Strains Of Papillomavirus Research Articles

Development of combined DNA-based piezoelectric biosensors for the simultaneous detection and genotyping of high risk Human Papilloma Virus strains

BackgroundHuman Papilloma Virus (HPV) is a DNA virus belonging to the Papovavirus family. Genital HPV types have been subdivided into medium-low risk, and high-risk (HPV 16 and 18), frequently associated with cervical cancer. Three DNA-based piezoelectric biosensors were here developed for a quick detection and genotyping of HPV. MethodsWe developed a method for the detection and genotyping of HPV in human cervical scraping samples based on coupling DNA piezoelectric sensors with Polymerase Chain Reaction (PCR). The novelty of this work was the design and immobilisation of a degenerate probe (chosen in a conserved region of the viral genome) for the simultaneous detection of 16 virus strains and of two specific probes (chosen in a less-conserved region of the viral genome) for genotyping. ResultsThe three biosensors were optimised with synthetic oligonucleotides with good reproducibility (HPVdeg CV% av 9%, HPV16 CV%av 9%; HPV18 CV%av 11%) with a detection limit of 50 nM. Cervical scraping samples after PCR amplification (in 40–200 nM range), were tested without the need of label with high selectivity and reproducibility. The results were in agreement with a reference method used in routinary analysis. ConclusionPiezoelectric biosensors have proven to be suitable for detection and genotyping of HPV.

Molecular dynamics of the DNA‐binding domain of the papillomavirus E2 transcriptional regulator uncover differential properties for DNA target accommodation

Papillomaviruses are small DNA tumor viruses that infect mammalian hosts, with consequences from benign to cancerous lesions. The Early protein 2 is the master regulator for the virus life cycle, participating in gene transcription, DNA replication, and viral episome migration. All of these functions rely on primary target recognition by its dimeric DNA-binding domain. In this work, we performed molecular dynamics simulations in order to gain insights into the structural dynamics of the DNA-binding domains of two prototypic strains, human papillomavirus strain 16 and the bovine papillomavirus strain 1. The simulations underline different dynamic features in the two proteins. The human papillomavirus strain 16 domain displays a higher flexibility of the beta2-beta3 connecting loop in comparison with the bovine papillomavirus strain 1 domain, with a consequent effect on the DNA-binding helices, and thus on the modulation of DNA recognition. A compact beta-barrel is found in human papillomavirus strain 16, whereas the bovine papillomavirus strain 1 protein is characterized by a loose beta-barrel with a large number of cavities filled by water, which provides great flexibility. The rigidity of the human papillomavirus strain 16 beta-barrel prevents protein deformation, and, as a consequence, deformable spacers are the preferred targets in complex formation. In contrast, in bovine papillomavirus strain 1, a more deformable beta-barrel confers greater adaptability to the protein, allowing the binding of less flexible DNA regions. The flexibility data are confirmed by the experimental NMR S2 values, which are reproduced well by calculation. This feature may provide the protein with an ability to discriminate between spacer sequences. Clearly, the deformability required for the formation of the Early protein 2 C-terminal DNA-binding domain-DNA complexes of various types is based not only on the rigidity of the base sequences in the DNA spacers, but also on the intrinsic deformability properties of each domain.

A National Survey of Pediatrician Knowledge and Attitudes Regarding Human Papillomavirus Vaccination

