Utilization of the bovine papillomavirus type 1 late-stage-specific nucleotide 3605 3 ' splice site is modulated by a novel exonic bipartite regulator but not by an intronic purine-rich element

Bovine papillomavirus type 1 (BPV-1) late gene expression is regulated at b oth transcriptional and posttranscriptional levels. Maturation of the capsi d protein (L1) pre-mRNA requires a switch in 3' splice site utilization. Th is switch involves activation of the nucleotide (nt) 3605 3' splice site, w hich is utilized only in fully differentiated keratinocytes during late sta ges of the virus life cycle, Our previous studies of the mechanisms that re gulate BPV-1 alternative splicing identified three cis-acting elements betw een these two splice sites. Two purine-rich exonic splicing enhancers, SE1 and SE2, are essential for preferential utilization of the nt 3225 3' splic e site at early stages of the virus life cycle. Another cis-acting element, exonic splicing suppressor 1 (ESS1), represses use of the nt 3225 3' splic e site, In the present study, we investigated the late-stage-specific nt 36 05 3' splice site and showed that it has suboptimal features characterized by a nonconsensus branch point sequence and a weak polypyrimidine track wit h interspersed purines. In vitro and in vivo experiments showed that utiliz ation of the nt 3605 3' splice site was not affected by SE2, which is intro nically located with respect to the nt 3605 3' splice site. The intronic lo cation and sequence composition of SE2 are similar to those of the adenovir us IIIa repressor element, which has been shown to inhibit use of a downstr eam 3' splice site. Further studies demonstrated that the nt 3605 3' splice site is controlled by a novel exonic bipartite element consisting of an AC -rich exonic splicing enhancer (SE4) and an exonic splicing suppressor (ESS 2) with a UGGU motif. Functionally, this newly identified bipartite element resembles the bipartite element composed of SE1 and ESS1, SE4 also functio ns on a heterologous 3' splice site. In contrast, ESS2 functions as an exon ic splicing suppressor only in a 3'-splice-site-specific and enhancer-speci fic manner. Our data indicate that BPV-1 splicing regulation is very comple x and is likely to be controlled by multiple splicing factors during kerati nocyte differentiation.