Human papillomaviruses trophic for anogenital epithelia cause benign warts, and certain genotypes are closely associated with cervical neoplasia. By using our modifications of the epithelial raft culture system, we were able to recapitulate and modulate the infectious program of a papillomavirus in vitro for the first time. Small pieces of a condyloma containing human papillomavirus type 11 were explanted onto a dermal equivalent consisting of a collagen matrix with fibroblasts and were cultured at the medium-air interface. The infected stem cells proliferated rapidly across the matrix, stratified, and differentiated, as judged by histology. The results correlated with the state of epithelial differentiation, which, in turn, was dependent on the type of fibroblast in the matrix. Under conditions where the epithelial outgrowth underwent terminal differentiation, the entire productive program took place, leading to virion assembly. In contrast, using an alternative condition where the outgrowth failed to achieve terminal differentiation, only the E-region RNAs from the E1 promoter accumulated to any appreciable extent. The proliferating cell nuclear antigen was induced in the differentiated suprabasal cells in the productive cyst growth, which also exhibited high copy viral DNA and abundant E6--E7 RNAs. Comparable cells in the nonproductive cyst outgrowth were negative for all three. These results suggest that the E6 and E7 proteins may play a role in establishing a cellular environment conducive to vegetative viral replication. The culture conditions described should be useful for genetic analysis of this family of important human pathogens and for testing potential pharmacological agents.

The Epstein-Barr virus BamHI-F promoter (Fp) is one of three used to transcribe the EBNA latency proteins, in particular, EBNA-1, the only viral gene product needed for episomal replication. Fp is distinguished by possession of the only EBNA-1 binding sites (the Q locus) in the Epstein-Barr virus genome outside oriP. Activity of Fp is negatively autoregulated by interaction of EBNA-1 at two sites in the Q locus, which is situated downstream of the RNA start site. We demonstrate in transient assays that this EBNA-1-mediated repression of Fp can be overcome by an E2F transcription factor which interacts with the DNA at a site centered between the two EBNA-1 binding sites within the Q locus. An E2F-1 fusion protein protects the sequence 5'-GGATGGCGGGTAATA-3' from DNase I digestion, and a DNA probe containing this sequence binds an E2F-specific protein complex from cell extracts, although this region is only loosely homologous with known consensus binding sites for E2F transcription factors. In mobility shift assays, E2Fcan displace the binding of EBNA-1 from the Q locus but not from oriP, where the E2F binding site is not present. E2F also activates expression of Fp in epithelial cells. These findings identify a potentially new binding site for members of the E2F family of transcription factors and suggest that such a factor is important for expression of EBNA-1 in lymphoid and epithelial cells by displacing EBNA-1 from the Q locus. In addition, the possibility that Fp activity is under cell cycle control is raised. Since the supply of functional E2F varies during the cell cycle and since in these assays overexpression of E2F can overcome repression of Fp by EBNA-1, control of transcription of EBNA-1 mRNA by cell cycle regulatory factors may help to bring about ordered replication of episomes.Epstein-Barr virus (EBV) is associated with the development of malignancies (lymphomas and carcinomas of the nasopharynx) in both of the cell types it infects (10,45,59). In each of these tumors, the viral genome exists as a circular episome, characteristic of EBV latency (42). The key gene required for initiation and maintenance of latent infection encodes Epstein-Barr nuclear antigen 1 (EBNA-1). By binding to specific repeated DNA sequence motifs at the origin of plasmid replication (oriP) (38), EBNA-1 activates both episomal replication and the oriP transcriptional enhancer (46,47,60,65) In this paper, we show that while Fp is repressed by EBNA-1, it can also be activated by an E2F-like transcription factor which binds to DNA within the Q locus and that the activity of the promoter is reciprocally regulated by these cellular and viral proteins. MATERUILS AND METHODSCell lines. Raji (44) is a latently infected EBV-positive Burkitt's lymphoma B-cell line, and Jurkat is an EBV-negative human T-cell line. SVK is a human keratinocyte cell line (35). All cells were maintained at 37°C in a 5% CO2 environment. Lymphoid cells were grown in RPMI medium supplemented with 10% fetal calf serum.Recombinant phagemid constructs. A series ...

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