Peh, Woei Ling; (2003) The role of the papillomavirus E4 protein during the late stage of the viral life cycle using in vivo model systems. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Papillomaviruses make up a large family of viruses that can infect the epithelium of a range of human and animal hosts, and are capable of inducing a spectrum of different cutaneous and mucosal epithelial diseases. These viruses are highly tissue and host specific, and are dependent on epithelia differentiation for the completion of their life cycle. The late stages of the viral life cycle have been shown to occur only in differentiated epithelial cells which support viral DNA amplification and the expression of viral late proteins (E4, L1 and L2). These late events can be detected using in situ hybridisation and immunodetection methods in infected tissue sections. Specific E4 antibodies were made against 4 animal types (COPV, CRPV, ROPV and BPV-1) and 1 human type (HPV-11) papillomaviruses. Using specific E4 antibodies and viral DNA probes, the late stage of the viral life cycle were examined in tissue sections of naturally and experimentally induced infections. Different patterns of viral late events are described and compared. The usefulness of animal models as an alternative approach to study of the papillomavirus life cycle is also discussed. The expression of the viral E4 protein and vegetative DNA replication had been shown to correlate exactly in vivo. However, the function of the E4 protein is still unclear. A series of E4 C- terminal truncation mutants were constructed along the E4 open reading frame by the introduction of stop codons using site-directed mutagenesis. Five different E4 truncation mutants were made in the CRPV and HPV-16 viral genomes. The shortest mutant for CRPV (labelled CRst9/0) and HPV-16 (labelled W12st15/6) was predicted to express an 8 or 14 amino acid E1^E4 protein respectively. The CRPV wildtype and E4 mutant DNA were prepared and inoculated onto the backs of wild cottontail (natural) and New Zealand (experimental) rabbit hosts. This thesis presents the data obtained from the study of the E4 mutants in the CRPV-rabbit model systems. It is the first time the papillomavirus E4 protein has been shown to have an essential role in the initiation of viral DNA amplification in vivo.

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