Control of HPV 18 DNA replication by cellular and viral transcription factors

Abstract

Papillomavirus replication in vivo requires the interaction of the virally encoded proteins El and E2 with the origin of replication which is localised in the regulatory region (long control region or LCR) of the viral genome. In genital human papillomaviruses (HPVs), the origin overlaps promoter elements of early transcription. In this study, we analysed the replication of HPV18 DNA using the complete LCR containing mutations in transcription regulatory elements. We found that each of the three E2 binding sites proximal to the AT-rich sequence of the origin contributes to the replication rate of DNA, although not identically. In addition, two sequences important for early transcription, an Spi binding site and the TATA box, were also found to play a role in replication. In contrast, two AP1 binding sites required for the enhancer-mediated activation of early transcription did not affect the replication, while other upstream sequences in the LCR did contribute to the replication efficiency. Our results indicate that besides a core origin of replication containing an AT-rich sequence and three E2 binding sites, auxiliary elements affect HPVl 8 DNA replication in the context of the full length LCR, some of which are important for transcription.

Papillomavirus E2 induces p53-independent apoptosis in HeLa cells

“…For replication experiments an E1 expression plasmid was constructed from the originally described E1/E2 expression plasmid EapCG (Demeret et al, 1995) by inserting a TTL (Translation Termination Linker) in the restriction sites SexA1 in the E2 ORF, and Blp1 in the E7 ORF. The origin-containing plasmid, poril8, was described in (Demeret et al, 1995).…”

“…Replication assays were performed as previously described in (Demeret et al, 1995). Brie¯y, 2 mg of the pori18 plasmid were cotransfected with 2 mg of the E1 expression plasmid and 1 mg of plasmids expressing either wt or mutant E2 proteins in 293 cells.…”

Mitotic Chromosome-Binding Activity of Latency-Associated Nuclear Antigen 1 Is Required for DNA Replication from Terminal Repeat Sequence of Kaposi's Sarcoma-Associated Herpesvirus

“…In the case of papillomavirus a viral helicase, E1 (67), in concert with a viral auxiliary protein, E2, unwinds the viral origin of replication and initiates DNA synthesis (13,17,18,62). However, long-term maintenance of a viral oriP-con- FIG.…”

Point mutations in SP1 motifs in the upstream regulatory region of human papillomavirus type 18 isolates from cervical cancers increase promoter activity.

“…In an extension of this work presented here, the functional significance of two naturally occurring mutations in the Sp1 site proximal to the HPV-18 E6\E7 promoter was assessed. Sp1 is a key activator of RNA polymerase II dependent promoters in HPV (HoppeSeyler & Butz, 1992) as in other systems (Kadonga et al, 1986), and has been recently identified as an accessory element in papillomavirus replication (Demeret et al, 1995).…”

“…The viral E2 protein plays an important role in promoter repression, through binding to its two recognition sites (BS1 and BS2) in the promoter proximal region of the HPV-18 URR (Steger & Corbach, 1997). The binding of E2 protein to these sites has been shown in vitro to interfere with the binding of Sp1 to its cognate sequence lying nearby (Demeret et al, 1995). This suggests that stronger binding of Sp1 may decrease the ability of the E2 protein to repress promoter activity.…”