Regulation of human papillomavirus type 31 polyadenylation during the differentiation-dependent life cycle

The L1 and L2 capsid genes of human papillomavirus type 31 (HPV-31) are expressed late in the differentiation-dependent life cycle from a promoter located in the E7 open reading frame (ORF) of the early region. These late HPV genes are transcribed by RNA polymerase II which reads through the region containing early polyadenylation signals and proceeds to a poly(A) site downstream of L1. In this study, we have investigated the mechanisms regulating differentiation-dependent polyadenylation and read-through in HPV-31. HPV-31 early transcripts were found to utilize a heterogeneous series of polyadenylation sites in undifferentiated cells. The sites for polyadenylation extended over a range of 100 nucleotides from within the E5 ORF to upstream of L2. Upon differentiation, the transcription of early genes increased, but no change in the heterogeneous distribution of 3' ends was detected. The early polyadenylation region was found to contain a single consensus hexanucleotide sequence, AAUAAA, as well as three weak binding sites for the cleavage stimulatory factor, CstF. In contrast to the heterogeneity at the early site, the 3' ends of late transcripts encoding L1 and L2 were localized to a narrow region downstream of the late AAUAAA element. The late polyadenylation signal was found to contain a single high-affinity site for CstF, as well as one consensus hexanucleotide sequence. By using a reporter assay, it was determined that the HPV-31 early polyadenylation sequences allowed significant levels of read-through into the late region in undifferentiated cells. Upon differentiation, this read-through was increased by approximately 50%, indicating that use of the early site decreased. Differentiation was also found to induce a 40% reduction in the levels of CstF subunits, which may contribute to the increased read-through of the early sequence. The insertion of the late high-affinity binding site for CstF into the early polyadenylation region significantly reduced the level of read-through, suggesting that these factors modulate read-through activity. Our studies demonstrate that HPV-31 late gene expression is regulated in a large part by posttranscriptional mechanisms, including the polyadenylation of early transcripts.