The Papillomavirus E2 Proteins: Master Regulators of Viral Replication and Gene Expression

The papillomaviruses constitute a large family of DNA viruses which cause proliferative lesions (papillomas or warts) in higher vertebrates. These lesions generally remain benign, but can progress to malignancy in the case of certain viral types and give rise to for example, cervical carcinomas. There is considerable interest in investigating the mechanism of papillomaviral DNA replication in order to identify means to suppress or eradicate persistent infection. In addition, papillomaviruses provide a useful model to understand eukaryotic DNA replication, since their structure is relatively simple and they replicate their DNA only in the S-phase of the host cell cycle. Papillomavirus DNA replication is strictly dependent upon two viral proteins, the full-length products of the El and E2 early genes. As suggested by protein sequence homology with the SV 40 and polyoma T proteins, the E1 proteins have ATPase and helicase activities and the full-length E2 proteins are transcription regulatory proteins. Work with bovine papillomavirus has shown that the E2 protein can bind directly to the El protein and enhance the origin ( ori) binding activity of El. Little is known about the mechanism of interaction between the human papillomavirus (HPV) El and E2 proteins. In this study, I have analysed in detail the role of the human papillomavirus type 16 (HPV-16) E2 protein in viral DNA replication. Initially, I investigated the association between El and E2 using a purified glutathione S-transferase-HPV-16 E1 fusion protein from E.coli and in vitro translated HPV-16E2 proteins, mutated in conserved regions of the N-terminal domain. This assay enabled me to identify two widely separate regions of the HPV- l 6E2 protein essential for binding to E1. I next tested the in viva activity of these HPV-l 6E2 mutants in transcriptional activation and stimulation of viral DNA replication. Several of these proteins revealed that the two functions of E2 can be separated. These studies define three widely spaced regions of the N-terminal domain which are important for DNA replication, two of which retain El-binding activity. These results point to an additional role of E2 in viral DNA replication other than simply localizing El to the origin of replication. Furthermore, I show that an important element for regulating viral DNA replication might be glucocorticoid hormones. In the last part of this work, I provide additional evidence for the importance of E2 as an auxiliary factor in viral DNA replication. An extensive set of mutations inactivating the cellular transcription factor binding sites present in the vicinity of the ori were tested. None of these mutations had a significant effect on replication, indicating that these sites and factors that bind to them, at least in the presence of E2, are dispensable for replication. Therefore, the HPV-l 6E2 protein plays a key role in papillomavirus transcription regulation and DNA replication. These two functions are clearly separable. The E2 protein appears to be the only auxiliary factor involved in stimulation of viral DNA replication and the cellular transcription factors which bind the origin do not affect transient DNA replication. These studies demonstrate that E2 has other activities in replication other than tether E1 to the origin.