Cryo EM Analysis Reveals Inherent Flexibility of Authentic Murine Papillomavirus Capsids.

Human papillomavirus (HPV) is a significant health burden and leading cause of virus-induced cancers. However, studies have been hampered due to restricted tropism that makes production and purification of high titer virus problematic. This issue has been overcome by developing alternative HPV production methods such as virus-like particles (VLPs), which are devoid of a native viral genome. Structural studies have been limited in resolution due to the heterogeneity, fragility, and stability of the VLP capsids. The mouse papillomavirus (MmuPV1) presented here has provided the opportunity to study a native papillomavirus in the context of a common laboratory animal. Using cryo EM to solve the structure of MmuPV1, we achieved 3.3 Å resolution with a local symmetry refinement method that defined smaller, symmetry related subparticles. The resulting high-resolution structure allowed us to build the MmuPV1 asymmetric unit for the first time and identify putative L2 density. We also used our program ISECC to quantify capsid flexibility, which revealed that capsomers move as rigid bodies connected by flexible linkers. The MmuPV1 flexibility was comparable to that of a HPV VLP previously characterized. The resulting MmuPV1 structure is a promising step forward in the study of papillomavirus and will provide a framework for continuing biochemical, genetic, and biophysical research for papillomaviruses.

Viruses Infecting the European Catfish (Silurus glanis).

In aquaculture, disease management and pathogen control are key for a successful fish farming industry. In past years, European catfish farming has been flourishing. However, devastating fish pathogens including limiting fish viruses are considered a big threat to further expanding of the industry. Even though mainly the ranavirus (Iridoviridea) and circovirus (Circoviridea) infections are considered well- described in European catfish, more other agents including herpes-, rhabdo or papillomaviruses are also observed in the tissues of catfish with or without any symptoms. The etiological role of these viruses has been unclear until now. Hence, there is a requisite for more detailed information about the latter and the development of preventive and therapeutic approaches to complete them. In this review, we summarize recent knowledge about viruses that affect the European catfish and describe their origin, distribution, molecular characterisation, and phylogenetic classification. We also highlight the knowledge gaps, which need more in-depth investigations in the future.

Papillomavirus assembly: An overview and perspectives.

Papillomavirus life cycle is tightly coupled to epithelial cell differentiation, which has hindered the investigation of many aspects of papillomavirus biology, including virion assembly. The development of in vitro production methods of papillomavirus pseudoviruses, and the production of "native" virus in raft cultures have facilitated the study of some aspects of the assembly process. In this paper we review the current knowledge of papillomavirus assembly, directions for future research, and the implications of these studies on new therapeutic interventions.

Biochemical fingerprints of human papillomavirus infection and cervical dysplasia using cervical fluids: Spectral pattern investigation.

The Pap smear is the primary screening tool for invasive cervical cancer resulting from a persistent infection with oncogenic human papillomavirus (HPV); however, there are the problems such as the inability to distinguish between HPV infection and cervical dysplasia and a low sensitivity remain. We present preliminary findings of a label-free method to detect and classify HPV infection and cervical dysplasia using human cervical fluids. Three experimental groups, defined as normal, HPV-positive, and cervical dysplasia, were evaluated through their Raman spectral patterns for noise-independence, high reproducibility, and uniformity. Clinical diagnosis was performed through liquid-based cervical cytology, HPV test, and cervical histologic examination. Healthy cervical fluids showed a strong Raman intensity at 877 cm-1 (symmetric C-C stretching), and at 963 cm-1 (phosphate), compared to a reference Raman peak at 1003 cm-1 (phenylalanine symmetric ring breath). The HPV-positive cervical fluids showed a strong intensity of a Raman peak at 1448 cm-1 corresponding to C-H deformation vibration mode and the highest similarity between the central and ring zones among the three groups. The cervical dysplasia fluids showed the presence of strong peaks compared to the control and HPV-positive groups. In addition, different Raman spectra were acquired according to HPV type. Therefore, all ranges of cervical fluid-induced Raman spectra could be used to detect the presence of cervical pre-cancer. Raman peak-gated assessment provides a label-free and nondestructive tool for the clinical diagnosis of HPV infection and cervical precancerous changes.

