Temporal usage of multiple promoters during the life cycle of human papillomavirus type 31b

The life cycles of human papillomaviruses (HPVs) are dependent Upon the disfferentiation of the epithelial cells they infect. HPV type 31b (HPV31b) virions can be purified following the growth of a latently HPV-infected cell line (CIN-612 9E) in the organotypic or raft system. Treatment of the CIN- 612 9E raft tissues with protein kinase C (PKC) activators is required for upregulation of late gene expression and efficient production of virions. We employed the raft culture system to study the temporal usage of HPV31b promoters during the viral life cycle. We compared monolayer cultures of CIN- 612 9E cells, untreated CIN-612 9E raft tissues, and PKC-induced CIN-612 9E raft tissues harvested at various time points during epithelial differentiation. We found that the HPV31b major early promoter precisely maps to nucleotide (nt) 99 (P₉₉). A transcriptional start site for both early and late gene transcripts mapped upstream of P₉₉ at nt 77 (P₇₇). The P₇₇ and P₉₉ promoters were used constitutively throughout the HPV31b life cycle; however, initiation from P₉₉ was much stronger than from P₇₇. Mapping of the differentiation-induced P₇₄₂ promoter revealed multiple start sites. These start Sites were difficult to detect in monolayer cultures, were induced in untreated rafts, and were greatest in PKC-induced raft tissues at 8 to 12 days. A constitutively active promoter, P₃₃₂₀, was also defined and is responsible for the transcription of unspliced and spliced RNAs containing E5a, E5b, L2, and L1 open reading frames.