Dennis J. McCance

The E6 protein of human papillomavirus type 16 binds to and inhibits co-activation by CBP and p300.

The co‐activators CBP and p300 are important for normal cell differentiation and cell cycle progression and are the targets for viral proteins that dysregulate these cellular processes. We show here that the E6 protein from the oncogenic human papillomavirus type 16 (HPV‐16) binds to three regions (C/H1, C/H3 and the C‐terminus) of both CBP and p300. The interaction of E6 with CBP/p300 was direct and independent of proteins known to bind the co‐activators, such as p53. The E6 protein from low‐risk HPV type 6 did not interact with C/H3 or the C‐terminus but associated with the C/H1 domain at 50% of the level of HPV‐16. HPV‐16 E6 inhibited the intrinsic transcriptional activity of CBP/p300 and decreased the ability of p300 to activate p53‐ and NF‐κB‐responsive promoter elements. Interestingly, some mutations in HPV‐16 E6 abrogated C/H3‐E6 interactions, but did not alter the ability of E6 to associate with the C/H1 domain, suggesting that these modified proteins could be used to delineate the functional significance of the C/H1 and C/H3 domains of CBP/p300.

The human papillomavirus type 16 E7 gene product interacts with and trans-activates the AP1 family of transcription factors.

The E7 gene product of human papillomavirus type 16 (HPV16) binds to the retinoblastoma gene product (pRb) and dissociates pRb‐E2F complexes. However, the observation that the ability of E7 to bind pRb is not required for the HPV16‐induced immortalization of primary keratinocytes prompted a search for other cellular factors bound by E7. Using a glutathione‐S‐transferase (GST) fusion protein system, we show that E7 complexes with AP1 transcription factors including c‐Jun, JunB, JunD and c‐Fos. The ability of E7 to complex with c‐Jun in vivo is demonstrated by co‐immunoprecipitation and the yeast two‐hybrid system. An analysis of E7 point mutants in the GST system indicates that the E7 zinc‐finger motif, but not the pRb binding domain, is involved in these interactions. Using c‐Jun deletion mutants, E7 binding maps between amino acids 224 and 286 of c‐Jun. E7 trans‐activates c‐Jun‐induced transcription from a Jun responsive promoter, and this activity correlates with the ability of E7 mutants to bind Jun proteins. Finally, a transcriptionally inactive c‐Jun deletion, which can bind E7, interferes with the E7‐induced transformation of rat embryo fibroblasts in cooperation with an activated ras, indicating that the Jun‐E7 interaction is physiologically relevant and that Jun factors may be targeted in the E7 transformation pathway.

Serological differentiation of human papillomavirus types 11, 16 and 18 using recombinant virus-like particles.

The L1 major capsid protein-coding sequences of human papillomavirus (HPV) types 11, 16 and 18 were expressed in the baculovirus system. Virus-like particles (VLPs) were purified from recombinant-infected Spodoptera frugiperda Sf9 cells and cell-free culture supernatants. Rabbits immunized with purified VLPs developed antibodies that reacted only with the specific VLP type used as the immunogen. In addition, rabbit antibodies raised against infectious HPV-11 virions only reacted with HPV-11 L1 VLPs and not with VLPs derived from either HPV-16 or HPV-18. These results suggest that HPV-11, HPV-16 and HPV-18 virions are antigenically distinct from one another. This observation should be considered in future studies of immune responses to HPV.

Rb binds c-Jun and activates transcription.

The retinoblastoma protein (Rb) acts as a critical cell‐cycle regulator and loss of Rb function is associated with a variety of human cancer types. Here we report that Rb binds to members of the AP‐1 family of transcription factors, including c‐Jun, and stimulates c‐Jun transcriptional activity from an AP‐1 consensus sequence. The interaction involves the leucine zipper region of c‐Jun and the B pocket of Rb as well as a C‐terminal domain. We also present evidence that the complexes are found in terminally differentiating keratinocytes and cells entering the G1 phase of the cell cycle after release from serum starvation. The human papillomavirus type 16 E7 protein, which binds to both c‐Jun and Rb, inhibits the ability of Rb to activate c‐Jun. The results provide evidence of a role for Rb as a transcriptional activator in early G1 and as a potential modulator of c‐Jun expression during keratinocyte differentiation.

