Background: Human bocavirus (HBoV), a parvovirus, is suspected to be an etiologic agent of respiratory disease and gastrointestinal disease in humans. All mRNAs of HBoV1 are transcribed from a single promoter.
Methods: In this study, we constructed EGFP and luciferase reporter gene vectors under the control of the HBoV1 full promoter (nt 1-252) and its mutated variants, respectively. Fluorescence microscopy was used to observe expression activities of the EGFP. Dual-luciferase reporter vectors were employed in order to evaluate critical promoter elements and the effect of NS1 protein on promoter activity.
Results: The HBoV1 promoter activity was about 2.2-fold and 1.9-fold higher than that of the CMV promoter in 293 T and HeLa cells, respectively. The putative transcription factor binding region of the promoter was identified to be located between nt 96 and nt 145. Mutations introduced in the CAAT box of the HBoV1 promoter reduced promoter activity by 34%, whereas nucleotide substitutions in the TATA box had no effect on promoter activity. The HBoV1 promoter activities in 293 T and HeLa cells, in the presence of NS1 protein, were 2- to 2.5-fold higher than those in the absence of NS1 protein.
Conclusion: The HBoV1 promoter was highly active in 293 T and HeLa cell lines, and the sequence from nt 96 to nt 145 was critical for the activity of HBoV1 promoter. The CAAT box, in contrast to the TATA-box, was important for optimum promoter activity. In addition, the transcriptional activity of this promoter could be trans-activated by the viral nonstructural protein NS1 in these cells.