Characterization of cis-acting and NS1 protein-responsive elements in the p6 promoter of parvovirus B19

Abstract

Parvovirus B19 infections are associated with diverse clinical manifestations, ranging from no symptoms to severe symptoms. The virus shows an extreme tropism for replication in erythroid progenitor cells, possibly due to the activity of the only functional promoter (p6) of the B19 virus genome in combination with both cell- and cell cycle-specific factors and the trans-activator protein NS1. As presented here, p6 promoter sequences derived from several B19 virus isolates proved to be highly conserved. Furthermore, mutations did not affect any of the potential binding sites for transcription factors. One variation of the base at position 223 was identified only in B19 virus isolates derived from patients with persistent infection or chronic arthritis. To determine promoter activity and to characterize regulatory elements, sequences spanning the total p6 promoter and subfragments of them were introduced into a eukaryotic expression vector upstream of the luciferase gene (from Photinus pyralis). After transfection into HeLa, CEM, BJAB, and K562 cells, the p6 promoter was found to be highly active. When introduced into the erythroid cell line K562, p6-controlled transcription exceeded that of the simian virus 40 promoter-enhancer used as a control by more than 25-fold. Sequence elements relevant for promoter activity 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 promoter, transcriptional activity mediated by the p6 promoter rose significantly after induction of NS1 expression. The region from nt 100 to 160 proved to be essential for NS1-mediated transcriptional activation. Furthermore, NS1-mediated transactivation was dependent on the presence of two GC-rich elements arranged in tandem upstream of the TATA box. These data indicate that NS1-mediated p6 transactivation is dependent on a multicomponent complex combining NS1 with ATF, NF-kappaB/c-Rel, and GC-box binding cellular factors.

FIG. 1

Map of the p6 promoter of parvovirus B19 with potential binding sites for transcription factors indicated (boxes). Inr, putative transcription start site. The position of the insertion of a T in one of the virus isolates and the locations of the exchange of a T for a G in five of the isolates sequenced are indicated (thick arrows).

FIG. 2

The p6 promoter fragments constructed and used for analysis of the promoter activity. Potential binding sites for transcription factors are indicated (shaded boxes). Inr, putative transcription start site. The lengths of the bars below the p6 promoter (top line) represent the lengths of the fragments.

FIG. 3

Mutant p6 promoter constructs tested. The binding sites for transcription factors which were altered (boxes A to G) are indicated, and the constructs and the boxes into which mutations were introduced are listed below the diagram.

FIG. 4

Comparison of the promoter strength of the p6 promoter from parvovirus B19 to that of the promoter-enhancer of SV40 in four cell lines. The values were calculated by dividing the amount of luciferase activity (normalized against the internal R. reniformis standard) of the p6 promoter by that of the SV40 promoter-enhancer in each cell line. The activity of the SV40 promoter-enhancer is therefore 1 in all cell lines tested.

FIG. 5

Comparison of the activities of the different wild-type (Fig. 2) (A) and mutant (Fig. 3) (B) p6 promoter constructs in cell lines HeLa, BJAB, CEM, and K562. Values are given in relation to that of construct p1/1, representing the unmodified p6 promoter, whose activity was arbitrarily set as 1 and whose standard deviation is therefore 0.

FIG. 6

Transactivation of the different p6 promoter constructs by the NS1 protein. Values were calculated as follows. The amount of transactivation by each construct was calculated by setting the luciferase activity of the respective construct in HeLa/NS-21 cells (normalized against the internal R. reniformis standard) relative to that in HeLa/pGRE cells. These values were then divided by the values obtained with construct p1/1, which represents the wild-type promoter. The value of transactivation of construct p1/1 is therefore 1 (with no standard deviation). pGL-3 Control contains the SV40 promoter-enhancer and was regarded as not transactivatable; therefore its value is the baseline.

FIG. 7

Model of the transactivating complex involving proteins bound to consensus sites for ATF/CREB, NF-κB/c-Rel, and Sp1 (GC boxes) which are bridged by NS1 monomer or oligomer. The DNA in between could easily be looped out. Inr, transcription start site.