The tripartite leader sequence is required for ectopic expression of HAdV-B and HAdV-E E3 CR1 genes

•HAdV-B- and E-specific E3 CR1 genes cannot be expressed using traditional vectors.•TPL is required for efficient expression of HAdV-B and E E3 CR1-encoded proteins.•TPL increases transcription and translation of HAdV-B- and E-specific E3 CR1 genes.

The unique repertoire of genes that characterizes the early region 3 (E3) of the different species of human adenovirus (HAdV) likely contributes to their distinct pathogenic traits. The function of many E3 CR1 proteins remains unknown possibly due to unidentified intrinsic properties that make them difficult to express ectopically. This study shows that the species HAdV-B- and HAdV-E-specific E3 CR1 genes can be expressed from vectors carrying the HAdV tripartite leader (TPL) sequence but not from traditional mammalian expression vectors. Insertion of the TPL sequence upstream of the HAdV-B and HAdV-E E3 CR1 open reading frames was sufficient to rescue protein expression from pCI-neo constructs in transfected 293T cells. The detection of higher levels of HAdV-B and HAdV-E E3 CR1 transcripts suggests that the TPL sequence may enhance gene expression at both the transcriptional and translational levels. Our findings will facilitate the characterization of additional AdV E3 proteins.