scholarly journals Novel Production of Bovine Papillomavirus Pseudovirions in Tobacco Plants

Pathogens ◽  
2020 ◽  
Vol 9 (12) ◽  
pp. 996
Author(s):  
Inge Pietersen ◽  
Albertha van Zyl ◽  
Edward Rybicki ◽  
Inga Hitzeroth
Keyword(s):  
Mammalian Cells ◽  
Bovine Papillomavirus ◽  
In Planta ◽  
Neutralising Antibodies ◽  
Native Virus ◽  
Cell Culture Systems ◽  
Virus Capsids ◽  
Plant Expression Systems ◽  
L1 And L2

Vaccine efficacy requires the production of neutralising antibodies which offer protection against the native virus. The current gold standard for determining the presence of neutralising antibodies is the pseudovirion-based neutralisation assay (PBNA). PBNAs utilise pseudovirions (PsVs), structures which mimic native virus capsids, but contain non-viral nucleic material. PsVs are currently produced in expensive cell culture systems, which limits their production, yet plant expression systems may offer cheaper, safer alternatives. Our aim was to determine whether plants could be used for the production of functional PsVs of bovine papillomavirus 1 (BPV1), an important causative agent of economically damaging bovine papillomas in cattle and equine sarcoids in horses and wild equids. BPV1 capsid proteins, L1 and L2, and a self-replicating reporter plasmid were transiently expressed in Nicotiana benthamiana to produce virus-like particles (VLPs) and PsVs. Strategies to enhance particle yields were investigated and optimised protocols were established. The PsVs’ ability to infect mammalian cells and express their encapsidated reporter genes in vitro was confirmed, and their functionality as reagents in PBNAs was demonstrated through their neutralisation by several different antibodies. This is the first report of BPV PsVs expressed in plants and demonstrates the potential for the development of therapeutic veterinary vaccines in planta.

scholarly journals L1 Interaction Domains of Papillomavirus L2 Necessary for Viral Genome Encapsidation

2001 ◽  
Vol 75 (9) ◽  
pp. 4332-4342 ◽  
Author(s):  
Martin M. Okun ◽  
Patricia M. Day ◽  
Heather L. Greenstone ◽  
Frank P. Booy ◽  
Douglas R. Lowy ◽  
...  
Keyword(s):  
Viral Genome ◽  
Bovine Papillomavirus ◽  
Interaction Domain ◽  
Binding Domains ◽  
C Terminus ◽  
Interaction Domains ◽  
L1 And L2 ◽  
Genome Encapsidation

ABSTRACT BPHE-1 cells, which harbor 50 to 200 viral episomes, encapsidate viral genome and generate infectious bovine papillomavirus type 1 (BPV1) upon coexpression of capsid proteins L1 and L2 of BPV1, but not coexpression of BPV1 L1 and human papillomavirus type 16 (HPV16) L2. BPV1 L2 bound in vitro via its C-terminal 85 residues to purified L1 capsomers, but not with intact L1 virus-like particles in vitro. However, when the efficiency of BPV1 L1 coimmunoprecipitation with a series of BPV1 L2 deletion mutants was examined in vivo, the results suggested that residues 129 to 246 and 384 to 460 contain independent L1 interaction domains. An L2 mutant lacking the C-terminal L1 interaction domain was impaired for encapsidation of the viral genome. Coexpression of BPV1 L1 and a chimeric L2 protein composed of HPV16 L2 residues 1 to 98 fused to BPV1 L2 residues 99 to 469 generated infectious virions. However, inefficient encapsidation was seen when L1 was coexpressed with either BPV1 L2 with residues 91 to 246 deleted or with BPV1 L2 with residues 1 to 225 replaced with HPV16 L2. Impaired genome encapsidation did not correlate closely with impairment of the L2 proteins either to localize to promyelocytic leukemia oncogenic domains (PODs) or to induce localization of L1 or E2 to PODs. We conclude that the L1-binding domain located near the C terminus of L2 may bind L1 prior to completion of capsid assembly, and that both L1-binding domains of L2 are required for efficient encapsidation of the viral genome.

