The Distal Promoter of the B438L Gene of African Swine Fever Virus Is Responsible for the Transcription of the Alternatively Spliced B169L

Abstract

The B169L protein (pB169L) of African swine fever virus (ASFV) is a structural protein with an unidentified function during the virus replication. The sequences of the B169L gene and the downstream B438L gene are separated by short intergenic regions. However, the regulatory mode of the gene transcription remains unknown. Here, we identified two distinct promoter regions and two transcription start sites (TSSs) located upstream of the open reading frame (ORF) of B438L. Using the promoter reporter system, we demonstrated that the cis activity of the ORF proximal promoter exhibited significantly higher levels compared with that of the distal promoter located in the B169L gene. Furthermore, transfection with the plasmids with two different promoters for B438L could initiate the transcription and expression of the B438L gene in HEK293T cells, and the cis activity of the ORF proximal promoter also displayed higher activities compared with the distal promoter. Interestingly, the B438L distal promoter also initiated the transcription of the alternatively spliced B169L mRNA (B169L mRNA2) encoding a truncated pB169L (tpB169L) (amino acids 92–169), and the gene transcription efficiency was increased upon mutation of the initiation codon located upstream of the alternatively spliced B169L gene. Taken together, we demonstrated that the distal promoter of B438L gene initiates the transcription of both the B438L mRNA and B169L mRNA2. Comprehensive analysis of the transcriptional regulatory mode of the B438L gene is beneficial for the understanding of the association of B438L protein and pB169L and the construction of the gene-deleted ASFV.