It is established that the host cell transcriptomes of natural lesions, organotypic rafts, and human papillomavirus (HPV)-immortalized keratinocytes are altered in the presence of HPV genomes. However, the establishment of HPV-harboring cell lines requires selection and immortalization, which makes it impossible to distinguish between alterations directly induced by HPV or indirectly by the need for immortalization or selection. To address direct effects of HPV infection on the host cell transcriptome, we have used our recently established infection model that allows efficient infection of primary keratinocytes with HPV16 virions. We observed only a small set of genes to be deregulated at the transcriptional level at 7 days postinfection (dpi), most of which fall into the category regulated by pocket proteins pRb, p107, and p130. Furthermore, cell cycle genes were not deregulated in cells infected with a virus lacking E7 despite the presence of episomal genome and viral transcripts. These findings imply that the majority of transcriptional changes are due to the E7 protein impairing pocket protein function. Additional pathways, such as the Fanconi anemia-BRCA pathway, became perturbed only after long-term culturing of infected cells. When grown as organotypic raft cultures, keratinocytes infected with wild-type but not E7 mutant virus had perturbed transcriptional regulation of pathways previously identified in natural lesions and in rafts derived from immortalized keratinocytes. We conclude that the HPV infection model provides a valuable tool to distinguish immediate transcriptional alterations from those induced by persistent infection and the need for selection and immortalization.IMPORTANCE To establish infection and complete the viral life cycle, human papillomavirus (HPV) needs to alter the transcriptional program of host cells. Until recently, studies were restricted to keratinocyte-derived cell lines immortalized by HPV due to the lack of experimental systems to efficiently infect primary keratinocytes. Need for selection and immortalization made it impossible to distinguish between alterations induced by HPV and secondary adaptation due to selection and immortalization. With our recent establishment of an extracellular matrix (ECM)-to-cell transfer system allowing efficient infection of primary keratinocytes, we were able to identify transcriptional changes attributable to HPV16 infection. Most perturbed genes fall into the class of S-phase genes, which are regulated by pocket proteins. Indeed, infection with viruses lacking E7 abrogated most transcriptional changes. It is important to note that many transcriptional alterations thought to be important for the HPV life cycle are actually late events that may reflect immortalization and, possibly, disease progression.