The study of the human pathogen papillomaviruses (HPVs) has been hampered by the inability to propagate the virus in tissue culture. The addition of 12-O-tetradecanoyl phorbol-13-acetate to the media of organotypic (raft) cultures increased expression of physiological markers of keratinocyte differentiation and concomitantly induced production of virions. Capsid production was detected in differentiated suprabasal cells. Virions approximately 54 nanometers in size were observed by electron microscopy in raft tissue cross sections in the suprabasal layers. Virions purified through isopycnic gradients were found to contain type 31b DNA and exhibited an icosahedral shape similar to that of papillomaviruses found in clinical samples.

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The expression of viral genes during the productive life cycle of human papillomaviruses (HPV) is tightly coupled to the differentiation program of epithelial cells. We have examined transcription of HPV as a function of differentiation in an in vitro organotypic raft culture system which allows for epithelial stratification at the air-liquid interface. When CIN612 cells, which contain episomal copies of HPV type 31b (HPV31b), were allowed to stratify in raft cultures, they differentiated in a manner which was histologically similar to that seen in a cervical intraepithelial neoplasia I biopsy lesion. In monolayer cultures of CIN612 cells, two major polycistronic HPV31b transcripts of 1.7 kb which encode (i) E6, E7, El^E4, and E5 and (ii) E6*, E7, El^E4, and E5 were identified. These RNAs initiated at a promoter, P97, in the upstream regulatory region of the virus. Following differentiation in raft cultures, the relative abundance of RNAs initiated at P97 was unchanged. In contrast, the expression of a 1.3-kb RNA encoding an El^E4 fusion protein and E5 was found to increase substantially following differentiation. This transcript was initiated at a novel promoter within the * Corresponding author.

The E6-E7 region of human papillomavirus types 16 and 18 is selectively retained and expressed in cervical carcinoma cells. In cultured human keratinocytes, expression of the E6 and E7 open reading frames of human papillomavirus type 18, under the control of its homologous promoter, resulted in high-frequency immortalization. Furthermore, by using a system that allows for stratification of keratinocytes in vitro (raft system), we observed that the morphological differentiation of these E6-E7 immortalized cells was altered such that parabasal cells extended throughout most of the epithelium, with abnormal nuclei present in the upper regions. Examination of E6-E7-expressing cell lines in the raft system at a later passage revealed that complete loss of morphological differentiation had occurred. E7 alone was a much less effective immortalizing agent than E6 and E7 together and acted only minimally to alter morphological differentiation in vitro. No such activities were found for E6 alone. High-frequency transformation of human epithelial cells thus appears to require expression of both E6 and E7 gene products.

Human papillomaviruses (HPVs) infect squamous epithelium and establish their genomes as episomes in proliferating basal cells. As infected cells differentiate, the viral DNA is amplified to high copy number and infectious virus is produced. Viral production has not yet been observed in vitro due to the inability of standard culture methods to duplicate most stages of epithelial differentiation. In this study, we have examined a cell line derived from a low-grade cervical lesion and found that it contained episomal copies of an HPV-31 subtype, HPV-31b, at approximately 50 copies per cell. When allowed to stratify at the air-liquid interface of in vitro raft cultures, this cell line differentiates in a manner which histologically resembles a low-grade cervical lesion in vivo. We have observed the amplification of HPV-31b genomes in distinct foci in the upper portion of the in vitro-stratified epithelium similar to that found in productive HPV infections in vivo. Although transcripts from the late region of HPV-31b were also detected specifically in stratified raft cultures, no capsid protein was found. These studies duplicate one important aspect of a productive HPV infection in vitro, the differentiationdependent amplification of papillomavirus genomes.

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The Transforming Growth Factor-beta superfamily member decapentaplegic (dpp) acts as an extracellular morphogen to pattern the embryonic ectoderm of the Drosophila embryo. To identify components of the dpp signaling pathway, we screened for mutations that act as dominant maternal enhancers of a weak allele of the dpp target gene zerknLllt. In this screen, we recovered new alleles of the Mothers against dpp (Mad) and Medea genes. Phenotypic analysis of the new Medea mutations indicates that Medea, like Mad, is required for both embryonic and imaginal disc patterning. Genetic analysis suggests that Medea may have two independently mutable functions in patterning the embryonic ectoderm. Complete elimination of maternal and zygotic Medea activity in the early embryo results in a ventralized phenotype identical to that of null dpp mutants, indicating that Medea is required for all dpp-dependent signaling in embryonic dorsal-ventral patterning. Injection of mRNAs encoding DPP or a constitutively activated form of the DPP receptor, Thick veins, into embryos lacking all Medea activity failed to induce formation of any dorsal cell fates, demonstrating that Medea acts downstream of the thick veins receptor. We cloned Medea and found that it encodes a protein with striking sequence similarity to human SMAD4. Moreover, injection of human SMAD4 mRNA into embryos lacking all Medea activity conferred phenotypic rescue of the dorsal-ventral pattern, demonstrating conservation of function between the two gene products.