Characterization of late gene transcripts expressed during vegetative replication of human papillomavirus type 31b

Abstract

Human papillomaviruses (HPVs) are etiologic agents of anogenital cancers. The lack of an efficient in vitro system with which to study the differentiation-dependent viral life cycle has impeded most investigations of viral transcription and gene expression. The CIN-612 clone 9E cell line latently maintains episomal copies of HPV type 31b (HPV31b). The complete replicative life cycle of HPV31b can be studied by using the organotypic (raft) culture system. A number of spliced HPV31b early gene transcripts and two late gene transcripts have been described in studies using the raft system. An HPV31b early promoter, P 97 , and a differentiation-induced promoter, P 742 , have been characterized by using this system. In this study, we used the raft system to analyze the temporal expression patterns of HPV31b late gene transcripts during the viral life cycle. The expression of late RNAs peaked at day 12 after lifting to the air-liquid interface; the levels then declined dramatically by day 16. The peak of late RNA expression was coincident with the appearance of virus particles in the raft tissues. We characterized transcripts with the potential to encode late gene products, including 19 RNAs containing the L1 region and 4 RNAs containing the E5b and L2 open reading frames. We also found evidence for two novel promoters. Transcription of both L1-and L2-containing RNAs initiated at a region upstream of the early promoter. In addition, late gene RNAs were also transcribed by using a promoter in the E4 reading frame.

Temporal Usage of Multiple Promoters during the Life Cycle of Human Papillomavirus Type 31b

“…Most cell lines used to study HPVs are derived from malignancies and contain integrated viral genomes with obstructed late functions. Continuing advances in organotypic or raft tissue culture systems have permitted the growth of differentiated keratinocytes in vitro and provided a permissive environment for the complete HPV life cycle (4,16,20,23,24,30,31,35). Recently, we used DNA transfection techniques coupled to the organotypic culture system to purify infectious stocks of HPV18 (31).…”

“…An understanding of the differentiation-dependent life cycles of high-risk HPVs has been enhanced greatly by the study of the latently infected CIN-612 9E cell line, which contains episomal copies of HPV31b (4,23,24,35). The growth of CIN-612 9E monolayer cells and raft tissues has permitted the identification of the HPV31b early promoter that maps to nucleotide (nt) 97 (P 97 ) and the P 742 differentiation-induced promoter (23).…”

“…The growth of CIN-612 9E monolayer cells and raft tissues has permitted the identification of the HPV31b early promoter that maps to nucleotide (nt) 97 (P 97 ) and the P 742 differentiation-induced promoter (23). At least 7 spliced, polycistronic early viral RNAs and 19 polycistronic late viral RNAs have been characterized with CIN-612 9E cells and raft tissues (23,24,35,36). A detailed analysis of HPV31b late gene transcripts indicated that late gene RNAs initiated from at least three distinct promoters; RNA start sites mapped in the region of P 97 , at multiple sites near P 742 , and close to the E4 splice acceptor site at nt 3295 (35).…”

The Effect of Productive HPV16 Infection on Global Gene Expression of Cervical Epithelium

“…The raft tissues were harvested after 10 days for microarray analysis and 20 days for qPCR, Western blot, and immunofluorescence staining. Viral gene expression has been shown to peak between 10 and 12 days (95) while virion maturity reaches maximum stability around 20 days (96).…”

An Overview on ERAP Roles in Infectious Diseases

“…Conversely, the expression of the viral genes essential for viral replication is highly accelerated in the more superficial layers of the epithelium, this results in viral genome amplification and in the production of thousands of viral copies per cell [78][79][80].…”