In vitro generation of HPV 16 infectious virions: A tool to study the HPV 16 life cycle

Human papillomaviruses (HPVs) are small double-stranded DNA viruses of approximately 8kb.  Out of the 100 HPV types identified, all are known to infect epithelial cells.  A subgroup of HPVs are known to infect anogenital epithelium, including that of the cervix, vulva and anus.  A subset of these are referred to as oncogenic HPV types due to their association with cancers.  HPV16 specifically is associated with 50% of all cervical cancers and 70% of all anal cancers.   HPV-16 is known to first infect basal cells and complete its life cycle through the fully differentiated layers of the epithelium.  We hypothesize that HPV can also initiate infection and complete its life cycle in the more differentiated layers, thus representing an alternative life cycle in the epithelium. To test this, HPV 16 infectious virions were first generated by co-transfecting 293FT cells with a plasmid carrying the HPV-16 L1/L2 capsid genes and the full-length circular HPV 16 genome. To determine if the HPV16 virions were infectious, the harvested virions were used to infect monolayer HaCat foreskin keratinocytes.  Transcripts for the E6 HPV 16 early gene were detected in HaCat cells by RT-PCR.  E6 expression was not detected following incubation of HPV 16 virions with HPV 16-specific neutralizing antibodies prior to HaCat infection.  These results demonstrate that we were successful in creating infectious HPV 16 virions.  Future work will involve the infection of in vitro polarized and three-dimensional epithelial models with HPV 16 infectious virions, which will allow us to investigate alternative HPV-16 life cycles.