Alan Storey

Mutations in the human papillomavirus type 16 E2 protein identify a region of the protein involved in binding to E1 protein

Papillomavirus DNA replication is primarily dependent upon two viral gene products, E1 and E2. Work with bovine papillomavirus has shown that the E2 protein can bind directly to the E1 protein and enhance the binding of E1 to the viral origin of replication. However, little is known about the mechanism of interaction between E1 and E2 proteins. In this study we have analysed in detail the association between human papillomavirus type 16 (HPV-16) E1 and E2 proteins. Using a purified glutathione S-transferase-HPV-16 E1 fusion protein from Escherichia coli and E2 proteins produced by in vitro transcription-translation, we have developed a rapid and simple method for investigating the association between E1 and E2 in vitro. The binding of E2 to E1 was found to be dependent on sequences in the N-terminal activation domain of the E2 protein. Truncated forms of E2, including a putative repressor form of E2 encoding the DNA binding domain, failed to associate with E1 in this assay. The region of E2 required for efficient binding to E1 was then localized using mutants in the activation domain of E2. These results demonstrated that only a short region of E2 was required for association with E1. This region of E2 was found to be highly conserved amongst all papillomaviruses, suggesting a conservation of E2 function and a common mechanism of interaction between these virally encoded proteins.

Regulation of human papillomavirus type 16 DNA replication by E2, glucocorticoid hormone and epidermal growth factor.

The E1 and E2 proteins are the only human papillomavirus (HPV) proteins required for transient replication of plasmids containing the viral origin. The E2 gene products play key roles in both viral transcription and replication. In this study we have analysed in further detail the nature of the association between E1 and E2 using a series of E2 proteins mutated in conserved regions of the N-terminal domain. These proteins were tested for their ability to activate transcription and to stimulate viral DNA replication. Several of these mutants revealed that the two functions of E2 can be separated, and that they define three widely spaced regions of the N-terminal domain which are important for DNA replication, two of which retain E1-binding activity. This suggests that E2 may have a role in viral DNA replication other than simply localizing E1 to the origin of replication. Additional important elements for regulating viral gene expression have been shown to be glucocorticoid hormones and epidermal growth factor (EGF). We show here that they may also be involved in regulating viral DNA replication. Our studies show that the addition of glucocorticoid hormone significantly stimulates viral DNA replication. In contrast, addition of EGF results in modest repression of viral DNA replication. These results have important implications for the pathogenesis of HPV infection and suggest that the relative levels of E2, glucocorticoid hormone and EGF may significantly affect the outcome of an HPV infection.

p53 polymorphism and risk of cervical cancer

Storey et al. reply — These reports assess the frequency of the p53Arg allele in different populations and conclude that homozygous p53Arg is not a risk factor for cancer associated with human papilloma virus (HPV). The functional differences between the p53 isoforms that we have described, provoked our initial epidemiological study. As we concluded then, it is crucial that investigations should be extended to different populations, and we are encouraged that such studies are underway.

Human Papilloma Viruses and Cancers of the Skin and Oral Mucosa

The major cellular component of skin and orogenital mucosa is the keratinocyte, which is characteristic of all stratified squamous epithelia. The keratinocyte is the target cell of the family of human papilloma viruses (HPVs), which may result in the development of benign “warts” (episomal infection) or the development of tumors (transformation of the tissue following viral integration). HPVs have an established role in anogenital carcinogenesis but their role in nonanogenital cancers of the skin (nonmelanoma skin cancer [NMSC]) or oral mucosa (oral squamous cell carcinomas [OSCC]) is less clear. Improving techniques for detection of HPV DNA have given rise to an increased understanding of the HPVs found in mucocutaneous sites and the viral—keratinocyte interactions. Thus we focus particularly on investigation of the expression and cellular functions of the subfamily of HPVs associated with epidermodysplasia verruciformis (EV HPVs).