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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.

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SUMMARYThe nucleotide sequence of bovine papillomavirus type 4 (BPV-4) was determined. The viral genome is 7261 base pairs long. Several overlapping open reading frames (ORFs) have been identified both on the basis of amino acid comparison with other papiUomaviruses and on their transcriptional pattern. Eight early ORFs (E 1 to 8) were recognized, coding for DNA replication and cell transformation functions, and three late ORFs (L1 to 3), coding for structural proteins. Like the E50RF of human papillomavirus type 6 the E50RF of BPV-4 is discontinuous. Unlike other papillomaviruses, the non-coding region upstream of the early ORFs (ncr-1) is short (385 base pairs), but there is another non-coding region (ncr-2) of nearly 500 base pairs between the L2 and L10RFs. Most of the putative regulatory sites are located in the ncr-1, although potential controlling elements are also found in other parts of the genome. Polyadenylation sites are present at the 3' end of both the early and the late transcription units. Comparison between the polypeptides of BPV-4 and other papillomaviruses showed that BPV-4 is evolutionarily closer to the epitheliotropic human and rabbit viruses than to BPV-1.

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.

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SUMMARYNIH 3T3 mouse fibroblasts were transformed in vitro by transfection with viral DNA from bovine papillomavirus (BPV) types 4, 2 and 1. The viral DNAs were molecularly cloned in pAT153 to construct pBV4, a recombinant plasmid containing the whole genome of BPV4, pBV2 containing the entire genome of BPV2, and pBV1-D1 which contains the 69~ transforming DNA fragments of BPV1. The transformed cells lost contact inhibition, were anchorage-independent, required low serum and were tumourigenic in nude mice. This is the first report of cell transformation in vitro by BPV4 DNA. The recombinant plasmids transformed NIH 3T3 cells with an efficiency of 200 to 300 foci/~tg DNA. Cleavage of the recombinant plasmids with enzymes that separate the viral DNA from pAT153 DNA did not significantly alter the efficiency of transformation. In all the transformed cells analysed, the recombinant plasmids were found as multiple copies of non-integrated monomers.

SUMMARYA cDNA library was synthesized using RNA from bovine papillomavirus type 4 (BPV-4)-induced papillomas. The major viral transcript was characterized by sequencing of its cDNA, primer extension mapping and S 1 nuclease protection studies. The transcript initiates at multiple sites between nucleotide (nt) 777 and nt 902, contains a splice junction between nt 1016 and nt 3376 and terminates at nt 4034, using the early polyadenylation signal at nt 4009. Sequencing of viral cDNAs and mRNA revealed deviations from the reported genomic sequence between nt 3412 and nt 3460. These resulted in a temporary frameshift, abolished the translation termination codon of the E5a open reading flame (ORF), and introduced a new termination codon in the E40RF. The new uninterrupted E50RF was found to have greater homology to the E40RFs of other papillomaviruses than to their E50RFs. The E50RF of BPV-4 is joined to a five codon ORF in the leader exon of the major transcript, in the same way as the E40RF in the major transcripts of BPV-1 and human papillomavirus type 11. On this basis, it has been redesignated E4, and the viral genomic sequence revised. Two novel transcriptional promoters of BPV-4 were defined: a putative controller of the multiple major RNA start sites around nt 870 and a minor TATA box at nt 691. In addition, minor early region transcripts were mapped which initiate between nt 3071 and nt 3152. None of these transcripts utilizes the splice site at nt 3376. These messages may express E2-encoded functions.