Objectif
Parvoviruses preferentially lyse transformed cells in vitro, as a possible result of the stimulation of the production and/or activity of cytotoxic viral nonstructural (NS) proteins. The genes encoding the NS proteins of parvovirus MVMp are under the control of the P4 promoter. The activity of natural and mutated P4 promoters has been compared in transfected cells using plasmids containing the reporter chloramphenicol acteyltransferase (CAT) gene downstream from this promoter. Mutations causing a dramatic decrease (between 80 and 90%) in CAT expression driven by the P4 promoter were introduced in either a guanine cytosine (GC) box located at -39 from the transcription start site or in a purine rich element adjacent to the GC box. Deletions in the GC box or in the purine rich element showed that the formation of all retarded complexes required the integrity of the GC box. Mutations (point mutations or deletions) in the purine rich element did affect the presence of some of the retarded bands. The latter complexes may thus be composed of deoxyribonucleic acid (DNA) binding proteins interacting indirectly (via a DNA binding protein) with the GC box could be present in these complexes. In this case the results obtained with the mutations in the purine rich sequence could be the consequence of a distal effect on the GC box of this region. The latter hypothesis is under investigation.
In the P4 promoter, 2 upstream regulatory elements have been identified by using specific antibodies and electrophoretic mobility shift assay (EMSA). These are recognition sites for the major late transcription factor (MLTF), 104 from the start site and for the NF-Y factor which binds to a sequence homologous to the Y box, 100 from the start site. These factors proved to interact with partially overlapping sequences. Their relative contribution to the expression directed by the P4 promoter will be determined.