| Infections by the high-risk human papillomavirus type 18 (HPV-18) or type 16 (HPV-16) can lead to cancers of the anogenital tract. Because the propagation of HPVs is restricted to differentiated human squamous epithelia, experimental studies of the productive program are conducted in organotypic cultures. The current methods are of low efficiency and time consuming and have relied on immortalized epithelial cell lines that do not support efficient viral DNA amplification or virion production, compromising HPV genetic analyses and obscuring normal virus-host cell interactions. I used Cre-loxP site-specific recombination to develop a relatively simple, fast and highly efficient system to produce HPV-18 genomic plasmids in primary human keratinocytes (PHKs). The transfected primary human keratinocytes can be quickly developed into organotypic raft cultures, obviating the need for immortalized cells or the immortalization function of the high-risk HPV types. Upon keratinocyte differentiation and early viral gene expression, the HPV-18 replicons amplified to high copy numbers in raft cultures. The major capsid protein (L1) expression was widely detected in superficial cells and in cornified layers on and after day 14 or 16. Electron microscopy examination revealed HPV-18 particles were packed in paracryatalling arrays in shrunken nuclei with condensed chromatins. These virions infected PHKs efficiently and were passaged in fresh PHK raft cultures, a feat not achieved until now. Thus, this system should be a useful tool to study HPV mutants or low-risk HPV genotypes. I further verified the utility of this system in HPV genetic analyses. Indeed, I was able to analyze the phenotypes of the HPV-18 E6*I and E6-null mutant replicons which had not been studied previously in raft cultures due to their inability to immortalize PHKs or failure to be maintained in transfected PHKs as plasmids. The mutant genomes were highly deficient in L1 production but were complemented in trans by a retrovirus expressing wild type HPV-18 E6. In support of translating basic scientific capabilities into clinical outcomes, this system should also be highly informative for testing agents that can prevent or treat human papillomavirus infections.;Keywords. Organotypic raft cultures of primary human keratinocytes, Cre-loxP site specific recombination, HPV-18 infection cycle, viral DNA amplification, HPV-18 E6*I and E6-null mutants, genetic complementation in trans.. |
