Regulatory interactions of transcription factor YY1 with control sequences of the E6 promoter of human papillomavirus type 8.

The varicella-zoster virus (VZV) origin of DNA replication (oriS) contains a 46-bp AT-rich palindrome and three consensus binding sites for the VZV origin binding protein (OBP) encoded by VZV ORF51. All three OBP binding sites are upstream of the palindrome in contrast to the sequence of the herpes simplex virus oriS, which has required OBP binding sites upstream and downstream of the AT-rich region. We are investigating the roles that sequences downstream of the palindrome play in VZV oriS-dependent DNA replication. Computer analysis identified two GC boxes, GC box 1 and GC box 2, in the downstream region which were predicted to be binding sites for the cellular transcription factor Sp1. Electrophoretic mobility shift assay and supershift assays showed that two members of the Sp family (Sp1 and Sp3) stably bind to GC box 1, but not to GC box 2. A predicted binding site for the cellular factor Yin Yang 1 (YY1) that overlaps with GC box 2 was also identified. Supershift and mutational analyses confirmed the binding of YY1 to this site. Mutation of GC box 1 resulted in loss of Sp1 and Sp3 binding and an increase in origin-dependent replication efficiency in DpnI replication assays. In contrast, mutation of the YY1 site had a statistically insignificant effect. These results suggest a model where origin-dependent DNA replication and viral transcription are coupled by the binding of Sp1 and Sp3 to the downstream region of the VZV replication origin during lytic infection. They may also have implications regarding establishment or reactivation of viral latency.Varicella-zoster virus (VZV) is a member of the Alphaherpesvirus subfamily of the Herpesviridae family. VZV is the causative agent of two types of disease, chicken pox (varicella) upon primary infection and shingles (zoster) after reactivation from latency (1, 2). The VZV genome consists of 125 kbp which encodes approximately 69 open reading frames (ORFs). The viral genome is made up of long and short unique segments designated U L and U S, respectively, both of which are bounded by inverted repeat sequences (11). The linear sequence of VZV genes is similar to that of herpes simplex virus type 1 (HSV-1) and includes the coding sequences for orthologues of the seven HSV-1 proteins required for origin-dependent DNA replication (9).The VZV genome contains two origins of DNA replication (oriS) within the internal repeats (IRs) and terminal repeats (TRs) bounding the U S segment at sites analogous to those within the HSV-1 genome. VZV, however, lacks the third HSV-1 origin (oriL), which is located near the center of the HSV-1 U L region (10,11,48,49). HSV-1 oriL has been shown to be dispensable for replication of HSV DNA (3, 53) but has been implicated in HSV pathogenesis and reactivation from latency (4). The equivalent region in the VZV genome is comprised of a bidirectional promoter that regulates the transcription of the VZV DNA polymerase catalytic subunit (ORF28) and DNA binding protein (ORF29) genes (32, 54).The architecture of the VZV and HSV-1 oriS regions ...

Section: Identification Of Potential Binding Sites For Cellular Factomentioning

confidence: 93%

Cellular Transcription Factors Sp1 and Sp3 Suppress Varicella-Zoster Virus Origin-Dependent DNA Replication

Khalil

Hay

Ruyechan

2008

“…This part of p300 and the homologous segment in CBP are recognized by a number of important transcriptional regulators, including p53 (2, 14, 26), c-fos (3,36), YY1, TFIIB, E2F, RNA helicase A, and p/CAF (6). A decreased recruitment of p300⌬1770-1814 due to the lack of the interaction domain for cellular DNA binding factors binding to the HPV 8NCR, such as the members of the AP1 complex, p53 and YY1 (1,16,37), or for components of the preinitiation complex may be the basis for this reduced activity of p300⌬1770-1814. Although the deletion of amino acids 1529 to 1572, located within the HAT domain and containing a second, here-identified low-affinity 8E6 binding site, affected p300 activity as well, it did not impair the coactivation by 8E6.…”

Section: Discussionmentioning

confidence: 99%

The E6 Protein of the Cutaneous Human Papillomavirus Type 8 Can Stimulate the Viral Early and Late Promoters by Distinct Mechanisms