Ultrastructural study of bovine papillomavirus during outbreaks in Brazil / Estudio ultraestructural del virus papiloma bovino durante brotes en Brasil

Papillomaviruses, belonging to the Papillomaviridae family, are small oncogenic viruses, causing papillomas and fibropapillomas in the mucosal and cutaneous epithelia of several animals. In bovine species, thirteen types (BPV 1-13) were characterized to date. In this study, the occurrence of papillomatosis in four outbreaks in cattle herds, coming from Brazilian states were registered. The papillomatous lesions were found located in the teats, udders, head and neck. Under the transmission electron microscope, by the negative staining technique, it was possible to visualized rounded-format papillomavirus, with icosahedral symmetry, characterized as "full" and "empty" particles, measuring on average 60 nm in diameter, in all the 40 samples observed of skin lesion fragments. The ultrathin sections revealed the presence of groups of viral, intranuclear, rounded particles measuring 35 nm in diameter and tubular particles with a diameter of 35-39 nm. At immunoelectron microscopy technique, positivity obtained was marked by the presence of aggregates of viral particles formed by the antigen-antibody interaction. In the immunocytochemistry technique, the antigen-antibody reaction showed colloidal gold particles evenly distributed over the surface of the virus. These results showed the importance of the transmission electron microscopy techniques in the diagnosis of bovine papillomatosis that can be used in routine procedures to identify viral agent of this important disease.

Los virus del papiloma que pertenecen a la familia Papillomaviridae son pequeños virus oncogénicos que causan papilomas y fibropapilomas en epitelio cutáneo y mucoso de distintas especies de animales. En el ganado vacuno, trece tipos (BPV- 1-13) se caracterizaron hasta el momento. En este estudio, se documenta la ocurrencia de cuatro brotes de papilomatosis en los rebaños de ganado, procedentes de estados brasileños. Las lesiones papilomatosas se localizaron en los pezones, la ubre, la cabeza y el cuello. Al microscopio electrónico de transmisión, en la técnica de tinción negativa fueran visualizadas partículas del virus del papiloma redondeadas, con simetría icosaédrica, caracterizadas como "llenas" y "vacías", midiendo unos 60 nm de diámetro en todas las 40 muestras de fragmentos de lesión de piel estudiado. Los cortes ultra finos mostraron la presencia de grupos de partículas virales, intranucleares redondeadas con 35 nm de diámetro y tubulares 35-39 nm de diámetro. En la técnica de microscopía inmunoelectrónica, la positividad obtenida se caracterizó por la presencia de agregados de partículas virales formadas por la interacción antígeno-anticuerpo. En la aplicación de la técnica de inmunocitoquímica, la reacción antígeno-anticuerpo mostró partículas de oro coloidal distribuidos de manera uniforme sobre la superficie del virus. Estos resultados muestran la importancia de las técnicas de microscopía electrónica de transmisión en el diagnóstico de papilomatosis bovina, que pueden ser utilizados en los procedimientos de rutina para la identificación del agente viral causante de esta importante enfermedad.

Disassembly and reassembly of human papillomavirus virus-like particles produces more virion-like antibody reactivity.

