The 5′-terminal 32 basepairs conserved between genome segments A and B contain a major promoter element of infectious bursal disease virus

The regions of the infectious bursal disease virus (IBDV) genome with regulatory function are not known. In the present study, progressively deleted lengths of the 5′ noncoding region of segment A were constructed in pGL3 vectors having SV40 enhancer or promoter, and a luciferase (LUC) reporter gene. Transient transfections of the constructs made in a promoter-less pGL3-Enhancer vector when transfected in Vero cells and the lysates assayed for LUC expression, allowed the localization of maximal activity to the 32-nucleotide stretch (precursor polyprotein ORF positions −131 to −100), which is highly conserved at the 5′ end of both genome segments. This fragment, when evaluated in parallel in an enhancer-less pGL3-Promoter vector demonstrated no activity. To determine if this region is recognized by IBDV replicative proteins, we engineered modifications in an enhancer-less pGL3-Promoter vector where the terminal 32-bp fragment, the full-length noncoding region, or the noncodig region with the 32-bp fragment deleted was positioned in either the plus-sense or the minus-sense orientation immediately downstream of the SV40 promoter and upstream of the LUC gene. Transfections of these constructs in IBDV-infected and uninfected Vero cells resulted in the endogenous generation of recombinant viral-LUC RNAs containing the 5′ terminal viral RNA sequences in either the plus-sense or the minus-sense orientation. LUC assays of the infected cell lysates showed up-regulated expression of LUC only with constructs containing the 32-bp fragment in the minus-sense orientation. Deletion of this 32-bp fragment abolished such LUC expression. We therefore conclude that the 5′-terminal 32 base pairs of genomic segment A contain a major promoter element in IBDV. In addition, our results show that IBDV replicative proteins recognize and transcribe single-stranded RNA in vivo.