Robust production and passaging of infectious HPV in squamous epithelium of primary human keratinocytes.
Using Cre-loxP-mediated recombination, we established a highly efficient and reproducible system that generates autonomous HPV-18 genomes in primary human keratinocytes (PHKs), the organotypic raft cultures of which recapitulated a robust productive program. While E7 promoted S-phase re-entry in numerous suprabasal differentiated cells, HPV DNA unexpectedly amplified following a prolonged G2 arrest in mid- and upper spinous cells. As viral DNA levels intensified, E7 activity diminished and then extinguished. These cells then exited the cell cycle to undergo virion morphogenesis. High titers of progeny virus generated an indistinguishable productive infection in naïve PHK raft cultures as before, never before achieved until now. An immortalization-defective HPV-18 E6 mutant genome was also characterized for the first time. Numerous cells accumulated p53 protein, without inducing apoptosis, but the productive program was severely curtailed. Complementation of mutant genomes by E6-expressing retrovirus restored proper degradation of p53 as well as viral DNA amplification and L1 production. This system will be invaluable for HPV genetic dissection and serves as a faithful ex vivo model for investigating infections and interventions.
Wang
Hsu-Kun
HK
Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, Alabama 35294, USA.
Duffy
Aaron A
AA
Broker
Thomas R
TR
Chow
Louise T
LT
D017209:Apoptosis; D018929:Cell Culture Techniques / Q000379:methods*; D002478:Cells, Cultured; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000378:metabolism; D004848:Epithelium / Q000821:virology; D016195:G2 Phase; D016679:Genome, Viral / Q000235:genetics; D052161:Human papillomavirus 18 / Q000235:genetics / Q000502:physiology* / Q000648:ultrastructure; D006801:Humans; D015603:Keratinocytes / Q000166:cytology / Q000821:virology*; D046529:Microscopy, Electron, Transmission; D009856:Oncogene Proteins, Viral / Q000378:metabolism; D030361:Papillomavirus Infections / Q000821:virology*; D010957:Plasmids / Q000235:genetics; D013997:Time Factors; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism; D014771:Virion / Q000378:metabolism / Q000648:ultrastructure; D014779:Virus Replication / Q000502:physiology*
D004268:DNA-Binding Proteins; C052633:E7 protein, Human papillomavirus type 18; D009856:Oncogene Proteins, Viral; D016159:Tumor Suppressor Protein p53