BACKGROUND: Human papillomavirus (HPV) vaccines based on major capsid protein L1 are licensed in over 100 countries to prevent HPV infections. The yeast-derived recombinant quadrivalent HPV L1 vaccine, GARDASIL(R), has played an important role in reducing cancer and genital warts since its introduction in 2006. The L1 proteins self-assemble into virus-like particles (VLPs). RESULTS: VLPs were subjected to post-purification disassembly and reassembly (D/R) treatment during bioprocessing to improve VLP immunoreactivity and stability. The post-D/R HPV16 VLPs and their complex with H16.V5 neutralizing antibody Fab fragments were visualized by cryo electron microscopy, showing VLPs densely decorated with antibody. Along with structural improvements, post-D/R VLPs showed markedly higher antigenicity to conformational and neutralizing monoclonal antibodies (mAbs) H16.V5, H16.E70 and H263.A2, whereas binding to mAbs recognizing linear epitopes (H16.J4, H16.O7, and H16.H5) was greatly reduced. Strikingly, post-D/R VLPs showed no detectable binding to H16.H5, indicating that the H16.H5 epitope is not accessible in fully assembled VLPs. An atomic homology model of the entire HPV16 VLP was generated based on previously determined high-resolution structures of bovine papillomavirus and HPV16 L1 pentameric capsomeres. CONCLUSIONS: D/R treatment of HPV16 L1 VLPs produces more homogeneous VLPs with more virion-like antibody reactivity. These effects can be attributed to a combination of more complete and regular assembly of the VLPs, better folding of L1, reduced non-specific disulfide-mediated aggregation and increased stability of the VLPs. Markedly different antigenicity of HPV16 VLPs was observed upon D/R treatment with a panel of monoclonal antibodies targeting neutralization sensitive epitopes. Multiple epitope-specific assays with a panel of mAbs with different properties and epitopes are required to gain a better understanding of the immunochemical properties of VLPs and to correlate the observed changes at the molecular level. Mapping of known antibody epitopes to the homology model explains the changes in antibody reactivity upon D/R. In particular, the H16.H5 epitope is partially occluded by intercapsomeric interactions involving the L1 C-terminal arm. The homology model allows a more precise mapping of antibody epitopes. This work provides a better understanding of VLPs in current vaccines and could guide the design of improved vaccines or therapeutics.

The papillomavirus virion: a machine built to hide molecular Achilles' heels.

Papillomaviruses are a diverse group of DNA viruses that infect the skin and mucosal tissues of vertebrates. More than 100 distinct human papillomavirus (HPV) genotypes have so far been identified. A subset of HPVs is known to cause human cancer. Although recently developed vaccines protect vaccinated individuals from the two most carcinogenic HPV types, there is a pressing need for next-generation vaccines that might offer broad-spectrum protection against the full range of cancer-causing HPVs. The ongoing development of such vaccines will be facilitated by a deeper understanding of the mechanics of the assembly of the nonenveloped papillomavirus virion, as well as the machine-like structural changes that occur in the virion during the process of infectious entry into host cells. This chapter reviews the field's current knowledge of these two aspects of papillomavirus biology and speculates about areas where further work is needed.

[Role of immune alterations induced by papillomavirus in development of cervical cancer ]. / Rôle des altérations immunitaires induites par le papillomavirus humain dans le développement du cancer du col utérin.

Squamous cell cancer of the uterine cervix is associated with a high morbidity and mortality worldwide and in Belgium. The link between cervical cancer and HPV has generated, in recent years, a great interest for studies aiming to better understand the role of the immune system in the control of these infections and for the development of prophylactic anti-HPV vaccines. The purpose of this work was to analyse the immune co-factors implicated in the promotion of the neoplastic process. We have shown that both virus-induced immune alterations and squamous metaplasia in the transformation zone of the uterine cervix play a role to create an immunotolerogenic microenvironment during the cervical carcinogenesis.

Translational comparison of HPV58 long and short L1 mRNAs in yeast (Saccharomyces cerevisiae) cell-free system.

