Characterization of the structural gene promoter of Aedes aegypti densovirus

Aedes aegypti densonucleosis virus (AeDNV) has two promoters that have been shown to be active by reporter gene expression analysis (B. N. Afanasiev, Y. V. Koslov, J. O. Carlson, and B. J. Beaty, Exp. Parasitol, 79: 322-339, 1994). Northern blot analysis of cells infected with AeDNV revealed two tra nscripts 1,200 and 3,500 nucleotides in length that are assumed to express the structural protein (VP) gene and nonstructural protein genes, respectiv ely. Primer extension was used to map the transcriptional start site of the structural protein gene. Surprisingly, the structural protein gene transcr ipt began at an initiator consensus sequence, CAGT, 60 nucleotides upstream from the map unit 61 TATAA sequence previously thought to define the promo ter. Constructs with the P-galactosidase gene fused to the structural prote in gene were used to determine elements necessary for promoter function. De letion or mutation of the initiator sequence, CAGT, reduced protein express ion by 93%, whereas mutation of the TATAA sequence at map unit 61 had littl e effect. An additional open reading frame was observed upstream of the str uctural protein gene that can express beta -galactosidase at a low level (2 0% of that of VP fusions), Expression of the AeDNV structural protein gene was shown to be stimulated by the major nonstructural protein NS1 (Afanasie v et al,, Exp, parasitol., 1994). To determine the sequences required for t ransactivation, expression of structural protein gene-beta -galactosidase g ene fusion constructs differing in AeDNV genome content was measured with a nd without NS1. The presence of NS1 led to an 8- to 10-fold increase in exp ression when either genomic end was present, compared to a 2-fold increase with a construct lacking the genomic ends. An even higher (37-fold) increas e in expression occurred with both genomic ends present; however, this was in part due to template replication as shown by Southern blot analysis. The se data indicate the location and importance of various elements necessary for efficient protein expression and transactivation from the structural pr otein gene promoter of AeDNV.