The role of E1^E4 in the differentiation-dependent life cycle of oncogenic human papillomaviruses.

摘要

Anogenital human papillomaviruses (HPVs) are the causative agent of ninety-nine percent of cervical carcinomas with HPV types 16, 18, 31, and 45 being the most prevalent. Understanding the role of each of the viral proteins in the HPV life cycle is essential for treatment and prevention of this disease. Many of the HPV proteins have been extensively studied and a number of their roles within the viral life cycle have been acknowledged. However, the role of E1^E4 proteins has not been well established. In my studies, the role ofE1^E4 in the HPV life cycle was investigated by mutating the E1^E4 proteinin the context of the complete viral genome of HPV type 31. Normal human foreskin keratinocytes were transfected with either HPV31 wild type or E1^E4 mutantgenomes. Episomal maintenance, replication and early gene transcription of viral genomes, were observed in undifferentiated cells in monolayer cultures with no differences detected between wild type and mutant genes. However, when HPV31 wild type and E1^E4 mutant transfected keratinocytes were inducedto differentiate in semi-solid media or in organotypic rafts cultures, viral DNA amplification and late gene transcription were significantly reduced in keratinocytes harboring E1^E4 mutant genomes. From these results,I concluded that HPV31 E1^E4 is essential for the late productive phase ofthe viral life cycle. Furthermore, I observed that HPV31 E1^E4 contributesto HPV induced S phase maintenance and prevents anucleation of differentiated keratinocytes in organotypic raft cultures. In addition, I found that E1^E4altered expression and phosphorylation of specific cell cycle regulators involved in the G2/M transition of the cell cycle. Finally, to determine if HPV E1^E4exhibited similar roles in other high risk HPV viral types, an E1E4 mutantwas constructed in the context of the entire HPV18 genome. I found that like HPV31 E1E4, HPV18 E1E4 was essential for viral DNA amplification and expressionof late transcripts. Since similar roles for both HPV types 31 and 18 E1E4were identified, a conserved role for E1E4 in the viral life cycle amonghigh risk HPV types is likely.