rendered both HPV16 and HPV18 E7 unable to repress differentiation-related gene expression. We then confirmed that the HPV18 E7 variant that could not bind PTPN14 was also impaired in repressing differentiation when expressed from the complete HPV18 genome. Finally, we found that the ability of HPV18 E7 to extend the lifespan of primary human keratinocytes required PTPN14 binding. CRISPR/Cas9 knockout of PTPN14 rescued keratinocyte lifespan extension in the presence of the PTPN14 binding-deficient HPV18 E7 variant. These results support the model that PTPN14 degradation by high-risk HPV E7 leads to repression of differentiation and contributes to its carcinogenic activity. IMPORTANCEHuman papillomavirus (HPV)-positive carcinomas account for nearly 5% of the global human cancer burden. The E7 oncoprotein is a primary driver of HPV-mediated carcinogenesis. HPV E7 binds and degrades the putative tumor suppressor, PTPN14. However, the impact of PTPN14 binding by E7 on cellular processes is not well defined. Here, we show that PTPN14 binding is mediated by a conserved C-terminal arginine residue of HPV E7 in vivo. Additionally, we found that PTPN14 binding contributes to the carcinogenic activity of HPV18 E7 (the second most abundant HPV type in cancers). Finally, we determined that PTPN14 binding by HPV16 E7 and HPV18 E7 represses keratinocyte differentiation. HPV-positive cancers are frequently poorly differentiated and the HPV life cycle is dependent upon the keratinocyte differentiation program. The finding that PTPN14 binding by HPV E7 impairs differentiation has significant implications for both HPV-mediated carcinogenesis and the HPV life cycle.