Recently, we developed a yeast cell-free system for translation of human papillomavirus (HPV) 58 short L1 mRNA and VLP assembly in vitro. In the present study, we examined the translation efficiency of HPV58 long and short L1 mRNAs in our established system. Production of HPV 58 long and short L1 proteins was significantly dependent on the amounts of the L1 mRNAs used and the time length of translation reaction. Compared with the long L1 mRNA, the short L1 mRNA consistently exhibited higher translation efficiency in all the experiments conducted. Supplementation of exogenous amino acids significantly enhanced translation of the long L1 mRNA in this system. Furthermore, a single amino acid leucine can be rate-limiting for translation of the long L1 mRNA in this system. Electron microscopy demonstrated that HPV 58 virus-like particles (VLPs) were assembled from both long and short L1 capsid proteins in the yeast cell-free system.

Papillomavirus pseudovirions packaged with the L2 gene induce cross-neutralizing antibodies.

BACKGROUND: Current vaccines against HPVs are constituted of L1 protein self-assembled into virus-like particles (VLPs) and they have been shown to protect against natural HPV16 and HPV18 infections and associated lesions. In addition, limited cross-protection has been observed against closely related types. Immunization with L2 protein in animal models has been shown to provide cross-protection against distant papillomavirus types, suggesting that the L2 protein contains cross-neutralizing epitopes. However, vaccination with L2 protein or L2 peptides does not induce high titers of anti-L2 antibodies. In order to develop a vaccine with the potential to protect against other high-risk HPV types, we have produced HPV58 pseudovirions encoding the HPV31 L2 protein and compared their capacity to induce cross-neutralizing antibodies with that of HPV L1 and HPV L1/L2 VLPs. METHODS: The titers of neutralizing antibodies against HPV16, HPV18, HPV31 and HPV58 induced in Balb/c mice were compared after immunization with L2-containing vaccines. RESULTS: Low titers of cross-neutralizing antibodies were detected in mice when immunized with L1/L2 VLPs, and the highest levels of cross-neutralizing antibodies were observed in mice immunized with HPV 58 L1/L2 pseudovirions encoding the HPV 31 L2 protein. CONCLUSIONS: The results obtained indicate that high levels of cross-neutralizing antibodies are only observed after immunization with pseudovirions encoding the L2 protein. HPV pseudovirions thus represent a possible new strategy for the generation of a broad-spectrum vaccine to protect against high-risk HPVs and associated neoplasia.

Subunit interactions in bovine papillomavirus.

Papillomaviruses, members of a group of dsDNA viruses associated with epithelial growths and tumors, have compact capsids assembled from 72 pentamers of the protein L1. We have determined the structure of bovine papillomavirus by electron cryomicrosopy (cryoEM), at approximately 3.6 A resolution. The density map, obtained from single-particle analysis of approximately 4,000 particle images, shows the trace of the L1 polypeptide chain and reveals how the N- and C-terminal "arms" of a subunit (extensions from its beta-jelly-roll core) associate with a neighboring pentamer. Critical contacts come from the C-terminal arm, which loops out from the core of the subunit, forms contacts (including a disulfide) with two subunits in a neighboring pentamer, and reinserts into the pentamer from which it emanates. This trace corrects one feature of an earlier model. We discuss implications of the structure for virion assembly and for pathways of infectious viral entry. We suggest that it should be possible to obtain image reconstructions of comparable resolution from cryoEM images of asymmetric particles. From the work on papillomavirus described here, we estimate that such a reconstruction will require about 1.5 million images to achieve the same number of averaged asymmetric units; structural variability will increase this number substantially.

The lift technique: forty years of experience at the department of pathology, Children's Hospital of Pittsburgh.

Despite the widespread utilization of immunohistochemical stains in pathologic practice, virus identification remains a challenge. In our institution, electron microscopy combined with the lift technique has been utilized for 40 years as a reliable diagnostic tool where a question of viral infection is raised by light microscopic observation and could not be otherwise confirmed. The combination of light microscopic and ultrastructural methods has allowed us to examine individual cells suspicious for harboring viral particles previously identified on a hematoxylin-eosin stained tissue section. In this review we describe the lift technique in detail with our modifications in the hope that the use of lift technique in these uncommon but specific circumstances will prove clinically valuable.

