HUMAN PAPILLOMAVIRUS TYPE 31B LATE GENE-EXPRESSION IS REGULATED THROUGH PROTEIN-KINASE C-MEDIATED CHANGES IN RNA PROCESSING

Expression of the human papillomavirus (HPV) capsid genes, L1 and L2, as well as amplification of viral DNA and virion assembly occur in the terminally differentiated layers of infected stratified squamous epit helium in vivo, These processes can be duplicated in the laboratory th rough the use of organotypic or raft cultures, When CIN612 cells, whic h contain episomal copies of the high-risk HPV type 31b, are allowed t o differentiate in raft cultures, the expression of transcripts encodi ng the early genes E1(boolean AND) E4 and E5 is induced, These transcr ipts are initiated at the differentiation-dependent P742 promoter loca ted in the middle of the E7 open reading frame. Exposure of raft cultu res to activators of protein kinase C, such as phorbol esters, results in the further induction of late gene expression as well as virion as sembly. In this study, we have investigated the mechanism by which act ivators of protein kinase C induce late gene expression, The major L1 transcript was found to be encoded by a bicistronic E1(boolean AND) E4 , L1 RNA which initiated at the differentiation-dependent promoter P74 2, Additional low-level expression of L1-containing RNAs was also obse rved from the early-region promoter, P97, The major L2 transcripts wer e found to be encoded by E1(boolean AND) E4, ES, L2, L1 RNAs which wer e also initiated in the early region, probably at the differentiation- specific promoter P742. While early and late RNAs were found to be exp ressed from the same promoter, they differed in utilization of splicin g and polyadenylation sites, Raft cultures treated with activators of protein kinase C induced expression of late genes, but no change in th e abundance of early RNAs initiated at the P742 promoter was observed, Thus, the increase in late gene expression was likely due to changes in RNA processing or stabilization rather than an increase in the rate of transcription from P742. Regulation of HPV late gene expression th erefore occurs at two levels: differentiation-dependent induction of t he P742 promoter, which can be mimicked in vitro by growth in raft cul tures, and posttranscriptional changes that can be induced by activati on of protein kinase C. These posttranscriptional changes may occur th rough inactivation or down-regulation of splicing factors which inhibi t use of the late region polyadenylation site, resulting in increased stability of late region transcripts.