The human papillomavirus (HPV) life cycle is linked to the differentiation state of the host cell. In virus-infected undifferentiated basal epithelial cells, HPV genomes are maintained as episomes at low copy number. Upon differentiation, a concomitant increase in viral copy number and an induction of late gene expression from a differentiation-specific promoter is seen. To investigate whether late gene expression was dependent on the amplification of the viral genome, inhibitors of DNA replication and in vitro systems for epithelial differentiation were used in conjunction with cells that stably maintain HPV31 episomes. Treatment of cells induced to differentiate in methylcellulose with the DNA synthesis inhibitor cytosine ␤-arabinofuranoside (AraC) blocked viral DNA amplification but did not prevent induction of late transcription. This suggests that late gene expression does not strictly require amplification of the viral genome and that differentiation signals alone are sufficient to activate transcription from the late promoter. However, DNA amplification does appear to be necessary for maximal induction of the late promoter. In order to examine the cis-acting elements that contribute to the activation of the late promoter, a transient reporter assay was developed. In these assays, an induction of late gene expression was seen upon differentiation that was specific to the late promoter. Mapping studies localized important regulatory elements to the E6/E7 region and identified short sequences that could serve as binding sites for transcription factors. Elements within the upstream regulatory region were also found to positively and negatively influence transcription from the late promoter. These results identify mechanisms important for the differentiation-dependent activation of late gene expression of high-risk papillomaviruses.

The polyomavirus hr-t class of mutants has served as a major prototype to study the function of middle T + small T in the virus lytic cycle, Biochem. Biophys. Acta 695 (2), 69-95). The properties of these middle T + small T defective mutants were defined by comparisons with "wild-type" strains reconstructed by marker rescue. Similar comparisons in the A2 genetic background have revealed a number of differences, J. Virol. 75, 8380-8389). Here we describe a major divergence in their effects on cell-cycle progression of both permissive mouse NIH3T3 cells and semipermissive Fischer rat FR3T3 cells. Infection of NIH3T3 or FR3T3 cells in serum-rich medium with wild-type A2 (WTA2) or WTA2-derived middle T + small T-defective mutants did not perturb cell cycling, tested up to entry into the third cycle. In contrast, infection with four hr-t mutants analyzed, examined in detail with mutant B2, resulted in an accumulation of cells in G2/M in a dose-dependent and serum-independent manner. The arrest began in the first cell cycle. At multiplicities of infection above 10 PFU/cell, 50-80% of the cell population became arrested by the end of the second cycle. FR3T3 arrested cells detached from the monolayer with a rounded up morphology. Three other hr-t mutants investigated were also found to arrest cells in G2/M. Expression of middle T and/or small T either in trans or in cis did not abrogate this cell-cycle arrest, as demonstrated in the latter case with the middle T + small T expressing strain "wtB2" obtained by repair of the B2 deletion. In FR3T3 cells, the induction of a cell-cycle arrest by wtB2 was accompanied by a severe delay and reduction in neoplastic transformation relative to WTA2 used at equal dose. Mutation(s) in the C-terminal domain of large T antigen, upstream of the site-specific DNA binding activity, is necessary for the cell-cycle block. The possible causes for the cell-cycle block are discussed.