scholarly journals Bovine Papillomavirus Type 1 Infection Is Mediated by SNARE Syntaxin 18

2007 ◽  
Vol 81 (14) ◽  
pp. 7435-7448 ◽  
Author(s):  
Valerie Laniosz ◽  
Kha C. Nguyen ◽  
Patricio I. Meneses
Keyword(s):  
Viral Infections ◽  
Major Capsid Protein ◽  
Target Cells ◽  
Bovine Papillomavirus ◽  
Snare Protein ◽  
Golgi Compartment ◽  
L1 And L2

ABSTRACT Events that lead to viral infections include the binding of the virus to the target cells, internalization of the virus into the cells, and the ability of the viral genome to be expressed. These steps are mediated by cellular and viral proteins and are temporally regulated. The papillomavirus capsid consists of two virally encoded capsid proteins, L1 and L2. Much is known about the role of the major capsid protein L1 compared to what is known of the role of the L2 protein. We identified the interaction of the L2 protein with SNARE protein syntaxin 18, which mediates the trafficking of vesicles and their cargo between the endoplasmic reticulum, the cis-Golgi compartment, and possibly the plasma membrane. Mutations of L2 residues 41 to 44 prevented the interaction of L2 protein with syntaxin 18 in cotransfection experiments and resulted in noninfectious pseudovirions. In this paper, we describe that syntaxin 18 colocalizes with infectious bovine papillomavirus type 1 (BPV1) pseudovirions during infection but does not colocalize with the noninfectious BPV1 pseudovirions made with an L2 mutant at residues 41 to 44. We show that an antibody against BPV1 L2 residues 36 to 49 (αL2 36-49) binds to in vitro-generated BPV1 pseudoviral capsids and does not coimmunoprecipitate syntaxin 18- and BPV1 L2-transfected proteins. αL2 36-49 was able to partially or completely neutralize infection of BPV1 pseudovirions and genuine virions. These results support the dependence of syntaxin 18 during BPV1 infection and the ability to interfere with infection by targeting the L2-syntaxin 18 interaction and further define the infectious route of BPV1 mediated by the L2 protein.

Exploring DNA damage responses in human cells with recombinant adenoviral vectors

2007 ◽  
Vol 26 (11) ◽  
pp. 899-906
Author(s):  
Melissa G. Armelini ◽  
Keronninn M. Lima-Bessa ◽  
Maria Carolina N. Marchetto ◽  
Alysson R. Muotri ◽  
Vanessa Chiganças ◽  
...  
Keyword(s):  
Dna Damage ◽  
Mammalian Cells ◽  
Excision Repair ◽  
Adenoviral Vectors ◽  
Dna Lesions ◽  
Human Cells ◽  
Polymerase Eta ◽  
Cell Culture Systems

Recombinant adenoviral vectors provide efficient means for gene transduction in mammalian cells in vitro and in vivo. We are currently using these vectors to transduce DNA repair genes into repair deficient cells, derived from xeroderma pigmentosum (XP) patients. XP is an autosomal syndrome characterized by a high frequency of skin tumors, especially in areas exposed to sunlight, and, occasionally, developmental and neurological abnormalities. XP cells are deficient in nucleotide excision repair (affecting one of the seven known XP genes, xpa to xpg) or in DNA replication of DNA lesions (affecting DNA polymerase eta, xpv). The adenovirus approach allows the investigation of different consequences of DNA lesions in cell genomes. Adenoviral vectors carrying several xp and photolyases genes have been constructed and successfully tested in cell culture systems and in vivo directly in the skin of knockout model mice. This review summarizes these recent data and proposes the use of recombinant adenoviruses as tools to investigate the mechanisms that provide protection against DNA damage in human cells, as well as to better understand the higher predisposition of XP patients to cancer. Human & Experimental Toxicology (2007) 26, 899—906

scholarly journals Functional interaction of a novel cellular protein with the papillomavirus E2 transactivation domain.

