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

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.

Papillomaviruses in ruminants: An update

“…Due to the complex and the difficulty of tissue culture systems for the in vitro propagation of the PV (Bienkowska‐haba et al., ), detailed genetic and molecular information has been accumulated, especially in human and bovine papillomavirus, using cloning and Sanger sequencing. Lately, the new technology of high efficiency sequencing has enabled the characterization of new and putative new BPVs (Da Silva et al., ; Daudt, da Silva, Streck, et al., ; Munday et al., ).…”

Air‐liquid interface cell culture: From airway epithelium to the female reproductive tract

“…The HPV life cycle is dependent on the terminal differentiation process of keratinocytes. Therefore, organotypic rafts have been used to analyze HPV infection, regulation of virus propagation and oncogenesis (Bienkowska-Haba et al, 2018;Meyers & Laimins, 1994). They also proved useful to study virus-host cell interactions for other viruses such as herpes-, adeno-, parvo-, and poxviruses (reviewed in Andrei, Duraffour, Van den Oord, & Snoeck, 2010).…”

An epithelial organoid model with Langerhans cells for assessing virus-host interactions

“…While many past studies have focused on the cultivation of epithelial organoids (figure 1), including detailed methodology papers that serve as a good foundation and provide more thorough descriptions of the process, as well as video tutorials [4], it will now be important to expand the perspective to different experimental design considerations (such as multi-component rafts) and a variety of endpoints and time-courses. Recently, novel methods for studying the HPV life cycle have been described elsewhere [57].…”