Productive replication of human papillomavirus type 16 (HPV16) occurs only in differentiated keratinocyte cells. In addition to the viral E2 activator protein, HPV16 and related HPV types express transcripts coding for an E8^E2C fusion protein, which limits genome replication in undifferentiated keratinocytes. To address E8^E2C's role in productive replication of HPV16, stable keratinocyte cell lines containing wild-type (wt), E8^E2C knockout (E8؊), or E8 KWK mutant (mt) genomes, in which conserved E8 residues were inactivated, were established. Copy numbers of E8؊ and E8 KWK mt genomes and amounts of early and late viral transcripts were greatly increased compared to those for the wt in undifferentiated keratinocytes, suggesting that HPV16 E8^E2C activities are highly dependent upon the E8 part. Upon differentiation in organotypic cultures, E8 mt genomes displayed higher early viral transcript levels, but no changes in cellular differentiation or virus-induced cellular DNA replication in suprabasal cells were observed. E8 mt genomes were amplified to higher copy numbers and showed increased L1 transcripts compared to wt genomes. Furthermore, the number of cells expressing the viral late protein E4 or L1 or amplifying viral genomes was greatly increased in E8 mt cell lines. In wild-type cells, E8^E2C transcript levels did not decrease by differentiation. Our data indicate that the E8^E2C repressor limits viral transcription and replication throughout the complete life cycle of HPV16. P ersistent infections with high-risk (HR) human papillomaviruses (HPV) are a necessary risk factor for the development of cancer of the cervix uteri and are also responsible for a fraction of anal, vaginal, penile, and oropharyngeal cancers (1-3). Notably, HPV16 is an extremely powerful human carcinogen that is responsible for approximately 50% of cervical cancers and is also represented in other HR-HPV-related cancers at high frequencies (2).HPV have a double-stranded DNA genome that is covalently closed and organized in nucleosomes. HPV possess eight to nine open reading frames (E1, E2, E4 to E8, L1, and L2) that are transcribed into polycistronic messages which are further processed by alternative splicing (4). It is currently assumed that HR-HPV encode at least 10 different viral proteins.The HR-HPV replication cycle is tightly linked to the differentiation state of the infected epithelium. In undifferentiated basalcell-like cells, HR-HPV genomes are maintained in the nucleus at 10 to 100 extrachromosomal copies per cell, and only early genes, mainly transcribed from the major early viral promoter located immediately upstream of the E6 gene (P97 for HPV16), but not the late viral L1 and L2 genes are expressed (5). Replication of the viral genome in undifferentiated cells requires the viral E1 and E2 proteins (6). E1 and E2 are sequence-specific DNA binding proteins which form a complex that recognizes the viral origin of replication (6). E1 assembles into a hexamer that acts as the replicative helicase and unwinds the viral DNA...