1997 ◽  
Vol 17 (12) ◽  
pp. 7208-7219 ◽  
Author(s):  
D E Breiding ◽  
F Sverdrup ◽  
M J Grossel ◽  
N Moscufo ◽  
W Boonchai ◽  
...  
Keyword(s):  
Amino Acid ◽  
Dna Replication ◽  
Transcriptional Activation ◽  
Mammalian Cells ◽  
Point Mutations ◽  
Cellular Protein ◽  
Bovine Papillomavirus ◽  
Transactivation Domain

The transactivation domain (AD) of bovine papillomavirus type 1 E2 stimulates gene expression and DNA replication. To identify cellular proteins that interact with this 215-amino-acid domain, we used a transactivation-defective mutant as bait in the yeast two-hybrid screen. In vitro and in vivo results demonstrate that the cDNA of one plasmid isolated in this screen encodes a 37-kDa nuclear protein that specifically binds to an 82-amino-acid segment within the E2 AD. Mutants with point mutations within this E2 domain were isolated based on their inability to interact with AMF-1 and were found to be unable to stimulate transcription. These mutants also exhibited defects in viral DNA replication yet retained binding to the viral E1 replication initiator protein. Overexpression of AMF-1 stimulated transactivation by both wild-type E2 and a LexA fusion to the E2 AD, indicating that AMF-1 is a positive effector of the AD of E2. We conclude that interaction with AMF-1 is necessary for the transcriptional activation function of the E2 AD in mammalian cells.

scholarly journals Efficient Intracellular Assembly of Papillomaviral Vectors

2004 ◽  
Vol 78 (2) ◽  
pp. 751-757 ◽  
Author(s):  
Christopher B. Buck ◽  
Diana V. Pastrana ◽  
Douglas R. Lowy ◽  
John T. Schiller
Keyword(s):  
Dna Sequences ◽  
Mammalian Cells ◽  
Neutralizing Antibodies ◽  
Production Efficiency ◽  
Gradient Centrifugation ◽  
Cesium Chloride ◽  
Structural Proteins ◽  
Bovine Papillomavirus ◽  
Early Protein ◽  
L1 And L2

ABSTRACT Although the papillomavirus structural proteins, L1 and L2, can spontaneously coassemble to form virus-like particles, currently available methods for production of L1/L2 particles capable of transducing reporter plasmids into mammalian cells are technically demanding and relatively low-yield. In this report, we describe a simple 293 cell transfection method for efficient intracellular production of papillomaviral-based gene transfer vectors carrying reporter plasmids. Using bovine papillomavirus type 1 (BPV1) and human papillomavirus type 16 as model papillomaviruses, we have developed a system for producing papillomaviral vector stocks with titers of several billion transducing units per milliliter. Production of these vectors requires both L1 and L2, and transduction can be prevented by papillomavirus-neutralizing antibodies. The stocks can be purified by an iodixanol (OptiPrep) gradient centrifugation procedure that is substantially more effective than standard cesium chloride gradient purification. Although earlier data had suggested a potential role for the viral early protein E2, we found that E2 protein expression did not enhance the intracellular production of BPV1 vectors. It was also possible to encapsidate reporter plasmids devoid of BPV1 DNA sequences. BPV1 vector production efficiency was significantly influenced by the size of the target plasmid being packaged. Use of 6-kb target plasmids resulted in BPV1 vector yields that were higher than those with target plasmids closer to the native 7.9-kb size of papillomavirus genomes. The results suggest that the intracellular assembly of papillomavirus structural proteins around heterologous reporter plasmids is surprisingly promiscuous and may be driven primarily by a size discrimination mechanism.