A human papillomavirus vaccine was licensed in June 2006. The vaccine is quadrivalent, protecting against 2 human papillomavirus strains that cause cervical cancer and 2 that cause genital warts. The objective of this study was to determine physician characteristics, knowledge, and attitudes associated with an intention to recommend human papillomavirus vaccination. Between August and October 2005, a cross-sectional survey was administered to a national network of 431 pediatricians. The network was developed from a random sample of American Academy of Pediatrics members and was designed to be representative of the organization's membership with respect to urban/rural location, practice type, and region. The survey was conducted before human papillomavirus vaccine licensure and therefore focused on a candidate quadrivalent human papillomavirus vaccine and a range of potential vaccination recommendations. The main outcome measure was intention to recommend a quadrivalent human papillomavirus vaccine to young adolescent (10- to 12-year-old) females. Survey response rate was 68%. If endorsed by national health organizations, 46% of respondents would recommend vaccination for 10- to 12-year-old females, 77% for 13- to 15-year-old females, and 89% for 16- to 18-year-old females. Corresponding rates for males were 37%, 67%, and 82%, respectively. Whereas 60% of respondents thought that parents would be concerned that human papillomavirus vaccination may encourage risky sexual behaviors, 11% reported that they themselves had this concern. Respondents who believed that other new adolescent immunization recommendations (eg, meningococcal, pertussis) would facilitate human papillomavirus vaccine implementation were more likely to intend to recommend vaccination. Although a national sample of pediatricians expressed a high level of acceptance of human papillomavirus vaccination in older adolescent females, fewer than one half anticipated giving human papillomavirus vaccine to younger female patients. Provider concerns about parental vaccine acceptance will need to be addressed to optimize human papillomavirus vaccination implementation.

Papillomavirus Proteins and their Potential as Drug Design Targets

The papillomaviruses are a family of small, double-stranded DNA viruses that infect the basal cells of cutaneous and mucosal epithelium. While a large percentage of the population is benignly infected with various strains of human papillomavirus (HPV), long-term infection by a subset of HPV strains is associated with malignant transformation. The prospects for prophylaxis against HPV infection have recently received an enormous boost with the approval by the US FDA of a vaccine targeted against the most common cancer-associated HPV strains. However, the large number of people already infected, the high cost of the vaccination regimen (particularly in poorer countries) and the HPV infections that these vaccines do not protect against underscore the need for therapeutic strategies. The elucidation of molecular details underlying fundamental processes in the viral life cycle, such as virus replication, transcription and HPV-induced carcinogenesis, is required to meet this aim. This article provides an overview of high-resolution structures of papillomavirus proteins and their functional complexes, with particular reference to mechanistic and structural features that could be exploited in the rational design of antiviral agents.

Formation of well-defined soluble aggregates upon fusion to MBP is a generic property of E6 proteins from various human papillomavirus species

Protein aggregation is a main barrier hindering structural and functional studies of a number of interesting biological targets. The E6 oncoprotein of Human Papillomavirus strain 16 (E6 16) is difficult to express under a native soluble form in bacteria. Produced as an unfused sequence, it forms inclusion bodies. Fused to the C-terminus of MBP, it is mainly produced in the form of soluble high molecular weight aggregates. Here, we produced as MBP-fusions seven E6 proteins from other HPV strains (5, 11, 18, 33, 45, 52, and 58) belonging to four different species, and we compared their aggregation state to that of MBP-E6 16. Using a fast mutagenesis method, we changed most non-conserved cysteines to the isosteric residue serine to minimize disulfide bridge-mediated aggregation during purification. Static and dynamic light scattering measurements, ultracentrifugation and electron microscopy demonstrated the presence in all MBP-E6 preparations of soluble high-molecular weight aggregates with a well-defined spherical shape. These aggregated particles are relatively monodisperse but their amount and their size vary depending on the conditions of expression and the strain considered. For all strains, minimal aggregate formation occurs when the expression is performed at 15 °C. Such observations suggest that the assembly of MBP-E6 aggregates takes place in vivo during protein biosynthesis, rather than occurring during purification. Finally, we show that all MBP-E6 preparations contain two zinc ions per protein monomer, suggesting that E6 domains within the high molecular weight aggregates possess a native-like fold, which enables correct coordination to the metal center.

