Down Regulation of the Interleukin-8 Promoter by Human Papillomavirus Type 16 E6 and E7 through Effects on CREB Binding Protein/p300 and P/CAF

Human papillomaviruses (HPVs) are small DNA tumor viruses which exhibit distinct tropism for stratified epithelia in different parts of the body. Several genotypes infecting genital mucosa are known to cause cancers, and among them types 16, 18, 31, and 33 are found in more than 90% of cervical cancers. Two major viral proteins, E6 and E7, are found to exert a significant impact on cellular control mechanisms. E6 of HPV type 16 is 151 amino acids (aa) long with two zinc fingers comprised of four Cys-X-X-Cys motifs between aa 30 to 56 and 103 to 139. E6 has been shown to bind to p53 and promote p53 degradation through a ubiquitin-dependent pathway (63). It has also been demonstrated that E6 of HPV type 16 can bind to coactivators CBP and p300 and disrupt CBP/p300-dependent transcription (57, 83). E6 from high-risk HPV alone is sufficient to transform mammary epithelial cells (64), though efficient immortalization of primary human keratinocytes requires both E6 and E7 (25). E7 of HPV type 16 consists of 98 aa with an approximate molecular size of 13 kDa. It is comprised of three conserved regions, namely CR1, CR2, and CR3. Binding partners of E7 consist of many important regulatory proteins and transcription factors, such as the pRb family and AP-1 factors (2-4, 11, 47, 67). It has been previously determined that E7 has an important role during the G1 phase of the cell cycle and in facilitating cells to overcome the G1/S boundary (10, 24, 58). The coactivator p300 was first identified through its direct interaction with adenovirus E1A protein (12, 80; Z. Arany, W. R. Sellers, D. M. Livingston, and R. Eckner, Letter, Cell 77:799-800, 1994). Its homologue, CBP (CREB binding protein), was initially described as a binding partner of the phosphorylated form of the cyclic AMP-responsive transcription factor CREB (8). There are several distinct regions within the protein sequence of CBP and p300 (20, 66). For example, two cystine-histidine-rich regions (C/H1 and C/H3) and a C-terminal domain adjacent to the C/H3 domain contribute to a large extent to the binding of CBP/p300 to transcription factors, basal transcription machinery elements, and viral proteins such as E1A and simian virus 40 large T protein and other cofactors (8, 9, 12-14, 19, 27, 37, 53, 61, 66, 81). The extreme N terminus binds to nuclear receptors such as retinoic acid receptor and estrogen receptor (26, 29). Finally, an intrinsic histone acetyltransferase (HAT) domain also exists between the C/H2 and C/H3 domains. Both CBP and p300 are essential components of the transcription machinery. Firstly, CBP/p300 binds directly to both the basal transcription machinery and to some transcription factors, serving as a bridge to connect the two major elements of transcription (1, 33, 48). Secondly, as well as having intrinsic HAT activity, CBP/p300 can also recruit other HATs, such as the p300/CBP-associated factor (P/CAF), to modify the tail of histone proteins forming the nucleosome by adding an acetyl group. This will eventually lead to the uncoiling of chromatin structure and facilitate the entry of transcription factors to promoter regions (75). Finally, by utilizing either their own HAT domain or recruited HAT, CBP and p300 are able to acetylate transcription factors directly, resulting in optimal transcription (6, 21, 40, 60, 68). One of the transcription factors activated by CBP/p300 is RelA/p65, which belongs to the NF-κB family. NF-κB transcriptional activation is essential for the synthesis of a variety of cytokines, chemokines, and inflammatory effectors which are crucial in initiating immune responses against viral or bacterial infections. Therefore, it would be beneficial for an infectious agent to evade the host immune system by down-regulating NF-κB transcriptional activation. Such disruption of NF-κB transcriptional activation can occur at several levels of NF-κB regulation. For instance, some enteric organisms have been shown to elicit immunosuppressive effects by down-regulating the synthesis of inflammatory effectors by preventing IκB-α degradation, thus avoiding the subsequent translocation of NF-κB family members to the nucleus (51). The interference of NF-κB transcription can also take place at the formation of the transcriptional complex. For example, SNIP1, a nuclear protein shown to be an inhibitor of the transforming growth factor β signal transduction pathway, inhibits NF-κB signaling by competing with RelA/p65 for binding to the C/H1 domain of CBP/p300 (28). It is now known that SRC-1 and P/CAF, along with CBP/p300, are required for the transcriptional activation by RelA/p65 and are recruited to an NF-κB-responsive promoter (65). Therefore, CBP/p300 serves as a platform to recruit these essential components to facilitate the transcription by p65. However, as shown recently by Chen et al. (7), the level of NF-κB transcriptional regulation is thought to be more complicated. They demonstrated for the first time that p65 is acetylated and that CBP/p300 and histone deacetylase-3 regulate the acetylation state of p65, which in turn determines the nuclear localization of p65 (7). As mentioned above, NF-κB is involved in the transcriptional control of a number of cytokines and chemokines, including interleukin-8 (IL-8). IL-8 belongs to the CXC chemokine family and was originally identified as a potent activator and chemoattractant for neutrophils, basophils, and T lymphocytes (56), although T lymphocytes have been shown to be much more sensitive to IL-8 chemotaxis than neutrophils (34). IL-8 is secreted predominantly as a 72-aa protein and is strongly induced during viral infection and bacterial invasion. Moreover, IL-8 is secreted in a stimulus-specific manner, such as treatment with tumor necrosis factor alpha (TNF-α), by a wide variety of cell types, including keratinocytes, and is regulated at the level of gene transcription (16, 49, 69, 70, 76). The transcriptional control of IL-8 is mediated by the 100 bp of the 5′-flanking sequence upstream of the TATA box in its promoter. This region contains cis-acting elements for the binding of AP-1 factors, CCAAT/enhancer binding protein β (C/EBP-β), and NF-κB transcription factors. Previous reports have demonstrated that the IL-8 promoter is predominantly activated by the induction of NF-κB complex containing p65, although AP-1 factors and C/EBP-β may also play supporting roles (31, 32, 46, 55, 71). HPVs may persist in their host for months, years, or even decades. To achieve this, the virus must have mechanisms to avoid inducing an effective immune response. Since we have previously shown that HPV-16 E6 inhibits the activity of an NF-κB consensus promoter (57), we explored the possibility that HPV-16 E6 down-regulates a more physiologically relevant NF-κB-induced promoter. In view of the fact that many cytokine and chemokine genes are regulated by NF-κB, we have chosen the promoter of IL-8, a chemokine predominantly regulated by NF-κB and induced by viral or bacterial infection in keratinocytes, as the subject of this study. In addition, since E7 is also known to modulate transcription and is synthesized concurrently with HPV-16 E6 during the course of viral infection, we also examined the effect of HPV-16 E7 on the IL-8 promoter. In this report, we show that E6 and E7 of HPV type 16 cooperatively repress IL-8 transcription. Furthermore, we offer a potential mechanism by which these viral proteins disrupt optimal NF-κB transcriptional activation, and the implications of these findings may help us understand how papillomaviruses cause persistent infections.