Intranuclear localization of human papillomavirus 16 E7 during transformation and preferential binding of E7 to the Rb family member p130

Abstract

To study intracellular pathways by which the human papillomavirus 16 oncogene E7 participates in carcinogenesis, we expressed an inducible chimera of E7 by fusion to the hormone-binding domain of the estrogen receptor. The chimeric protein (E7ER) transformed rodent fibroblast cell lines and induced DNA synthesis on addition of estradiol. In coimmunoprecipitation experiments, E7ER preferentially bound p130 when compared to p107 and pRb. After estradiol addition, E7ER localization changed to a more intense intranuclear staining. Induction of E7 function was not correlated with binding to p130 or pRb but rather with intranuclear localization and modest induction of binding to p107.Human papillomavirus (HPV) infection of genital tract epithelium is associated with dysplastic changes in epithelial growth and is an important prerequisite for the development of cervical cancer. Two viral genes, E6 and E7, mediate transformation by HPV (1-4). The majority of cervical carcinomas contain truncated forms of HPV, which retains E6 and E7, integrated into the cellular genome (5-9). HPV16 E7 induces growth of cells in soft agar (1), stimulates DNA synthesis (10, 11), cooperates with ras to transform primary cells (12, 13), and overcomes G 1 arrest induced by serum deprivation, actinomycin D, or overexpression of p21 (14-17). The protein encoded by E7 has a region of shared sequence homology with both adenovirus E1A and SV40 large T antigen, which mediates binding to the retinoblastoma gene product pRb (13,18). By binding to pRb, E7 alters the interaction of pRb with the transcription factor E2F-1, resulting in activation of E2F-responsive genes (19). E7 protein from high-risk HPV types, HPV16 and HPV18, binds with a higher affinity to pRb than E7 protein from low-risk HPV types such as HPV6 and HPV11 (20). Therefore, the prevailing model is that E7 protein, by binding to pRb, promotes progression through the cell cycle. The role of E7 binding to other Rb-family members such as p107 and p130 has not been entirely elucidated. However, it is known that induction of B-myb transcription by E2F-1 is the result of E7 binding to p107 rather than to pRb (21).To assess the relationship between transformation and E7 binding to Rb-family members, we have constructed an inducible chimeric molecule consisting of HPV16 E7 fused in-frame to the hormone-binding domain of the estrogen receptor (ER), an approach used to render other oncogenes steroiddependent (22)(23)(24)(25)(26)(27). By using this inducible chimera of E7, we demonstrate that HPV16 E7 activity is correlated with intranuclear localization of the chimera. In addition, E7 binds to greater proportions of intracellular p130 and p107 than pRb in quiescent cells.

Interaction between the HPV E7 oncoprotein and the transcriptional coactivator p300

“…This pattern of staining was more evident when p300 was overexpressed (Figure 5b) from the exogenous plasmid. In agreement with previous studies (Smith-McCune et al, 1999;Guccione et al, 2002), HPV-16E7 in U2-OS cells shows predominantly nuclear localization with nucleolar exclusion (Figure 5a, b). Merged images showed multiple regions and dot-like structures of E7 and p300 colocalization in the case of endogenous p300 (Figure 5a), and a similar pattern of colocalization is also seen when both proteins are overexpressed (Figure 5b).…”

Reconstitution of telomerase activity combined with HPV-E7 expression allow human preadipocytes to preserve their differentiation capacity after immortalization

“…20 The fact that Chub-S7 cells retained the capacity to differentiate can be explained by the greater binding of HPV-E7 on p107 than pRb, whereas SV40 T-Ag interacts equivocally with pRb, p107, and p130. 21 This hypothesis is reinforced by the observation that unlike mouse preadipocytes lacking pRb, 20 C/EBPa gene, an important transcription factor involved in the process of adipocyte differentiation, was expressed during Chub-S7 cell differentiation. Finally, another pathway, than those involving the pRb family, could explain the preserved adipogenic capacity of Chub-S7 cells.…”

Repression of human papillomavirus oncogenes in HeLa cervical carcinoma cells causes the orderly reactivation of dormant tumor suppressor pathways

“…The high-risk HPV E6 protein binds to p53 and targets it for accelerated ubiquitin-mediated degradation, and the high-risk HPV E7 protein binds to hypophosphorylated members of the retinoblastoma family, resulting in their destabilization and the disruption of Rb͞E2F repressor complexes (14)(15)(16)(17)(18)(19)(20)(21). Therefore, levels of p53 and hypophosphorylated Rb are typically low in cells expressing the E6 and E7 proteins.…”