Potent Anti-Tumor Effect Generated by a Novel Human Papillomavirus (HPV) Antagonist Peptide Reactivating the pRb/E2F Pathway

Human papillomavirus type 16 (HPV16) E7 is a viral oncoprotein believed to play a major role in cervical cancer. In this study, an antagonist peptide against HPV16E7 protein was first identified from screening the c7c phage display peptide library. The binding specificity and affinity of the selected peptide to HPV16E7 were tested by competitive enzyme-linked immunosorbent assay (ELISA). The antagonist peptide showed obvious anti-tumor efficacy both in cell lines and animal tumor models. Significant cell proliferation inhibition with high specificity was noted when HPV16-positive cells were treated with the peptide. This anti-tumor efficacy was resulted from overriding the activities of HPV16E7 and reactivating the pRb/E2F pathway, as shown by a series of experiments. Flow cytometry analysis revealed that the selected peptide induced G1 arrest in a dose-dependent manner. Competitive ELISA, pull down, and Co-IP experiments indicated that the selected peptide disrupted the interaction between HPV16E7 and pRb proteins both in vitro and in vivo. Luciferase reporter assay verified that transcription activities of E2F were suppressed by the peptide through restoration of pRb. RT-PCR and Western blot revealed that it reduced cyclins A, D1, and E1 expression, and led to HPV16E7 protein degradation, but pRb protein stabilization. The current study suggests that this specific peptide may serve as a potential therapeutic agent for HPV16-positive cervical cancer

Dengue Virus Inhibits Immune Responses in Aedes aegypti Cells

The ability of many viruses to manipulate the host antiviral immune response often results in complex host-pathogen interactions. In order to study the interaction of dengue virus (DENV) with the Aedes aegypti immune response, we have characterized the DENV infection-responsive transcriptome of the immune-competent A. aegypti cell line Aag2. As in mosquitoes, DENV infection transcriptionally activated the cell line Toll pathway and a variety of cellular physiological systems. Most notably, however, DENV infection down-regulated the expression levels of numerous immune signaling molecules and antimicrobial peptides (AMPs). Functional assays showed that transcriptional induction of AMPs from the Toll and IMD pathways in response to bacterial challenge is impaired in DENV-infected cells. In addition, Escherichia coli, a Gram-negative bacteria species, grew better when co-cultured with DENV-infected cells than with uninfected cells, suggesting a decreased production of AMPs from the IMD pathway in virus-infected cells. Pre-stimulation of the cell line with Gram-positive bacteria prior to DENV infection had no effect on DENV titers, while pre-stimulation with Gram-negative bacteria resulted in an increase in DENV titers. These results indicate that DENV is capable of actively suppressing immune responses in the cells it infects, a phenomenon that may have important consequences for virus transmission and insect physiology

The Canine Papillomavirus and Gamma HPV E7 Proteins Use an Alternative Domain to Bind and Destabilize the Retinoblastoma Protein

The high-risk HPV E6 and E7 proteins cooperate to immortalize primary human cervical cells and the E7 protein can independently transform fibroblasts in vitro, primarily due to its ability to associate with and degrade the retinoblastoma tumor suppressor protein, pRb. The binding of E7 to pRb is mediated by a conserved Leu-X-Cys-X-Glu (LXCXE) motif in the conserved region 2 (CR2) of E7 and this domain is both necessary and sufficient for E7/pRb association. In the current study, we report that the E7 protein of the malignancy-associated canine papillomavirus type 2 encodes an E7 protein that has serine substituted for cysteine in the LXCXE motif. In HPV, this substitution in E7 abrogates pRb binding and degradation. However, despite variation at this critical site, the canine papillomavirus E7 protein still bound and degraded pRb. Even complete deletion of the LXSXE domain of canine E7 failed to interfere with binding to pRb in vitro and in vivo. Rather, the dominant binding site for pRb mapped to the C-terminal domain of canine E7. Finally, while the CR1 and CR2 domains of HPV E7 are sufficient for degradation of pRb, the C-terminal region of canine E7 was also required for pRb degradation. Screening of HPV genome sequences revealed that the LXSXE motif of the canine E7 protein was also present in the gamma HPVs and we demonstrate that the gamma HPV-4 E7 protein also binds pRb in a similar way. It appears, therefore, that the type 2 canine PV and gamma-type HPVs not only share similar properties with respect to tissue specificity and association with immunosuppression, but also the mechanism by which their E7 proteins interact with pRb

Human Papillomavirus-16 E7 Interacts with Glutathione S-Transferase P1 and Enhances Its Role in Cell Survival