Decreased Migration of Langerhans Precursor-Like Cells in Response to Human Keratinocytes Expressing Human Papillomavirus Type 16 E6/E7 Is Related to Reduced Macrophage Inflammatory Protein-3α Production

ABSTRACT Infection with high-risk human papillomavirus (HPV) types, particularly types 16 and 18, contributes to 90% of cervical cancer cases. HPV infects cutaneous or mucosal epithelium, tissue that is monitored for microbial infection or damage by Langerhans cells. In lesions produced by HPV type 16, there is a reduction in numbers of immune cells, especially Langerhans cells. Langerhans precursor cells selectively express CCR6, the receptor for macrophage inflammatory protein 3α (MIP-3α), and function as potent immune responders to inflamed epithelium and initiators of the innate immune response. It has been reported that E6 and E7 of high-risk HPVs interfere with immune mediators in order to suppress the recruitment of immune cells and antiviral activities of infected cells. Here we show that, following proinflammatory stimulus, HPV-16 E6 and E7 inhibit MIP-3α transcription, resulting in suppression of the migration of immature Langerhans precursor-like cells. Interestingly, the E6 and E7 proteins from the low-risk HPV types also inhibited MIP-3α transcription. These results suggest that one mechanism by which HPV-infected cells suppress the immune response may be through the inhibition of a vital alert signal, thus contributing to the persistence of HPV infection.

Human Papillomavirus Type 16 E7 Maintains Elevated Levels of the cdc25A Tyrosine Phosphatase during Deregulation of Cell Cycle Arrest

ABSTRACT Essential to the oncogenic properties of human papillomavirus type 16 (HPV-16) are the activities encoded by the early gene product E7. HPV-16 E7 (E7.16) binds to cellular factors involved in cell cycle regulation and differentiation. These include the retinoblastoma tumor suppressor protein (Rb) and histone deacetylase (HDAC) complexes. While the biological significance of these interactions remains unclear, E7 is believed to help maintain cells in a proliferative state, thus establishing an environment that is conducive to viral replication. Most pathways that govern cell growth converge on downstream effectors. Among these is the cdc25A tyrosine phosphatase. cdc25A is required for G1/S transition, and its deregulation is associated with carcinogenesis. Considering the importance of cdc25A in cell cycle progression, it represents a relevant target for viral oncoproteins. Accordingly, the present study focuses on the putative deregulation of cdc25A by E7.16. Our results indicate that E7.16 can impede growth arrest induced during serum starvation and keratinocyte differentiation. Importantly, these E7-specific phenotypes correlate with elevated cdc25A steady-state levels. Reporter assays performed with NIH 3T3 cell lines and human keratinocytes indicate that E7 can transactivate the cdc25A promoter. In addition, transcriptional activation by E7.16 requires the distal E2F site within the cdc25A promoter. We further demonstrate that the ability of E7 to abrogate cell cycle arrest, activate cdc25A transcription, and increase cdc25A protein levels requires intact Rb and HDAC-1 binding domains. Finally, by using the cdk inhibitor roscovitine, we reveal that E7 activates the cdc25A promoter independently of cell cycle progression and cdk activity. Consequently, we propose that E7.16 can directly target cdc25A transcription and maintains cdc25A gene expression by disrupting Rb/E2F/HDAC-1 repressor complexes.

