Analysis of the corrected DNA sequence for the bovine papillomavirus type 4 (BPV4) genome revealed that there is no open reading frame (ORF) that might encode an E6 protein. The other two B subgroup bovine papillomaviruses, BPV3 and BPV6, were found to have the same arrangement of ORFs in this region as BPV4. Thus, we conclude that E6 functions are either not required by these viruses or are performed by another viral (or host) protein. Furthermore, the position that might be expected to be occupied by E6, between the long control region and the E7 ORF, contains the E8 ORF, which has the potential to encode a 42-residue polypeptide with considerable similarity to the E5 transforming protein of BPV1. Therefore, it appears that during the evolution of the B subgroup of BPVs, genomic rearrangements may have occurred resulting in the present layout of the early ORFs.

The long control region (LCR) of bovine papillomavirus type 4 demonstrated enhancer activity when cloned upstream of a bacterial chloramphenicol acetyltransferase reporter gene under thymidine kinase promoter control. Deletion analysis of the LCR revealed the presence of several positive and negative control elements, all of which could function independently of the viral E2 trans-activator. Each of the three positive elements present appeared to be paired with a negative element which modulated its activity. DNase I footprinting was used to identify protein binding sites within the LCR, which might represent these control elements. The results suggest a highly complex and finely tuned control of viral gene expression.