Characterization of the promoter elements and transcription profile of Periplaneta fuliginosa densovirus nonstructural genes

Abstract

Periplaneta fuliginosa Densovirus (PfDNV), an autonomous invertebrate parvovirus that infects the cockroach, is unusual in that alternative splicing is involved in the structural gene expression. The expression strategy for nonstructural (NS) genes has yet not been reported. Northern blot analysis of cockroach larvae infected with PfDNV revealed two transcripts for the NS genes, one of 2.6 kb, and the other of 1.9 kb. The two transcripts were shown to begin at a common initiator consensus sequence, CAGT, located in the terminus of ITR. The 1.9 kb transcript was produced by splicing out the ns3 gene from the 2.6 kb transcript. To understand the mechanism of transcriptional regulation of NS genes, the 5′-flanking sequence of ns3 gene (325 bp), which encompasses the region from the 5′-terminus of the viral genome to the initiator ATG codon of the ns3 gene, was cloned and fused to a luciferase reporter gene. The luciferase reporter assay showed that this sequence possessed promoter activity in Sf9, Ld652, Tn368, and S2 cell lines. Subsequent promoter deletion analysis showed that the promoter exhibited TATA-dependent and TATA-independent transcriptional activities. Moreover, we found that the promoter activity of the 325-bp fragment in S2 cells could be enhanced significantly by co-transfection of the nonstructural protein NS1 and that the NS1 binding element, (CAC) 4 repeat, mediated the promoter activity activated by NS1 protein.