Development of a photodiode array biochip using a bipolar semiconductor and its application to detection of human papilloma virus.

We describe a DNA microarray system using a bipolar integrated circuit photodiode array (PDA) chip as a new platform for DNA analysis. The PDA chip comprises an 8 x 6 array of photodiodes each with a diameter of 600 microm. Each photodiode element acts both as a support for an immobilizing probe DNA and as a two-dimensional photodetector. The usefulness of the PDA microarray platform is demonstrated by the detection of high-risk subtypes of human papilloma virus (HPV). The polymerase chain reaction (PCR)-amplified biotinylated HPV target DNA was hybridized with the immobilized probe DNA on the photodiode surface, and the chip was incubated in an anti-biotin antibody-conjugated gold nanoparticle solution. The silver enhancement by the gold nanoparticles bound to the biotin of the HPV target DNA precipitates silver metal particles at the chip surfaces, which block light irradiated from above. The resulting drop in output voltage depends on the amount of target DNA present in the sample solution, which allows the specific detection and the quantitative analysis of the complementary target DNA. The PDA chip showed high relative signal ratios of HPV probe DNA hybridized with complementary target DNA, indicating an excellent capability in discriminating HPV subtypes. The detection limit for the HPV target DNA analysis improved from 1.2 nM to 30 pM by changing the silver development time from 5 to 10 min. Moreover, the enhanced silver development promoted by the gold nanoparticles could be applied to a broader range of target DNA concentration by controlling the silver development time.

An epidermotropic canine papillomavirus with malignant potential contains an E5 gene and establishes a unique genus.

A novel canine papillomavirus, CfPV-2, was cloned from a footpad lesion of a golden retriever. Unlike the known canine oral papillomavirus (COPV), which has a double-stranded DNA genome size of 8607 bps, the genome of CfPV-2 is 8101 bps. Some of this size difference is due to an abbreviated early-late region (ELR), which is 1200 bps shorter than that of COPV. However, CfPV-2 has other differences from COPV, including the presence of an E5 ORF between the E2 gene and the ELR and an enlarged E4 ORF (one of the largest PV E4 open reading frames). The genome of CfPV-2 shares low homology with all the other papillomaviruses and, even in the most highly conserved ORF of L1, the nucleotide sequence shares only 57% homology with COPV. Due to this highly divergent DNA sequence, CfPV-2 establishes a new PV genus, with its closest phylogenetic relatives being amongst the Xi and Gamma genuses. CfPV-2 also has unique biological features; it induces papillomas on footpads and interdigital regions which, if infection is persistent, can progress to highly metastatic squamous cell carcinoma. CfPV-2 does not induce oral papillomas in immunocompetent animals and antibodies generated against COPV and CfPV-2 are type-specific. The availability of a new canine papillomavirus with differing genetic and biological properties now makes it possible to study type-specific host immune responses, tissue tropism and the comparative analysis of viral gene functions in the dog.

Papillomavirus virus-like particles as vehicles for the delivery of epitopes or genes.

Papillomaviruses (PVs) are simple double-strand DNA viruses whose virion shells are T = 7 icosahedrons and composed of major capsid protein L1 and minor capsid protein L2.L1 alone or together with L2 can self-assemble into virus-like particles (VLPs) when expressed in eukaryotic or prokaryotic expression systems. Although the VLPs lack the virus genome DNA, their morphological and immunological characteristics are very similar to those of nature papillomaviruses. PV VLP vaccination can induce high titers of neutralizing antibodies and can effectively protect animals or humans from PV infection. Moreover, PV VLPs have been good candidates for vehicles to deliver epitopes or genes to target cells. They are widely used in the fields of vaccine development, neutralizing antibody detection, basic virologic research on papillomaviruses, and human papillomavirus (HPV) screening. Besides the structural biology and immunological basis for PV VLPs used as vehicles to deliver epitopes or genes, this review details the latest findings on chimeric papillomavirus VLPs and papillomavirus pseudoviruses, which are two important forms of PV VLPs used to transfer epitopes or genes.