Indirect Readout of DNA Sequence by Papillomavirus E2 Proteins Depends Upon Net Cation Uptake

The papillomavirus E2 proteins bind with high affinity to palindromic DNA sequences consisting of two highly conserved four base-pair sequences flanking a variable "spacer" of identical length (ACCG NNNN CGGT). While intimate contacts are observed between the bound proteins and conserved DNA in the available co-crystal structures, no contact is seen between the proteins and the spacer DNA. The ability of human papillomavirus strain 16 (HPV-16) E2 and bovine papillomavirus strain 1 (BPV-1) E2 to discriminate among binding sites with different spacer sequences is dependent on their sensitivity to the unique conformational and/or dynamic properties of the spacer DNA in a process termed "indirect readout". Differential sequence-specific K(+) uptake in low ionic strength solutions lacking Mg(2+) is observed upon E2 protein binding to sites containing the AATT, TTAA or ACGT spacer sequences. In contrast, the cation displacement typical of protein-DNA complex formation is observed at high K(+) concentrations or in the presence of Mg(2+). These results are interpreted to reflect the sequence-specific stabilization of bent DNA conformations by cations localized within the narrowed minor grooves of the protein-bound DNA and the intrinsic structure and flexibility of the DNA target. Mg(2+) differentially affects the binding of the HPV-16 E2 DNA binding domain (HPV16-E2/D) and the BPV-1 E2 DNA binding domain (BPV1-E2/D) to sites bearing different spacer sequences. This study suggests that monovalent and divalent cations contribute to the discrimination of DNA structure and flexibility that could in turn contribute to the specificity with which HPV16-E2/D and BPV1-E2/D mediate DNA replication and gene transcription.

The human papillomavirus 16 E6 protein can either protect or further sensitize cells to TNF: effect of dose

High-risk strains of human papillomavirus, including HPV 16, cause human cervical carcinomas, due in part to the activity of their E6 oncogene. E6 interacts with a number of cellular proteins involved in host-initiated apoptotic responses. Paradoxically, literature reports show that E6 can both protect cells from and sensitize cells to tumor necrosis factor (TNF). To examine this apparent contradiction, E6 was transfected into U2OS cells and stable clones were treated with TNF. Intriguingly, clones with a high level of E6 expression displayed an increased sensitivity to TNF by undergoing apoptosis, while those with low expression were resistant. Furthermore, TNF treatment of cells in which the expression of E6 was regulated by the addition of doxycycline demonstrated clearly that while low levels of E6 protect cells from TNF, high levels sensitize cells. Together, these results demonstrate that virus-host interactions can be complex and that both quantitative and qualitative aspects are important in determining outcome.

Molecular Diagnostic Assays for Cervical Neoplasia: Emerging Markers for the Detection of High-Grade Cervical Disease

The accurate detection and diagnosis of cervical carcinoma and its malignant precursors (collectively referred to as high-grade cervical disease) represents one of the current challenges in clinical medicine and cytopathology. The advent of molecular diagnostics and the use of whole-genome profiling using DNA microarrays promises to yield improved understanding of the disease process with the subsequent development of more accurate diagnostic procedures based upon these discoveries. Recent reports describing a variety of experimental approaches have identified a series of candidate genes that are overexpressed in cervical carcinoma. In this article, representative examples of these markers and the resulting translational research will be reviewed within the context of improved cervical disease detection. An emerging class of markers, the minichromosome maintenance protein family of DNA licensing factors (MCM-2, MCM-6, MCM-7), shows promise for the specific detection of high-grade cervical disease using simple antibody-based immunochemistry formats. These proteins are overexpressed in cervical disease as a result of infection by oncogenic strains of human papillomavirus (HPV) and subsequent uncontrolled activation of gene transcription and aberrant S-phase induction, mediated through the E2F transcription factor pathway. This behavior appears to be a hallmark of high-grade cervical disease and provides the link between oncogenic HPV infections and the molecular behavior of cervical neoplasia (CN). The use of these molecular descriptors of CN in simple immunochemistry formats compatible with conventional cytology preparations is anticipated to improve the screening and detection of cervical disease within the healthcare system.