Müller‐Schiffmann

Beckmann

Steger

2006

The expression of the proteins encoded by human papillomaviruses (HPVs) is tightly linked to the differentiation program of the infected keratinocytes. The late promoter, expressing the structural proteins, becomes activated in the differentiated keratinocytes, while the early promoter is also active in the basal layers. We have shown previously that the viral transcriptional regulator E2 and the cellular coactivator p300 cooperate in activation of gene expression of HPV8, which infects the skin and is associated with epidermodysplasia verruciformis. Here we demonstrate that this activation is further stimulated after overexpression of the E6 oncoprotein of HPV8 (8E6). RNase protection experiments revealed that 8E6 efficiently cooperates with 8E2 and p300 in activation of the late promoter. In addition, the early promoter, which did not respond to 8E2 and/or p300, was stimulated more than fourfold by 8E6. Our data suggest that both promoters are activated via distinct mechanisms, since the activation of the early promoter was achieved by the N-terminal moiety of 8E6; in contrast, its C-terminal half was sufficient for late promoter activation. This was markedly reduced by the deletion of amino acids 132 to 136 of 8E6, which also abolished the binding to p300, indicating that a direct interaction between 8E6 and p300 is involved. Moreover, a 45-amino-acid segment within the C/H3 region of p300 is required for 8E6 to stimulate the coactivator function of p300. Our results demonstrate for the first time that an E6 oncoprotein of HPV directly contributes to the regulation of HPV gene expression.Human papillomaviruses (HPVs) are small double-stranded DNA viruses causing a variety of benign hyperproliferative lesions of the skin or the mucosa. Infections with certain HPV types have a high risk of undergoing malignant progression. In more than 95% of cervical cancers, the DNA of genital highrisk types such as HPV16 and HPV18 can be found, indicating that infection with these types is a prerequisite for cancer progression (34). Among the HPVs infecting the skin, HPV5 and HPV8 are classified as high-risk types. This is based on the observation that HPV-induced skin lesions in epidermodysplasia verruciformis patients, which often develop into squamous cell carcinomas after a long latency preferred in sun exposed areas, mainly contain HPV5 or HPV8 DNA (41). The oncogenic potential of HPV8 was confirmed by the ability of the early HPV8 region, encoding the regulatory proteins, to induce benign and malignant skin tumors in transgenic mice (43). Additionally, there is limited evidence for the carcinogenicity of epidermodysplasia verruciformis-associated HPVs in nonmelanoma skin carcinomas of the immunocompetent population in general (40,41).The E6 and E7 proteins of mucosal HPVs transform cells by interfering with cell cycle regulation and counteracting apoptosis.

“…This implies that cellular factors binding to the NCR of HPV8 play a role in mediating cooperativity between 8 E2 and p300. The NCR of HPV8 encodes binding sites for a variety of cellular transcription factors, like AP1, NF1, YY1, RUNX1, and papillomavirus-binding factor (5,14,39,45). Most of these factors also use CBP/p300 as coactivators (26; reviewed in references 46 and 62).…”

Section: Discussionmentioning

confidence: 99%

Cooperative Activation of Human Papillomavirus Type 8 Gene Expression by the E2 Protein and the Cellular Coactivator p300

Müller

Ritzkowsky

Steger

2002

The E2 proteins of papillomaviruses (PV) bind to the coactivator CBP/p300 as do many other transcription factors, but the precise role of CBP/p300 in E2-specific functions is not yet understood. We show that the E2 protein of human PV type 8 (HPV8) directly binds to p300. Activation of HPV8 gene expression by low amounts of HPV8 E2 was stimulated up to sevenfold by coexpression of p300. The interaction between E2 and p300 may play a role in differentiation-dependent activation of PV gene expression, since we can show that the expression level of p300 increases during keratinocyte differentiation. Surprisingly, sequence-specific binding of E2 to its recognition sites within the regulatory region of HPV8 is not necessary for this cooperation, indicating that E2 can be recruited to the promoter via protein-protein interaction. HPV8 E2 binds via its N-terminal activation domain (AD), its C-terminal DNA binding domain (DBD), and its internal hinge region to p300 in vitro. Transient-transfection assays revealed that the AD is necessary and sufficient for cooperative activation with p300. However, we provide evidence that the interaction of the hinge and the DBD of HPV8 E2 with p300 may contribute. Our data suggest an important role of p300 in regulation of HPV8 gene expression and reveal a new mechanism by which E2 may be recruited to a promoter to activate transcription without sequence specific DNA binding.Papillomaviruses (PV) infect the basal cells of the skin or the mucosa, causing proliferative lesions like warts or dysplasias. PV require differentiating keratinocytes to replicate their DNA. The expression of the structural proteins is restricted to some of the most-differentiated keratinocytes. This cell tropism is due to the involvement of transcription factors specifically expressed in these cells. In addition to ubiquitously expressed and keratinocyte-specific cellular transcription factors, viral gene expression is also modulated by the viral E2 protein. The E2 protein binds via its carboxy (C)-terminal DNA binding domain (DBD) and dimerization domain to the 12-bp palindromic sequence ACCN 6 GGT mostly located within the regulatory region, also called the long control region or noncoding region (NCR) of the PV genome. In the case of bovine PV type 1 (BPV1), the long control region contains 12 E2 binding sites, which mediate a strong activation of several BPV1 promoters by E2 (48-50). Human PV (HPV) types infecting the genital mucosa contain four E2 binding sites in conserved positions. Here, only a moderate activation by E2 could be detected, and E2 repressed HPV gene expression in most cases. This repression is mediated by binding to two promoter-proximal E2 binding sites, as revealed by transient transfections of cervical carcinoma cell lines and immortalized skin keratinocytes (10,52,58,59). Repression of HPV gene expression by the E2 activator protein occurs when E2 binding sites are overlapping with the binding sites for cellular transcription factors necessary for promoter activity. In contrast to nat...