Characterization of the conserved chiA and v-cath bidirectional promoter of Autographa californica multiple nucleopolyhedrovirus (AcMNPV)
In the AcMNPV genome, ~28% of the genes are arranged divergently on opposite strands with an intergenic region of <1 kbp. In this configuration, a bidirectional promoter generally drives expression of both genes. However, no baculovirus bidirectional promoters have been characterized in any detail. We chose the AcMNPV chiA/v-cath intergenic region to serve as a model to characterize transcriptional regulation of bidirectional gene pairs during AcMNPV infection. We sequentially truncated putative upstream regulatory regions of chiA and v-cath to identify sequences essential for transcriptional initiation. Forty bp of the chiA gene 5’-flanking region was sufficient to support chiA transcription at half the level of the AcΔCC+CC repair virus. Interestingly, v-cath transcription from viruses containing only 40 bp of their upstream 5’-flanking region was found to be higher by 4-fold relative to the level of native expression. Linker-scanning mutagenesis that inserted 5 bp linkers spanning the chiA/v-cath intergenic region identified nucleotides critical for the transcriptional activation of both genes. From this, nucleotides -36 to -45, of the v-cath gene were found to negatively regulate v-cath mRNA expression. Quantitative RT-PCR studies revealed a 2-4 fold higher chiA mRNA expression relative to v-cath possibly explaining why translation of CHIA can be detected 6 hours earlier than V-CATH. This study identifies upstream regions of viral chiA and v-cath required for initiation of transcription and provides the first insight into baculovirus mechanisms for transcriptional regulation of interdependent gene pairs.