Epidemiology and Natural History of Anal HPV Infection and ASIL and Cancer in the General Population

Carcinoma of the anal canal is an increasingly prevalent problem that has been causally associated with infection by oncogenic strains of the human papillomavirus. Concurrent infection with HPV and the human immunodeficiency virus is the most significant risk factor; however, among HIV-negative men and women, risk factors include receptive anal intercourse, the presence of condyloma, multiplicity of HPV serotype infection, injection drug abuse, and the presence of cervical, vulvar, or penile neoplasia. Although there is limited data on the natural history of anal HPV infection and the progression to anal cancer, a number of epidemiologic and associative studies have demonstrated that anal squamous intraepithelial lesions represent an intermediate step in the progression to cancer. This relationship is analogous to the pathogenesis of invasive cervical carcinoma that is similarly asociated with cervical squamous intraepithelial lesions. Further study may elucidate the factors responsible for the pattern of regression or persistence after HPV infection.

Human papillomavirus infections in children.

The human papillomavirus is a ubiquitous 55-nm DNA virus that causes a variety of clinical disease states in children, commonly referred to as warts. The natural history of warts is spontaneous regression through the development of a complex blend of cell-mediated and humoral immunity. Although spontaneous immunity can develop, as many as one third of children will have persistent human papillomavirus infection beyond 2 years. Therapeutic modalities are manifold, primarily because no therapy is universally effective. The purpose of this review is to update the reader with the latest information on the human papillomavirus and its therapeutics in children. Recently, encouraging research has been conducted in human papillomavirus, including destructive and immunologic therapies. Vaccines tailored to genital human papillomavirus strains are just coming into clinical use. Manipulation of the immune system through medications or vaccination will likely help contain human papillomavirus in the future and prevent secondary human papillomavirus oncogenesis of the skin and cervix.

The Human Papillomavirus 16 E6 Protein Binds to Fas-associated Death Domain and Protects Cells from Fas-triggered Apoptosis

High risk strains of human papillomavirus (HPV), such as HPV 16, cause human cervical carcinoma. The E6 protein of HPV 16 mediates the rapid degradation of the tumor suppressor p53, although this is not the only function of E6 and cannot completely explain its transforming potential. Previous work in our laboratory has demonstrated that E6 can protect cells from tumor necrosis factor-induced apoptosis by binding to the C-terminal end of tumor necrosis factor R1, thus blocking apoptotic signal transduction. In this study, E6 was shown to also protect cells from apoptosis induced via the Fas pathway. Furthermore, use of an inducible E6 expression system demonstrated that this protection is dose-dependent, with higher levels of E6 leading to greater protection. Although E6 suppresses activation of both caspase 3 and caspase 8, it does not affect apoptotic signaling through the mitochondrial pathway. Mammalian two-hybrid and in vitro pull-down assays were then used to demonstrate that E6 binds directly to the death effector domain of Fas-associated death domain (FADD), with deletion and site-directed mutants enabling the localization of the E6-binding site to the N-terminal end of the FADD death effector domain. E6 is produced in two forms as follows: a full-length version of approximately 16 kDa and a smaller version of about half that size corresponding to the N-terminal half of the full-length protein. Pull-down and functional assays demonstrated that the full-length version, but not the small version of E6, was able to bind to FADD and to protect cells from Fas-induced apoptosis. In addition, binding to E6 leads to degradation of FADD, with the loss of cellular FADD proportional to the amount of E6 expressed. These results support a model in which E6-mediated degradation of FADD prevents transmission of apoptotic signals via the Fas pathway.

Vaccine Prevents HPV Infection and Early Cervical Lesions

Persistent infection with oncogenic strains of human papilloma virus (HPV), especially two prevalent high-risk types (HPV-16 and HPV-18), causes a large proportion of cervical malignancies. With funding and coordination from the vaccine manufacturer, researchers in North America and Brazil assessed the efficacy of a bivalent HPV-16/18 vaccine against new and persistent HPV infection, abnormal …

Pelvics, Paps, and HPV

Chimere is a 15 year old who has come for followup for pelvic inflammatory disease (PID) for which she was treated in the emergency department (ED) 5 days ago. As part of her history, she is asked when her last Pap test was and she responds they did one in the ED. The chances of a Pap test being done in an ED are extremely remote, but Chimere's response is quite common. Most teens equate a pelvic exam with a Pap test and assume it was normal because they did not hear otherwise. The close relationship between certain strains of human papillomavirus (HPV) and cervical cancer makes that erroneous assumption potentially harmful to these young women and their future reproductive health.