Human Papillomavirus Type 16 E7 Up-regulates AKT Activity through the Retinoblastoma Protein

Human papillomaviruses (HPV) are small DNA tumor viruses causally associated with cervical cancer. The early gene product E7 from high-risk HPV is considered the major transforming protein expressed by the virus. Although many functions have been described for E7 in disrupting normal cellular processes, we describe in this study a new cellular target in primary human foreskin keratinocytes (HFK), the serine/threonine kinase AKT. Expression of HPV type 16 E7 in HFK caused inhibition of differentiation, hyperproliferation, and up-regulation of AKT activity in organotypic raft cultures. The ability of E7 to up-regulate AKT activity is dependent on its ability to bind to and inactivate the retinoblastoma (Rb) gene product family of proteins. Furthermore, we show that knocking down Rb alone, with short hairpin RNAs, was sufficient to up-regulate AKT activity in differentiated keratinocytes. Up-regulation of AKT activity and loss of Rb was also observed in HPV-positive cervical high-grade squamous intraepithelial lesions when compared with normal cervical tissue. Together, these data provide evidence linking inactivation of Rb by E7 in the up-regulation of AKT activity during cervical cancer progression.

Acetylation regulates the differentiation-specific functions of the retinoblastoma protein

The retinoblastoma tumor‐suppressor protein (pRb) is known to induce growth arrest and cellular differentiation. The molecular determinants of pRb function include protein–protein interactions and post‐translational modifications such as phosphorylation. Recently, the co‐activator p300 was found to acetylate pRb. The biological significance of pRb acetylation, however, remains unclear. In the present study, we provide evidence that pRb undergoes acetylation upon cellular differentiation, including skeletal myogenesis. In addition to p300, the p300‐Associated Factor (P/CAF) can mediate pRb acetylation as pRb interacts directly with the acetyltransferase domain of P/CAF in vitro and can associate with P/CAF in differentiated cells. Significantly, by using a C terminal acetylation‐impaired mutant of pRb, we reveal that acetylation does not affect pRb‐dependent growth arrest or the repression of E2F transcriptional activity. Instead, acetylation is required for pRb‐mediated terminal cell cycle exit and the induction of late myogenic gene expression. Based on these results, we propose that acetylation regulates the differentiation‐specific function(s) of pRb.

Human Papillomavirus Type 16 E6 and E7 Cause Polyploidy in Human Keratinocytes and Up-Regulation of G2-M-phase Proteins

Human papillomavirus type 16 proteins E6 and E7 have been shown to cause centrosome amplification and lagging chromosomes during mitosis. These abnormalities during mitosis can result in missegregation of the chromosomes, leading to chromosomal instability. Genomic instability is thought to be an essential part of the conversion of a normal cell to a cancer cell. We now show that E6 and E7 together cause polyploidy in primary human keratinocytes soon after these genes are introduced into the cells. Polyploidy seems to result from a spindle checkpoint failure arising from abrogation of the normal functions of p53 and retinoblastoma family members by E6 and E7, respectively. In addition, E6 and E7 cause deregulation of cellular genes such as Plk1, Aurora-A, cdk1, and Nek2, which are known to control the G2-M-phase transition and the ordered progression through mitosis.

Induction of tetraploidy through loss of p53 and upregulation of Plk1 by human papillomavirus type-16 E6.

Cancer cells are insensitive to many signals that inhibit growth of untransformed cells. Here, we show that primary human epithelial cells expressing human papillomavirus (HPV) type-16 E6/E7 bypass arrest caused by the DNA-damaging drug adriamycin and become tetraploid. To determine the contribution of E6 in the context of E7 to the resistance of arrest and induction of tetraploidy, we used an E6 mutant unable to degrade p53 or RNAi targeting p53 for knockdown. The E6 mutant fails to generate tetraploidy; however, the presence of E7 is sufficient to bypass arrest while the p53 RNAi permits both arrest insensitivity and tetraploidy. We published previously that polo-like kinase 1 (Plk1) is upregulated in E6/E7-expressing cells. We observe here that abnormal expression of Plk1 protein correlates with tetraploidy. Using the p53 binding-defective mutant of E6 and p53 RNAi, we show that p53 represses Plk1, suggesting that loss of p53 results in tetraploidy through upregulation of Plk1. Consistent with this hypothesis, overexpression of Plk1 in cells generates tetraploidy but does not confer resistance to arrest. These results support a model for transformation caused by HPV-16 where bypass of arrest and tetraploidy are separable consequences of p53 loss with Plk1 required only for the latter effect.