Human papillomavirus type 16 E2 protein has no effect on transcription from episomal viral DNA

Abstract

The human papillomavirus (HPV) E2 protein plays an important role in viral DNA replication. Many studies with high-risk HPVs have demonstrated that the E2 protein can also repress transcription of the E6 and E7 oncogenes. This conclusion, based on experiments carried out with cervical cancer cells bearing integrated HPV genomes, is currently assumed to be applicable to the normal HPV life cycle, in which the viral genomes are episomal. Here, we have tested experimentally whether this assumption is correct. We made use of a pair of isogenic cell lines, W12 and S12. W12 cells contain episomal HPV16 genomes, whereas S12 cells, which are derived from the W12 line, contain HPV DNA as integrated copies. When we expressed E2 in S12 cells, we observed strong repression of E6 and E7 transcription. In contrast, no effect of E2 on the transcription of these genes was detected in W12 cells. While integration of the viral genome into the host DNA contributes to the difference between W12 and S12 cells, integration by itself is not sufficient to explain this difference. Instead, the chromatin structure in the region of the E6 and E7 promoter (p97), which we show to be very different in these two cell lines, is likely to be the cause of the different responsiveness of p97 to the E2 protein. Experiments with the histone deacetylase inhibitor trichostatin A (TSA) indicated that the episomal HPV16 DNA is in a relatively inaccessible state prior to TSA treatment. Our results, together with those of others, suggest that any effect of the E2 protein on the expression of the E6 and E7 genes during the normal viral life cycle is of secondary importance compared to the function of E2 in replication.

FIG. 1.

(A) Southern blot analysis of undigested HPV16 DNA from W12 cells after total DNA extraction. W12 cell DNA was compared to a supercoiled DNA marker (scDNA marker). (B) Southern blot analysis of HPV16 episomes isolated by the Hirt extraction method from W12 and S12 cells and digested with BamHI. (C) Southern blot analysis of HPV16 DNA in W12 and S12 cells. DNA was extracted from 6 × 10⁶ cells, and equal amounts of total DNA were digested with BamHI or PstI and subjected to electrophoresis and hybridization using a ³²P-labeled HPV16 specific DNA probe.

FIG. 2.

HPV16 E2 protein levels in W12 and S12 cells after nuclear and cytoplasmic extraction analyzed by Western blotting. Noninfected and rAd5-infected cell extracts were compared to rAdE2-infected cell extracts used as positive control for the presence of the E2 protein at 48 h postinfection.

FIG. 3.

Effects of expression of E2 on the E6 and E7 transcription in S12 and W12 cells (A) and CIN612 9E cells (B). At 48 h postinfection, RNA was isolated and equal amounts were analyzed by Northern blotting with a ³²P-labeled HPV31 E6-E7-specific DNA probe.

FIG. 4.

pRb and p53 proteins in rAd5- and rAdE2-infected cells analyzed by Western blotting at 48 h postinfection.

FIG. 5.

Effects of HPV16 E2 protein on the integrated p97 promoter. Different HaCat clones containing stably integrated pGl3b 822.4 plasmids were infected with rAd5 and rAdE2. Activity of the HPV16 p97 promoter was determined by luciferase assay and normalized according to the control samples infected with rAd5.

FIG. 6.

E6 and E7 RNA levels in W12 and S12 cells and L1 RNA levels in W12 cells after treatment with increasing amounts of TSA. RNA was harvested, and equal amounts were analyzed by Northern blotting with a ³²P-labeled HPV16 E6- and E7-specific probe or a ³²P-labeled HPV16 L1-specific DNA probe. A ³²P-labeled β-actin DNA sequence was used as control.