Cell-type-specific separate regulation of the E6 and E7 promoters of human papillomavirus type 6a by the viral transcription factor E2

Abstract

Gene expression of human papillomaviruses (HPV) is tightly controlled by cellular factors and by the virally encoded E2 protein through binding to distinct sites within the regulatory noncoding region. While for the high-risk genital papillomaviruses a single promoter drives the expression of all early genes, a second promoter present in the E6 open reading frame of the low-risk HPV type 6 (HPV6) would allow an independent regulation of E6 and E7 oncogene expression. In this report, we provide the first evidence that E2 regulates both early promoters of HPV6 separately and we show that promoter usage as well as E2 regulation is cell type dependent. Among the different epithelial cell lines tested, only RTS3b cells allowed an expression pattern similar to that observed in naturally infected benign condylomas. While the E6 promoter was repressed by E2 to 50% of its basal activity, the E7 promoter was simultaneously stimulated up to fivefold. Activation of the E7 promoter was mediated predominantly by the binding of E2 to the most promoter-distal E2 binding site. Repression of the E6 promoter depended on the presence of two intact promoter-proximal binding sites. Mutation of both of these repressor binding sites reversed the effect of E2 on the E6 promoter from repression to activation. In contrast, in HT3 cells we observed an E2-mediated activation of the E6 promoter in the context of the wild-type noncoding region. This indicated that repression of the E6 promoter by binding of E2 to both promoterproximal binding sites did not function in the cellular environment provided by HT3 cells. These data suggest that the separate regulation of the E6 and E7 promoters of HPV6 is mediated through successive occupation of binding sites with different affinities for E2 depending on the intracellular concentration of E2 and on the cellular environment provided by the infected cell.

Methylation status of HPV16 E2‐binding sites classifies subtypes of HPV‐associated oropharyngeal cancers

“…With consequently rising concentrations, E2 also binds to the lowaffinity proximal E2BS3 and E2BS4. This represses transcription from the p97 promoter and thereby maintains the levels of E6 and E7 at a low and autoregulated level as shown in Figure 1A 9,10,12 and as reviewed by von Knebel Doeberitz and Vinokurova 11 (note that the numbering of E2BSs in the study by von Knebel Doeberitz and Vinokurova 11 differs from that currently used).…”

“…7 The most important transcriptional regulator of E6 and E7 oncogene expression is the viral E2 protein, which binds to 4 E2-binding sites (E2BSs1, 2, 3, and 4) in the URR. 8 The current model of E2 function in the host cells during the normal viral life cycle implies that E2 binds with high affinity to the E2BS1, resulting in activation of the p97 early promoter even at low E2 concentrations, 9,10 as reviewed by von Knebel Doeberitz and Vinokurova. 11 This promoter activation results in enhanced expression of the early viral proteins, including the oncoproteins E6 and E7 and E2 itself.…”

Differential methylation of E2 binding sites in episomal and integrated HPV 16 genomes in preinvasive and invasive cervical lesions

“…If the concentration of the E2 protein rises beyond a certain level, E2 binds also to the low affinity proximal E2BSs (E2BS2, 3 and 4) and represses transcription from the early promoter p97 through displacement of Sp1 and TBP from their respective binding sites, thereby keeping the levels of E6 and E7 under control. [11][12][13] Accordingly, it has been suggested that deregulated expression of E6 and E7 oncogenes and initiation of the transformation process may result from loss of the E2 repressive functions. Viral integration into the host genome usually occurs downstream of the E6 and E7 genes, often in E1 and E2 regions 14,15 and results in a loss of negative-feedback control of oncogene expression.…”

HPV 16 E2 binding sites 1 and 2 become more methylated than E2 binding site 4 during cervical carcinogenesis

“…The binding of E2 protein to the different E2 binding sites will exert either transcriptional activation or repression on the E6/E7 promoter, depending on their distance from the TATA box of the promoter [Dostatni et al, ]. Binding to E2BS4 (the E2 binding site distal to TATA box of E6/E7 promoter) results in transcriptional activation whereas binding to E2BS1 or E2BS2 (the E2 binding sites proximal to TATA box of E6/E7 promoter) results in transcriptional repression through displacement of Sp1 and TBP from their binding sites [Dong et al, ; Demeret et al, ; Rapp et al, ; Steger and Corbach, ; Soeda et al, ]. Hence, at low concentration of E2, it is expected that E2 protein will bind to E2BS4, exerting transcriptional activation on the E6/E7 promoter and when the E2 protein concentration increases, more E2 protein will bind to E2BS1&2 relative to E2BS4, resulting in transcriptional repression [Doeberitz and Vinokurova, ].…”