Analyses of variant human papillomavirus type-16 E5 proteins for their ability to induce mitogenesis of murine fibroblasts.

BACKGROUND: Human papillomavirus type 16 (HPV-16) E5 protein co-operates with epidermal growth factor to stimulate mitogenesis of murine fibroblasts. Currently, little is known about which viral amino acids are involved in this process. Using sequence variants of HPV-16 E5 we have investigated their effects upon E5 transcription, cell-cycling and cell-growth of murine fibroblasts. RESULTS: We demonstrate that: (i) introduction of Thr64 into the reference E5 sequence of HPV-16 abrogates mitogenic activity: both were poorly transcribed in NIH-3T3 cells; (ii) substitution of Leu44Val65 or, Thr37Leu44Val65 into the HPV-16 E5 reference backbone resulted in high transcription in NIH-3T3 cells, enhanced cell-cycle progression and high cell-growth; and, (iii) inclusion of Tyr8 into the Leu44Val65 backbone inhibited E5 induced cell-growth and repression of p21 expression, despite high transcription levels. CONCLUSION: The effects of HPV-16 E5 variants upon mitosis help to explain why Leu44Val65 HPV-16 E5 variants are most prevalent in 'wild' pathogenic viral populations in the UK.

Detection of HPV transcripts by nested RT-PCR.

Human papillomaviruses (HPVs) are etiologic for the development of cervical cancer and its precursor lesions, cervical intraepithelial neoplasia (CIN). Nearly all cervical carcinomas (CaCx) harbor HPV DNA, but the presence of HPV alone is not indicative of the future development of neoplasia. While both normal and abnormal smears may harbor HPV DNA, the detection of HPV mRNA is associated, although not exclusively, with abnormal cytology. Recent observations suggest women with a high HPV viral load are at a significantly greater risk for CIN development--particularly those infected with high-risk (HR) HPV types, such as HPV type 16 (HPV 16). Thus, assays capable of detecting HPV transcripts may have useful prognostic value and could be utilized to identify biological markers for progression to high-grade cervical disease. This chapter describes the nested reverse transcriptase (nRT)-polymerase chain reaction (PCR) methods developed in our laboratory for the detection of the majority of all early region HPV-16 transcripts.

Buccal exposure to human papillomavirus type 16 is a common yet transitory event of childhood.

High-risk human papillomaviruses, such as type 16 (HPV-16), are established etiological agents for cervical carcinoma. In most cases, this virus is transmitted sexually, though can also be spread from mother to infant at delivery. We have demonstrated previously a high prevalence ( approximately 52%) of HPV-16 DNA in the mouths of prepubertal children, albeit with low levels of transcription [Rice et al., 2000]. We investigated whether childhood buccal infections with HPV-16 are persistent or transient and whether children became infected through contact with their immediate family members. Two groups of children were selected: one group were all initially HPV-16 E5 DNA-positive in sensitive polymerase chain reaction tests of swabs from their buccal mucosa (n = 20), and the other group consisted of children who were all HPV-16 E5-negative (n = 19). Thirty months later, a second oral swab was collected from each child and tested for HPV DNA. At this second visit, 40% of the HPV-16-positive group had no detectable HPV-16 DNA; conversely, 63% of children who were originally HPV-16-negative had now acquired the virus. Three months later, a third sample was collected from eight children and their immediate families (seven were HPV-16 E5 DNA-positive at the second visit). Amongst the family samples tested, in two families a single previously untested child was HPV-16 DNA-positive. It is concluded that HPV-16 DNA in the oral cavities of children is a transient event and is most probably acquired from their peers.