Characterization of an abundant, unique 1.7-kilobase bovine herpesvirus 4 (BHV-4) late RNA and mapping of a BHV-4 IE2 transactivator-binding site in its promoter-regulatory region

Abstract

We characterized an abundant late 1.7-kb cytoplasmic polyadenylated RNA (L1.7 RNA) transcribed from the bovine herpesvirus 4 (BHV-4) HindIII W fragment, in a region of the genome not conserved with Epstein-Barr virus and herpesvirus saimiri. L1.7 RNA contains only extremely short (<100 nucleotides) open reading frames followed by two repeat arrays. The first repeat array contains 11 copies of a 23-bp unit, TGGCACTA GTAGCATTTAACCCC. The second and third copies are each interrupted by 15- to 17-bp sequences that are identical to each other for the first 15 bp. In addition, the second and third copies of the repeat unit each contain two copies of nucleotides 5 to 9 (ACTAG) of the repeat unit, one at each end of the interruption. The second repeat array contains 12 copies of a 25-bp sequence, GCTGTGTATTATTGAGTATTTTTTA. The promoter-regulatory region of L1.7 was activated by the BHV-4 immediate-early gene 2 product (IE2), a homolog of the Epstein-Barr virus R transactivator, in cotransfection assays. We mapped an IE2 recognition site within a 167-bp fragment approximately 10 bp 5' to the start of L1.7 RNA transcription, using cotransfection assays and gel retardation assays. Using gel retardation assays, we mapped an IE2-binding site within this fragment to a 31-bp region from 56 to 86 bp 5' to the start of L1.7 RNA transcription. This IE2-binding site was able to transfer IE2 responsiveness to a heterologous promoter. However, IE2 responsiveness was affected by both position and orientation. Alignment of the L1.7 IE2-binding site sequence with sequences of two other BHV-4 IE2-binding sites resulted in a provisional IE2-binding site consensus sequence different from the Epstein-Barr virus R transactivator-binding site.