THE ENHANCER OF HUMAN PAPILLOMAVIRUS TYPE 16

Abstract

Human Papillomaviruses were once grouped into the virus family Papovaviridae, based on the nature of their nucleic acid and the shape of the icosahedric capsid. Nowadays the papillomaviruses are put into a separate group of viruses because their viral genome is larger than that of the polyoma- and simian viruses and is genetically organized in a completely differently manner. Also, regulation of gene expression differs from those of the polyoma- and simian viruses as shown in studies on bovine papillomaviruses. Until recently, not much was known about the regulation of gene expression of human papillomaviruses. The involvement of new techniques in molecular biology promised some insight on how the virus gene expression is regulated. The long control region (LCR), devoid of long open reading frames (ORF), free of nucleosomes, and located between the end of ORF L 1 and the start of ORF E6, was a good candidate to receive intra-and extracellular signals controlling the expression of the viral genes. The study presented in this thesis deals with the issue of how human papillomaviruses, in particular HPV-16 found in cervical cancers, receive signals via their LCR which then in turn can alter viral gene expression. To this end, a technique was used to visualize proteins that bind to the long control region of the virus, namely DNase I -footprinting. The localization and characterization of proteins binding to the LCR are prerequisites to the understanding how viral gene expression is regulated. This study of the HPV-16 LCR revealed 23 regions of protein-DNA interactions. 9 of these 'footprints' were located within a segment functional as cell type specific enhancer. 3 footprints were assigned to the major viral promoter, relevant for transcription starts at position 97 of the viral DNA. Characterization of the proteins binding to the enhancer, by their DNA recognition sequence requirements, showed that 7 out of the 9 regions were recognized by the same protein. According to the conserved sequence (the 5'-TTGGC-3' motif), it can be inferred that the protein binding to these 7 sites is nuclear factor 1 (NF1 ), a protein originally found involved in the replication of adenovirus. The test whether this cluster of 7 NF-1 binding sites is an idiosyncracy of HPV-16 or a common feature of the enhancer of papillomaviruses, a computer search for 5'-TTGGC-3' motifs in all available sequenced papillomaviruses was performed. It turned out that all human papillomaviruses had clusters of these motifs in their long control region, while bovine papillomaviruses 1 (BPV 1 ), polyomavirus and simian virus 40 (SV 40) don't have this sequence feature. To prove the relevance of this computer search, 4 different viral LCRs were chosen for the study of protein-DNA interactions. Out of 22 TTGGC-motifs tested in this study, all bound a protein in a manner indistinguishable whether a crude nuclear extract from Hela cells was used or purified nuclear factor 1. This observation that papillomaviruses had conserved this clustered appearance of NF-1 binding sites in their LCR led to questions about their function. On the one side, it could be shown by in-vivo experiments that the HPV-16 enhancer is activated by NF1 factors. On the other side, it could be shown that neither the NF-1 binding sites alone nor the neighbouring binding sites for other DNA binding proteins per se could function as a transcriptional enhancer in the case of HPV16. However, competition experiments showed that these clusters of NF-1 binding sites are indispensable for HPV-16 enhancer function. An extension of this footprint analysis showed that this cluster of NF1 sites embraces binding sites for additional transcription factors, and that a co-operation with factors mediating response to glucocorticoid hormones or phorbol esters may regulate HPV16 gene expression.