UBC Theses and Dissertations
Characterization of the promoter in a human B19 parvovirus Blundell, Matthew Charles
The nucleotide sequence of the B19-Wi isolate of an autonomous human parvovirus was determined and compared with the sequence of the closely related isolate, B19-Au. The B19-Wi genome was similar to the B19-Au genome, as shown from DNA sequence analyses. It had been previously suggested from the sequence of the B19-Au genome, that the termini may be imperfect inverted terminal repeats. The additional sequence present on the right-hand terminus of B19-Wi supported that supposition. The hairpin termini of the B19 genome were of the same type as those found in adeno-associated parvoviruses, and suggested B19 may be more closely related to AAV viruses than to the autonomous parvoviruses. In vitro transcription with HeLa nuclear extracts using B19-Wi DNA identified a single active RNA polymerase II promoter between map unit 5 and 6. A single promoter was unique; all other parvoviruses characterized to date have two or more active promoters. A series of ordered deletions, prepared in the region upstream of the initiation site of the promoter, indicated that multiple cis-activating motifs were required to maximize in vitro transcription. A transcription factor in HeLa cells specifically bound to and protected a GC rich sequence or GC-box upstream of the promoter, as shown by gel-shift competition assays, and DNAse I and DMS footprinting assays. The protected GC-box had the consensus sequence of a high affinity binding site for the trans-activating factor SP1. The GC-box also formed the distal element of a tandem SPl-like motif, 21 bp upstream of the active TATA box. Synthetic oligonucleotides containing the GC-box specifically bound a HeLa factor and also depressed in vitro transcription from the B19 promoter when added as a competitor. Although not conclusive, binding of the HeLa factor may be inhibited by methylation of a cytosine residue within the GC-box. In vitro transcriptional activity decreased when the GC-box upstream of the B19 promoter was modified by site specific mutagenesis. Preliminary identification of other cis-activating motifs, which include two sequences recognized by factors that are functional both in transcription and replication, suggested that the B19 promoter is a complex regulatory region.