Mutational analysis of the 3' open reading frames and the splice junction at nucleotide 3225 of bovine papillomavirus type 1

Abstract

Functional analysis of the 3' open reading frames (ORFs) of bovine papillomavirus type 1 (BPV-1) has been complicated by the organization of that part of the genome. A region between nucleotides (nt) 3173 and 3551 contains three overlapping ORFs (E2, E3, and E4), as well as a 3' splice junction at nt 3225 which is used by many of the BPV-1 transcripts. To more clearly assign functions to specific ORFs in this region, single-base substitution mutations were generated which introduced translational termination codons into each of the three ORFs; a fourth mutation substituted an A with a C at nt 3223, altering the 3' splice junction consensus sequence from AG to CG. The E3- and the E4-specific mutants were wild type in their abilities to transform susceptible mouse C127 cells, to replicate as stable plasmids, and to trans-activate the E2 conditional enhancer. The E2-specific termination mutant was defective for plasmid replication, transformation, and trans-activation and could not be complemented for efficient transformation of a flat cell line which expressed the full-length E2 gene product. The splice junction mutant was defective for transformation of C127 cells and of a flat cell line expressing the full-length E2 gene product. These data extend previous analyses of the 3' ORFs and suggest that a spliced E2 product is involved in cellular transformation. The splice junction mutant could replicate as a stable plasmid, indicating that there is no absolute requirement in plasmid replication for a viral gene product expressed solely from an mRNA using the 3' splice junction at nt 3225.