A new cell culture model to genetically dissect the complete human papillomavirus life cycle

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Abstract
Herein, we describe a novel infection model that achieves highly efficient infection of primary keratinocytes with human papillomavirus type 16 (HPV16). This cell culture model does not depend on immortalization and is amenable to extensive genetic analyses. In monolayer cell culture, the early but not late promoter was active and yielded a spliced viral transcript pattern similar to HPV16-immortalized keratinocytes. However, relative levels of the E8^E2 transcript increased over time post infection suggesting the expression of this viral repressor is regulated independently of other early proteins and that it may be important for the shift from the establishment to the maintenance phase of the viral life cycle. Both the early and the late promoter were strongly activated when infected cells were subjected to differentiation by growth in methylcellulose. When grown as organotypic raft cultures, HPV16-infected cells expressed late E1^E4 and L1 proteins and replication foci were detected, suggesting that they supported the completion of the viral life cycle. As a proof of principle that the infection system may be used for genetic dissection of viral factors, we analyzed E1, E6 and E7 translation termination linker mutant virus for establishment of infection and genome maintenance. E1 but not E6 and E7 was essential to establish infection. Furthermore, E6 but not E7 was required for episomal genome maintenance. Primary keratinocytes infected with wild type HPV16 immortalized, whereas keratinocytes infected with E6 and E7 knockout virus began to senesce 25 to 35 days post infection. The novel infection model provides a powerful genetic tool to study the role of viral proteins throughout the viral life cycle but especially for immediate early events and enables us to compare low- and high-risk HPV types in the context of infection. Current cell culture models for the study of the human papillomavirus (HPV) life cycle depend on immortalized keratinocytes harboring episomal HPV genomes. However, the requirement for immortalization restricts the study to only a few HPV types and does not allow investigating immediate early events of the viral life cycle. Despite many efforts, efficient infection of primary keratinocytes has not been achieved until now. Using pre-binding of virus to extracellular matrix deposited by keratinocytes, we now achieve very efficient infection of primary keratinocytes. The infection model allows studying the complete viral lifecycle. It could be extended to HPV types that do not immortalize keratinocytes and allows an extensive genetic screen of the contributions of viral factors throughout the viral lifecycle. It should aid the investigations of processes leading to HPV-induced immortalization.
Funding Information
  • National Institute of Allergy and Infectious Diseases (R01AI081809)
  • National Institute of Allergy and Infectious Diseases (R01AI118904)
  • National Cancer Institute (R01CA211576)
  • National Institute of Dental and Craniofacial Research (R01DE025565)
  • National Institute of General Medical Sciences (P30GM110703)
  • Feist-Weiller Cancer Center

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