Background:Human Papillomavirus (HPV)-16 is a paradigm for "high-risk" HPVs, the causative agents of virtually all cervical carcinomas. HPV E6 and E7 viral genes are usually expressed in these tumors, suggesting key roles for their gene products, the E6 and E7 oncoproteins, in inducing malignant transformation.Methodology/Principal Findings:By protein-protein interaction analysis, using mass spectrometry, we identified glutathione S-transferase P1-1 (GSTP1) as a novel cellular partner of the HPV-16 E7 oncoprotein. Following mapping of the region in the HPV-16 E7 sequence that is involved in the interaction, we generated a three-dimensional molecular model of the complex between HPV-16 E7 and GSTP1, and used this to engineer a mutant molecule of HPV-16 E7 with strongly reduced affinity for GSTP1.When expressed in HaCaT human keratinocytes, HPV-16 E7 modified the equilibrium between the oxidized and reduced forms of GSTP1, thereby inhibiting JNK phosphorylation and its ability to induce apoptosis. Using GSTP1-deficient MCF-7 cancer cells and siRNA interference targeting GSTP1 in HaCaT keratinocytes expressing either wild-type or mutant HPV-16 E7, we uncovered a pivotal role for GSTP1 in the pro-survival program elicited by its binding with HPV-16 E7.Conclusions/Significance:This study provides further evidence of the transforming abilities of this oncoprotein, setting the groundwork for devising unique molecular tools that can both interfere with the interaction between HPV-16 E7 and GSTP1 and minimize the survival of HPV-16 E7-expressing cancer cells. © 2009 Mileo et al

Presence of papillomavirus sequences in condylomatous lesions of the mamillae and in invasive carcinoma of the breast

BACKGROUND: Viruses including Epstein–Barr virus (EBV), a human equivalent of murine mammary tumour virus (MMTV) and human papillomavirus (HPV) have been implicated in the aetiology of human breast cancer. We report the presence of HPV DNA sequences in areolar tissue and tumour tissue samples from female patients with breast carcinoma. The presence of virus in the areolar–nipple complex suggests to us a potential pathogenic mechanism. METHODS: Polymerase chain reaction (PCR) was undertaken to amplify HPV types in areolar and tumour tissue from breast cancer cases. In situ hybridisation supported the PCR findings and localised the virus in nipple, areolar and tumour tissue. RESULTS: Papillomavirus DNA was present in 25 of 29 samples of breast carcinoma and in 20 of 29 samples from the corresponding mamilla. The most prevalent type in both carcinomas and nipples was HPV 11, followed by HPV 6. Other types detected were HPV 16, 23, 27 and 57 (nipples and carcinomas), HPV 20, 21, 32, 37, 38, 66 and GA3-1 (nipples only) and HPV 3, 15, 24, 87 and DL473 (carcinomas only). Multiple types were demonstrated in seven carcinomas and ten nipple samples. CONCLUSIONS: The data demonstrate the occurrence of HPV in nipple and areolar tissues in patients with breast carcinoma. The authors postulate a retrograde ductular pattern of viral spread that may have pathogenic significance

Human papillomavirus and oropharyngeal cancer in Greenland in 1994–2010

Background. Oropharyngeal squamous cell carcinoma (OPSCC) is associated with the sexually transmitted human papillomavirus (HPV), smoking and alcohol. In Greenland, a high rate of HPV-induced cervical cancer and venereal diseases are found, which exposes the population for high risk of HPV infection. In Greenland, only girls are included in the mandatory HPV vaccination program. Objective. To investigate the annual incidence of OPSCC and the proportion of HPV-associated OPSCC (HPV+ OPSCC) in Greenland in 1994–2010. Design. At Rigshospitalet, University of Copenhagen, we identified all Greenlandic patients diagnosed and treated for OPSCC from 1994 to 2010. Sections were cut from the patient's paraffin-embedded tissue blocks and investigated for p16 expression by immunohistochemistry. HPV analyses were performed with 2 sets of general HPV primers and 1 set of HPV16-specific primer. HPV+ OPSCC was defined as both >75% p16+ cells and PCR positive for HPV. Results. Of 26 Greenlandic patients diagnosed with OPSCC, 17 were males and 9 were females. The proportion of HPV+ OPSCC in the total study period was 22%, without significant changes in the population in Greenland. We found an increase in the proportion of HPV+ OPSCC from 14% in 1994–2001 to 25% in 2002–2010 (p=0.51). Among males from 20 to 27% (p=0.63) and in females from 0 to 20% (p=0.71). The annual OPSCC incidence increased from 2.3/100,000 (CI=1.2–4.2) in 1994–2001 to 3.8/100,000 (CI=2.4–6.2) in 2002–2010: among males from 2.4/100,000 (CI=1.0–5.7) to 5.0/100,000 (CI=2.9–8.9). Conclusion. Even though the population is at high risk of HPV infection, the proportion of 22% HPV+ OPSCC in the total study period is low compared to Europe and the United States. This might be explained by our small study size and/or by ethnic, geographical, sexual and cultural differences. Continuing observations of the OPSCC incidence and the proportion of HPV+ OPSCC in Greenland are needed

HPV31 E7 facilitates replication by activating E2F2 transcription through its interaction with HDACs

The E7 proteins of human papillomaviruses (HPVs) contribute to oncogenesis by associating with Rb family members as well class I histone deacetylases (HDACs). The binding of HDACs is also important for the maintenance of viral episomes during the differentiation-dependent productive life cycle. The effects of E7 and other viral proteins on E2F family members were examined in differentiating keratinocytes. E7 was found to specifically activate E2F2 transcription in suprabasal keratinocytes through its ability to bind HDACs. Chromatin immunoprecipitation assays demonstrated that, in differentiating cells, E7 acts to inhibit HDAC binding to the E2F2 promoter resulting in activation of expression. Reduction of E2F2 levels through the use of siRNA confirmed that E2F2 expression facilitated HPV replication but its loss did not affect cell proliferation. Our study demonstrates a mechanism by which binding of HDACs to E7 directly modulates viral replication and identifies E2F2 as a possible target for antiviral therapies