Human papillomaviruses (HPVs) amplify in differentiated strata of a squamous epithelium. The HPV E7 protein destabilizes the p130/retinoblastoma susceptibility protein family of tumor suppressors and reactivates S-phase reentry, thereby facilitating viral DNA amplification. The high-risk HPV E6 protein destabilizes the p53 tumor suppressor and many other host proteins. However, the critical E6 targets relevant to viral DNA amplification have not been identified, because functionally significant E6 mutants are not stably maintained in transfected cells. Using Cre-loxP recombination, which efficiently generates HPV genomic plasmids in transfected primary human keratinocytes, we have recapitulated a highly productive infection of HPV-18 in organotypic epithelial cultures. By using this system, we now report the characterization of four HPV-18 E6 mutations. An E6 null mutant accumulated high levels of p53 and amplified very poorly. p53 siRNA or ectopic WT E6 partially restored amplification, whereas three missense E6 mutations that did not effectively destabilize p53 complemented the null mutant poorly. Unexpectedly, in cis, two of the missense mutants amplified, albeit to a lower extent than the WT and only in cells with undetectable p53. These observations and others implicate p53 and additional host proteins in regulating viral DNA amplification and also suggest an inhibitory effect of E6 overexpression. We show that high levels of viral DNA amplification are critical for late protein expression and report several previously undescribed viral RNAs, including bicistronic transcripts predicted to encode E5 and L2 or an alternative form of E1^E4 and L1.human papillomavirus DNA amplification | trans complementation | HPV transcripts P apillomaviruses are small DNA viruses with a protein capsid harboring a double-stranded circular genome of ∼7,900 bp. Dozens of human papillomavirus (HPV) types are tropic for the anogenital tract (1). HPV-16 and -18 and closely related genotypes are high-risk (HR) types and at a low frequency, can cause cancers, in which the viral E6 and E7 oncogenes are invariably overexpressed. HPV-6 and -11 are low-risk (LR) types and induce benign anogenital warts and laryngeal papillomas (review in ref. 2). Typically, viral DNA is maintained as low copy nuclear plasmids in basal and parabasal keratinocytes, and vegetative amplification depends on squamous differentiation (review in ref.3). Because viral DNA replication requires the host DNA replication machinery, the role of the HPV E7 protein is to promote S-phase reentry in differentiated cells that have withdrawn from the cell cycle. It does so by destabilizing the p130 protein (4, 5), a pocket protein related to the retinoblastoma susceptibility protein, a major tumor suppressor. The HR but not the LR HPV E7 protein also destabilizes retinoblastoma susceptibility protein (6). Both HR and LR HPV E6 proteins inactivate the transcription regulatory activities of another major tumor suppressor, p53 (7-10), abrogating its control over cell cycle ar...