scholarly journals Identification of a Second Transforming Function in Bovine Papillomavirus Type 1 E6 and the Role of E6 Interactions with Paxillin, E6BP, and E6AP

2000 ◽  
Vol 74 (2) ◽  
pp. 812-816 ◽  
Author(s):  
Kingshuk Das ◽  
Joanna Bohl ◽  
Scott B. Vande Pol
Keyword(s):  
Calcium Binding ◽  
Mammalian Cells ◽  
Strong Association ◽  
Selection Strategy ◽  
Temperature Sensitive ◽  
Bovine Papillomavirus ◽  
Cellular Targets ◽  
Transformation Activity

ABSTRACT Papillomavirus E6 oncoproteins transform mammalian cells through interaction with cellular proteins. Bovine papillomavirus type 1 E6 (BE6) interacts with three previously described cellular targets: the E6AP E3 ubiquitin ligase, the calcium-binding protein E6BP (also known as ERC-55), and paxillin, which is a focal adhesion adapter protein. BE6 interacts strongly with each of these proteins in vitro, binding to similar peptide sequences found in E6AP, E6BP, and paxillin. To determine which BE6 interactions are necessary for transformation by BE6, we used a novel selection strategy for temperature-sensitive BE6 mutants in yeast that could discriminate in their interaction between E6AP, E6BP, and paxillin. All BE6 mutants that retained transforming ability retained association with paxillin, while some mutants that were transformation positive failed to interact with E6AP or E6BP. This study demonstrates that oncogene mutants that are temperature sensitive for transformation can be selected in yeast and that the induction of anchorage-independent cell proliferation by BE6 does not require strong association of BE6 with either E6AP or E6BP. Of particular interest is the identification of a BE6 mutant that interacts strongly with the acidic charged leucine motifs of E6AP, E6BP, and paxillin but is devoid of transformation activity, thereby genetically identifying a second essential transformation function in BE6 that is independent of interaction with acidic charged leucine motifs.

scholarly journals N-Glycosylation Modification of Plant-Derived Virus-Like Particles: An Application in Vaccines

2014 ◽  
Vol 2014 ◽  
pp. 1-8 ◽  
Author(s):  
Hyun-Soon Kim ◽  
Jae-Heung Jeon ◽  
Kyung Jin Lee ◽  
Kisung Ko
Keyword(s):  
Mammalian Cells ◽  
Structural Characteristics ◽  
Regulatory Elements ◽  
In Planta ◽  
Virus Like Particles ◽  
Therapeutic Delivery ◽  
Recombinant Glycoproteins ◽  
Animal Use ◽  
Plant Expression Systems ◽  
And Control

Plants have been developed as an alternative system to mammalian cells for production of recombinant prophylactic or therapeutic proteins for human and animal use. Effective plant expression systems for recombinant proteins have been established with the optimal combination of gene expression regulatory elements and control of posttranslational processing of recombinant glycoproteins. In plant, virus-like particles (VLPs), viral “empty shells” which maintain the same structural characteristics of virions but are genome-free, are considered extremely promising as vaccine platforms and therapeutic delivery systems. Unlike microbial fermentation, plants are capable of carrying outN-glycosylation as a posttranslational modification of glycoproteins. Recent advances in the glycoengineering in plant allow human-like glycomodification and optimization of desired glycan structures for enhancing safety and functionality of recombinant pharmaceutical glycoproteins. In this review, the current plant-derived VLP approaches are focused, andN-glycosylation and its in planta modifications are discussed.

