CHARACTERIZATION OF CIS-ACTING AND NS1 PROTEIN-RESPONSIVE ELEMENTS INTHE P6 PROMOTER OF PARVOVIRUS B19

Parvovirus B19 infections are associated with diverse clinical manifes tations, ranging from no symptoms to severe symptoms. The virus shows an extreme tropism for replication in erythroid progenitor cells, poss ibly due to the activity of the only functional promoter (p6) of the B 19 virus genome in combination with both cell-and cell cycle-specific factors and the trans-activator protein NS1, As presented here, p6 pro moter sequences derived from several B19 virus isolates proved to be h ighly conserved. Furthermore, mutations did not affect any of the pote ntial binding sites for transcription factors. One variation of the ba se at position 223 was identified only in B19 virus isolates derived f rom patients with persistent infection or chronic arthritis. To determ ine promoter activity and to characterize regulatory elements, sequenc es spanning the total p6 promoter and subfragments of them were introd uced into a eukaryotic expression vector upstream of the luciferase ge ne (from Photinus pyralis), After transfection into HeLa, CEM, BJAB, a nd K562 cells, the p6 promoter was found to be highly active. When int roduced into the erythroid cell line K562, p6-controlled transcription exceeded that of the simian virus 40 promoter-enhancer used as a cont rol by more than 25-fold, Sequence elements relevant for promoter acti vity mapped to the regions from nucleotides (nt) 100 to 190 and 233 to 298, Also, the segment from nt 343 to 400 downstream of the TATA box was important for transcriptional activity in HeLa and K562 cells. By transfecting the promoter-luciferase constructs into a HeLa cell line stably carrying the viral NS1 gene under the control of an inducible p romoter, transcriptional activity mediated by the p6 promoter rose sig nificantly after induction of NS1 expression. The region from nt 100 t o 160 proved to be essential for NS1-mediated transcriptional activati on. Furthermore, NS1-mediated transactivation was dependent on the pre sence of two GC-rich elements arranged in tandem upstream of the TATA box, These data indicate that NS1-mediated p6 transactivation is depen dent on a multicomponent complex combining NS1 with ATF, NF-kappa B/c- Rel, and GC-box binding cellular factors.