MUTATIONAL ANALYSES OF DIFFERENTIATION-DEPENDENT HUMAN PAPILLOMAVIRUSTYPE-18 ENHANCER ELEMENTS IN EPITHELIAL RAFT CULTURES OF NEONATAL FORESKIN KERATINOCYTES

Human papillomaviruses (HPVs) reproduce only in differentiated squamou s epithelia, Viral transcription is rather restricted in basal strata but increases dramatically in the spinous cells, Inopportune viral onc oprotein expression in the basal reserve cells can lead to dysplasias and carcinomas, Until now all studies to identify transcription factor binding sites within the upstream regulatory region (URR) that contro ls the expression of the oncogene have been conducted in proliferating cell cultures, We report the establishment of a reproducible and conv enient system to examine cis elements important for differentiation-de pendent transcriptional regulation. The bacterial lacZ gene under the control of the HPV URR-E6 promoter was transduced into primary human k eratinocytes from neonatal foreskin by using high titer recombinant re troviruses. Acutely infected PHKs were then grown into stratified and differentiated epithelium on collagen rafts. lacZ expression was almos t entirely restricted to the spinous cells, indicating that promoter a ctivity was differentiation dependent, as seen in vivo. Using this sys tem, we initiated a mutational analysis of previously identified promo ter and enhancer elements within the HPV-18 URR. Three categories of m utations were observed: those that caused severe, moderate, or very sm all reduction in lacZ expression. The results show both similarities a nd differences to previously published and present studies in prolifer ating primary human keratinocytes in monolayer cultures or in immortal ized or transformed cell lines, This system is applicable to study bot h host and viral promoters that require squamous differentiation for t heir activity.