Decision science and cervical cancer.

Mathematical modeling is an effective tool for guiding cervical cancer screening, diagnosis, and treatment decisions for patients and policymakers. This article describes the use of mathematical modeling as outlined in five presentations from the Decision Science and Cervical Cancer session of the Second International Conference on Cervical Cancer held at The University of Texas M. D. Anderson Cancer Center, April 11-14, 2002. The authors provide an overview of mathematical modeling, especially decision analysis and cost-effectiveness analysis, and examples of how it can be used for clinical decision making regarding the prevention, diagnosis, and treatment of cervical cancer. Included are applications as well as theory regarding decision science and cervical cancer. Mathematical modeling can answer such questions as the optimal frequency for screening, the optimal age to stop screening, and the optimal way to diagnose cervical cancer. Results from one mathematical model demonstrated that a vaccine against high-risk strains of human papillomavirus was a cost-effective use of resources, and discussion of another model demonstrated the importance of collecting direct non-health care costs and time costs for cost-effectiveness analysis. Research presented indicated that care must be taken when applying the results of population-wide, cost-effectiveness analyses to reduce health disparities. Mathematical modeling can encompass a variety of theoretical and applied issues regarding decision science and cervical cancer. The ultimate objective of using decision-analytic and cost-effectiveness models is to identify ways to improve women's health at an economically reasonable cost.

HPV? What's That?

Some strains of human papillomavirus are strongly associated with cervical cancer; in fact, HPV screening of women with abnormal Pap smear results has been proposed as a way to identify those who are at high risk for cervical cancer. But, are women well informed about HPV? A total of 1032 English-speaking women (mean age, 30) who attended a London well-woman clinic completed surveys …

Human Papillomavirus E2 Down-regulates the Human Telomerase Reverse Transcriptase Promoter

The transcriptional regulation of the human telomerase reverse transcriptase (hTERT) gene is a critical step in transformation and differentiation. Human papillomavirus E2 protein inhibits cell growth in HPV-infected cells and triggers apoptosis in HeLa cells. Because E2 induces cell growth suppression and senescence, we hypothesize that the protein may modulate cellular gene expression related to these processes. In this report, we demonstrate that E2 inhibits the hTERT promoter. The mapping of the E2-responsive region of hTERT reveals that Sp1 is important for E2-mediated repression of this promoter in 293T cells. Site-directed mutagenesis data on the hTERT promoter show that E2 does not abolish E-Box-mediated transcription and represses promoter activity via the Sp1 binding site. Furthermore, chromatin immunoprecipitation assays indicate that E2 is actively recruited to the hTERT promoter region. Our findings provide novel insights into the biological function of human papillomavirus E2.

The Human Papillomavirus 16 E6 Protein Binds to Tumor Necrosis Factor (TNF) R1 and Protects Cells from TNF-induced Apoptosis

High risk strains of human papillomavirus (HPV), such as HPV 16, cause human cervical carcinoma. The E6 protein of HPV 16 mediates the rapid degradation of p53, although this is not the only function of E6 and cannot completely explain its transforming potential. Previous work in our laboratory has demonstrated that transfection of HPV 16 E6 into the tumor necrosis factor (TNF)-sensitive LM cell line protects expressing cells from TNF-induced apoptosis in a p53-independent manner, and the purpose of this study was to determine the molecular mechanism underlying this protection. Caspase 3 and caspase 8 activation were significantly reduced in E6-expressing cells, indicating that E6 acts early in the TNF apoptotic pathway. In fact, E6 binds directly to TNF R1, as shown both by co-immunoprecipitation and mammalian two-hybrid approaches. E6 requires the same C-terminal portion of TNF R1 for binding as does TNF R1-associated death domain, and TNF R1/TNF R1-associated death domain interactions are decreased in the presence of E6. HA-E6 also blocked cell death triggered by transfection of the death domain of TNF R1. Together, these results provide strong support for a model in which HPV E6 binding to TNF R1 interferes with formation of the death-inducing signaling complex and thus with transduction of proapoptotic signals. They also demonstrate that HPV, like several other viruses, has developed a method for evading the TNF-mediated host immune response.

