Induction of the Human Papillomavirus Type 31 Late Promoter Requires Differentiation but Not DNA Amplification

Abstract

The human papillomavirus (HPV) life cycle is linked to the differentiation state of the host cell. In virus-infected undifferentiated basal epithelial cells, HPV genomes are maintained as episomes at low copy number. Upon differentiation, a concomitant increase in viral copy number and an induction of late gene expression from a differentiation-specific promoter is seen. To investigate whether late gene expression was dependent on the amplification of the viral genome, inhibitors of DNA replication and in vitro systems for epithelial differentiation were used in conjunction with cells that stably maintain HPV31 episomes. Treatment of cells induced to differentiate in methylcellulose with the DNA synthesis inhibitor cytosine ␤-arabinofuranoside (AraC) blocked viral DNA amplification but did not prevent induction of late transcription. This suggests that late gene expression does not strictly require amplification of the viral genome and that differentiation signals alone are sufficient to activate transcription from the late promoter. However, DNA amplification does appear to be necessary for maximal induction of the late promoter. In order to examine the cis-acting elements that contribute to the activation of the late promoter, a transient reporter assay was developed. In these assays, an induction of late gene expression was seen upon differentiation that was specific to the late promoter. Mapping studies localized important regulatory elements to the E6/E7 region and identified short sequences that could serve as binding sites for transcription factors. Elements within the upstream regulatory region were also found to positively and negatively influence transcription from the late promoter. These results identify mechanisms important for the differentiation-dependent activation of late gene expression of high-risk papillomaviruses.

Robust production and passaging of infectious HPV in squamous epithelium of primary human keratinocytes

“…3D; data not shown). Accordingly, we suggest that HPV DNA must amplify to a high copy number for efficient late protein expression, contrary to a previous report (Spink and Laimins 2005). Rarely we detected faint L1 antigen in cells not also having high viral DNA.…”

A highly efficient system to produce infectious human papillomavirus: Elucidation of natural virus-host interactions

“…This conclusion is contrary to a previous report that late gene expression is dependent on squamous differentiation but does not require viral DNA replication. 9 As a routine, we expose the raft cultures to bromodeoxyuridine (BrdU) for 6-12 hrs immediately prior to harvest so that cells in S phase can be identified by the incorporation of BrdU into chromosomal DNA. Using tissue sections from time course experiments, we have determined a temporal order of the HPV productive program by fluorescence in situ hybridization detection of viral DNA amplification (DNA-FISH), concurrently with antibody detection of BrdU incorporation and E7-induced cellular proteins or viral proteins, as well as by DAPI staining of nuclei.…”

Differentiation-Dependent Changes in Levels of C/EBPβ Repressors and Activators Regulate Human Papillomavirus Type 31 Late Gene Expression

“…In HPV-31, the late promoter is referred to as p742 (17), while it is called p670 in HPV-16 (15). Differentiation of HPV-positive keratinocytes alone has been shown to be sufficient to activate low levels of late transcription, while genome amplification increases the levels of late gene expression through increased template numbers (5,28). In contrast to the information available about the regulators of the early promoter, significantly less is known about the transcription factors that control late gene expression.…”

“…We initially wanted to determine whether LAP was capable of activating the HPV-31 late promoter and to identify reactive sequences. For this analysis, we made use of a luciferase reporter containing sequences from the 5= end of the HPV-31 URR through the E6/E7 open reading frames (p31-luc-7045-891) (28,32). Previous studies have shown that in this construct, the p97 early promoter contributes minimally to late gene expression upon keratinocyte differentiation (28).…”

“…For this analysis, we made use of a luciferase reporter containing sequences from the 5= end of the HPV-31 URR through the E6/E7 open reading frames (p31-luc-7045-891) (28,32). Previous studies have shown that in this construct, the p97 early promoter contributes minimally to late gene expression upon keratinocyte differentiation (28). A series of epithelial cell lines, including C33A, HaCaT, 293T, and HPV-31-positive CIN-612 cells, were transfected with p31-luc-7045-891, in the presence or absence of an LAP expression plasmid, and assayed for luciferase activity 48 h posttransfection as described previously (28).…”