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Methods in molecular biology (Clifton, N.J.)

Human keratinocyte cultures in the investigation of early steps of human papillomavirus infection.


PMID 24281871

Abstract

Human papillomaviruses (HPVs) are non-enveloped DNA viruses that are highly tropic for mucosal and cutaneous epithelia. The HPV life cycle is tightly linked to epithelial cell differentiation, where HPVs only infect the basal proliferating keratinocytes, and progeny virus assembly and release only occurs in differentiated upper-layer keratinocytes. Therefore, human keratinocyte monolayer cultures provide a useful model to study the early stages of HPV infection. However, previous reports have shown some conflicting results of virus-host interactions during HPV entry, which may be partly attributable to the different cell culture models used to examine these steps of HPV infection. Thus, there is a need to have a standardized in vitro model system to study virus-host interactions during HPV entry. Here, we describe the three most widely accepted keratinocyte models for studying HPV infection: primary human foreskin keratinocytes, normal immortalized keratinocytes, and transformed HaCaT keratinocytes. We also describe methods to genetically manipulate these cells, enabling the study of candidate host genes that may be important during HPV infection. Lastly, we outline simple and robust methods to assay HPV infectivity, which can be used to determine whether knockdown or overexpression of a particular gene affects HPV entry.