Performance of cytology and human papillomavirus testing in relation to the menstrual cycle.

Cervical smears prepared around the time of menses have been linked to unsatisfactory specimens and false negative results; however, it is unclear whether liquid-based cytology is similarly affected and data relating date of last menstrual period (LMP) to human papillomavirus (HPV) DNA testing are conflicting. Accordingly, we evaluated liquid-based cytology and HPV test results using Hybrid Capture 2 and PCR by LMP (days 0-10; 11-21; 22-28). We studied 5060 participants in ALTS, the Atypical Squamous Cells of Undetermined Significance (ASCUS) Low Grade Squamous Intraepithelial Lesion (LSIL) Triage Study. On average, women had 3.4 examinations (median 4, range 1-5) during a 2-year period of observation permitting an examination of intra-individual variation in cytology and HPV by LMP. Although uncommon, unsatisfactory cytology specimens were most likely on days 0-10. For satisfactory specimens, the frequency with which cytologic categories were reported varied by time since LMP, although differences were modest and did not affect the chance of abnormal cytology or its severity among women diagnosed with CIN2+. The frequency of positive HC2 tests did not vary with date of LMP. Among HPV infected women, independent of eventual diagnosis and the number of viral genotypes present, mid-cycle specimens yielded the highest frequency of LSIL cytologic interpretations and the highest HPV load; however, the magnitude of these effects were small. Intraindividual correlations of cytology or HPV by LMP were generally weak. We conclude that mid-cycle specimens yield slightly higher HPV DNA loads and slightly increased LSIL interpretations, but the clinical impact is marginal. Standardizing collection times would slightly improve interpretation of trends in HPV load. Finally, these data are consistent with the view that the biological properties of the HPV-infected cervix vary with the date of the LMP.

Generation and applications of HPV pseudovirions using vaccinia virus.

This chapter outlines the generation and application of human papillomavirus type 33 (HPV 33) pseudovirions. These pseudovirions are structurally indistinguishable from native virions and are therefore valuable tools for the study of papillomavirus/cell interactions. The method describes (1) the construction of vaccinia viruses recombinant for the major and minor HPV capsid proteins, L1 and L2, respectively, (2) the transfection of Cos7 cells with a marker plasmid replicating to high copy numbers, (3) the expression of L1 and L2 using the vaccinia virus expression system, (4) the extraction, purification, and analysis of HPV-33 pseudovirions, (5) pseudoinfection assays, (6) pre- and post-attachment neutralization of pseudovirions, and (7) the use of inhibitors for study of binding and uptake of pseudovirions. The methods described have been successfully adopted for HPV 16 and 18 and may thus be applied for other HPV types, too.

Pigmented cutaneous papillomatosis (pigmented epidermal nevus) in three pug dogs; histopathology, electron microscopy and analysis of viral DNA by the polymerase chain reaction.

Canine pigmented epidermal nevus (CPEN) is a skin disorder of some breeds of dog characterized by multiple black plaques of the haired and non-haired skin. Three cases of pigmented cutaneous papillomatosis (previously described also as CPEN) in pug dogs were investigated histopathologically, immunohistochemically and electron microscopically. Additionally, DNA analyses with the polymerase chain reaction (PCR) were performed in two cases. Many nuclei of the stratum granulosa were diffusely immunolabelled for specific structural antigens of bovine papillomavirus (subgroup A), but nuclear inclusion bodies were not detected by retrospective examination of haematoxylin and eosin-stained sections of the affected skin. Aggregates of small numbers of viral particles (ranging from 37 to 43 nm in diameter) with a hexagonal structure were sparsely scattered throughout the nuclei of some of the superficial keratinocytes. PCR amplification targeted for the L1 gene of papillomavirus cloned from a case of CPEN yielded an expected fragment of 194-bp in the two CPEN cases examined but not in a case of canine oral papilloma.