Suppression of pRB expression in normal human mammary epithelial cells is associated with resistance to all-trans-retinoic acid but not N-(4-hydroxylphenyl)-retinamide.

Despite the widespread clinical use of synthetic and naturally occurring retinoids, the down stream targets of retinoids have not been fully characterized. We observe that G(1/0)-phase arrest induced by all-trans-retinoic acid (ATRA) in normal human mammary epithelial cells (HMECs) is temporally associated with a significant decrease in the levels of hyperphosphorylated retinoblastoma protein (pRB). Suppression of pRB protein expression in HMECs by retroviral-mediated expression of the E7 protein of the human papillomavirus strain 16 (HPV-16) was associated with resistance to ATRA-mediated growth arrest but not to the synthetic retinoid N-(4-hydroxyphenyl) retinamide (4-HPR or fenretinide). 4-HPR but not ATRA induced apoptosis in HMECs independent of the level of pRB protein expression. These observations suggest that ATRA- but not 4-HPR-mediated growth arrest may be dependent on the coordinated expression of pRB and emphasize the chemotherapeutic potential of 4-HPR, particularly for suppressing growth of tumors lacking pRB function.

The Structural Basis of DNA Target Discrimination by Papillomavirus E2 Proteins

The papillomavirus E2 proteins regulate the transcription of all papillomavirus genes and are necessary for viral DNA replication. Disruption of the E2 gene is commonly associated with malignancy in cervical carcinoma, indicating that E2 has a role in regulating tumor progression. Although the E2 proteins from all characterized papillomaviruses bind specifically to the same 12-base pair DNA sequence, the cancer-associated human papillomavirus E2 proteins display a unique ability to detect DNA flexibility and intrinsic curvature. To understand the structural basis for this phenomenon, we have determined the crystal structures of the human papillomavirus-18 E2 DNA-binding domain and its complexes with high and low affinity binding sites. The E2 protein is a dimeric beta-barrel and the E2-DNA interaction is accompanied by a large deformation of the DNA as it conforms to the E2 surface. DNA conformation and E2-DNA contacts are similar in both high and low affinity complexes. The differences in affinity correlate with the flexibility of the DNA sequence. Preferences of E2 proteins from different papillomavirus strains for flexible or prevent DNA targets correlate with the distribution of positive charge on their DNA interaction surfaces, suggesting a role for electrostatic forces in the recognition of DNA deformability.

Microbial epitopes act as altered peptide ligands to prevent experimental autoimmune encephalomyelitis.

Molecular mimicry refers to structural homologies between a self-protein and a microbial protein. A major epitope of myelin basic protein (MBP), p87-99 (VHFFKNIVTPRTP), induces experimental autoimmune encephalomyelitis (EAE). VHFFK contains the major residues for binding of this self-molecule to T cell receptor (TCR) and to the major histocompatibility complex. Peptides from papilloma virus strains containing the motif VHFFK induce EAE. A peptide from human papilloma virus type 40 (HPV 40) containing VHFFR, and one from HPV 32 containing VHFFH, prevented EAE. A sequence from Bacillus subtilis (RKVVTDFFKNIPQRI) also prevented EAE. T cell lines, producing IL-4 and specific for these microbial peptides, suppressed EAE. Thus, microbial peptides, differing from the core motif of the self-antigen, MBPp87-99, function as altered peptide ligands, and behave as TCR antagonists, in the modulation of autoimmune disease.