Specific Labeling of Protein Domains with Antibody Fragments

1981 ◽  
Vol 39 ◽  
pp. 416-417
Author(s):  
U. Aebi ◽  
L.E. Buhle ◽  
W.E. Fowler
Keyword(s):  
Image Processing ◽  
Specific Protein ◽  
Antibody Fragments ◽  
Supramolecular Organization ◽  
Two Dimensional ◽  
Ordered Structures ◽  
Virus Capsids ◽  
Processing Techniques

Many important supramolecular structures such as filaments, microtubules, virus capsids and certain membrane proteins and bacterial cell walls exist as ordered polymers or two-dimensional crystalline arrays in vivo. In several instances it has been possible to induce soluble proteins to form ordered polymers or two-dimensional crystalline arrays in vitro. In both cases a combination of electron microscopy of negatively stained specimens with analog or digital image processing techniques has proven extremely useful for elucidating the molecular and supramolecular organization of the constituent proteins. However from the reconstructed stain exclusion patterns it is often difficult to identify distinct stain excluding regions with specific protein subunits. To this end it has been demonstrated that in some cases this ambiguity can be resolved by a combination of stoichiometric labeling of the ordered structures with subunit-specific antibody fragments (e.g. Fab) and image processing of the electron micrographs recorded from labeled and unlabeled structures.

Ultrastructural Changes in Three Different Mammalian Cells Following Ultraviolet Laser Irradiation

1972 ◽  
Vol 30 ◽  
pp. 350-351
Author(s):  
K. Shankar Narayan ◽  
Kailash C. Gupta ◽  
Tohru Okigaki
Keyword(s):  
Ultraviolet Light ◽  
Mammalian Cells ◽  
Biological Effects ◽  
Survival Rates ◽  
Chinese Hamster ◽  
Cell Types ◽  
Differential Sensitivity ◽  
Ultrastructural Changes ◽  
Ultraviolet Laser

The biological effects of short-wave ultraviolet light has generally been described in terms of changes in cell growth or survival rates and production of chromosomal aberrations. Ultrastructural changes following exposure of cells to ultraviolet light, particularly at 265 nm, have not been reported.We have developed a means of irradiating populations of cells grown in vitro to a monochromatic ultraviolet laser beam at a wavelength of 265 nm based on the method of Johnson. The cell types studies were: i) WI-38, a human diploid fibroblast; ii) CMP, a human adenocarcinoma cell line; and iii) Don C-II, a Chinese hamster fibroblast cell strain. The cells were exposed either in situ or in suspension to the ultraviolet laser (UVL) beam. Irradiated cell populations were studied either "immediately" or following growth for 1-8 days after irradiation.Differential sensitivity, as measured by survival rates were observed in the three cell types studied. Pattern of ultrastructural changes were also different in the three cell types.

Vacuoles Induced in Mammalian Cells by Helicobacter Cytotoxin are Acidic Organelles

1990 ◽  
Vol 48 (3) ◽  
pp. 168-169
Author(s):  
M. H. Chestnut ◽  
C. E. Catrenich
Keyword(s):  
Acridine Orange ◽  
Mammalian Cells ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Ulcer Disease ◽  
Gastroduodenal Disease ◽  
H Pylori

Helicobacter pylori is a non-invasive, Gram-negative spiral bacterium first identified in 1983, and subsequently implicated in the pathogenesis of gastroduodenal disease including gastritis and peptic ulcer disease. Cytotoxic activity, manifested by intracytoplasmic vacuolation of mammalian cells in vitro, was identified in 55% of H. pylori strains examined. The vacuoles increase in number and size during extended incubation, resulting in vacuolar and cellular degeneration after 24 h to 48 h. Vacuolation of gastric epithelial cells is also observed in vivo during infection by H. pylori. A high molecular weight, heat labile protein is believed to be responsible for vacuolation and to significantly contribute to the development of gastroduodenal disease in humans. The mechanism by which the cytotoxin exerts its effect is unknown, as is the intracellular origin of the vacuolar membrane and contents. Acridine orange is a membrane-permeant weak base that initially accumulates in low-pH compartments. We have used acridine orange accumulation in conjunction with confocal laser scanning microscopy of toxin-treated cells to begin probing the nature and origin of these vacuoles.

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