Characterization of patient-derived HPV16 E6 and E7 variant alleles

Human papillomavirus type 16 (HPV16) is the most prevalent carcinogenic HPV type. Many HPV16 sequence variants have been described, some of which cause protein-coding mutations. However, most experiments have characterized the viral proteins encoded by the HPV16 prototype clone, and some researchers have proposed that variants identified in high-grade lesions and cancers may have higher oncogenic potential than the prototype. To determine whether oncoproteins encoded by such variants have greater oncogenic potential than the prototype version, we performed a comparative biochemical analysis of selected naturally occurring HPV16 E6 and E7 variant alleles identified in cervical lesions and/or control samples. We examined interactions of prototype and HPV16 E7 variant alleles with the retinoblastoma tumor suppressor pRB, the ubiquitin ligase UBR4 (p600), and the tumor suppressor PTPN14. We also compared HPV16 E6 prototype and variant protein interactions with the p53 tumor suppressor, the UBE3A (E6AP) ubiquitin ligase, and the PDZ-domain protein SCRIB. Several E6 or E7 variant proteins exhibited reduced binding to these cellular targets, suggestive of partial or complete loss of function. Our findings reveal that protein-protein interactions that enable the oncogenic activities of HPV16 E6 and E7 can be missing from naturally occurring HPV16 variants and that bona fide loss-of-function variants can be detected in high-grade cervical lesions or cancers. We conclude that clinical association alone is insufficient to predict the pathogenic potential of HPV16 E6 or E7 variant proteins.IMPORTANCEHuman papillomavirus type 16 (HPV16) is the leading cause of HPV-associated cancers, and most functional studies have been performed with the prototype viral clone. Numerous naturally occurring HPV16 variants have been detected in cervical lesions and cancers, raising the possibility that some variants may possess enhanced transforming activity. E6 and E7 are the major oncoproteins encoded by HPV16. By comparing key molecular interactions of prototype and naturally occurring E6 and E7 variant proteins, we found that several variant proteins display reduced engagement with tumor suppressors and ubiquitin ligases that are important for oncogenic activities. Notably, some variants found in high-grade lesions and cancers show partial or complete loss of engaging these critical host proteins. These findings indicate that the presence of an HPV16 variant in a cancer does not necessarily imply increased oncogenic potency. Functional characterization is therefore essential to interpret the biological and clinical significance of HPV16 genetic diversity.