Ultrastructure and taxonomy of Paraphysomonas (Chrysophyceae) and related genera 2

This is the second of two accounts reporting on the occurrence and systematics of freshwater species of the colourless chrysophycean genus Paraphysomonas in the Cambridge area, using electron microscopy of whole mounts of both cells and scales and sections of scales. Twenty species are included here, eleven of which are new: P. cancellala, P. canistrum, P. homolepis, P. ignivoma, P. limbata, P. morchella, P. runcinifera, P. stelligera, P. stephanolepis, P. subquadrangularis and P. undulata. Five new subspecies are also established: P. circumvallata ssp. mediogranulata, P. poteriophora ssp. manubriata, P. punctata ssp. atrema , ssp. microlepis and ssp. simplicior. P. diademifera (Takahashi) comb. nov. [= Lepidochromonas diademifera (Takahashi) Kristiansen] has been included in Paraphysomonas since it has been found to be colourless and not pigmented as originally described and also to possess scales of a similar structure to those in other species of Paraphysomonas. One previously described species, P. inconspicua Takahashi, is here considered as a synonym of P. butcheri Pennick & Clarke. P. vacuolata Thomsen is removed from Paraphysomonas since it has been found to possess a chloroplast and will be included in a new genus in the third paper of this series. The 37 presently known species of Paraphysomonas have been arranged in 11 different groups according to similarities in scale structure. Consideration of the possible evolution of scale structure in Paraphysomonas and the possible interrelationships within the genus led to the conclusion that all species should be included in a single genus.     

Ultrastructure and taxonomy of Paraphysomonas (Chrysophyceae) and related genera 3

New observations are presented on the internal ultrastructure of the scale–bearing chrysophycean genera Chromophysomonas, Chrysosphaerella , the new genus Polylepidomonas and 15 species of Paraphysomonas. These data show that the pigmented genera Chromophysomonas, Chrysosphaerella and Polylepidomonas have a generally similar internal structure and that their taxonomic separation is based only on differences in scale structure. The structure of Paraphysomonas resembles that of these genera but the cells always possess a leucoplast rather than a chloroplast. In cell structure, the pigmented genera resemble the naked genus Ochromonas while Paraphysomonas resembles Spumella , the colourless counterpart of Ochromonas. Evaluation of the differences between these genera and the scale–bearing genera Mallomonas and Synura has led to the conclusion that Chromophysomonas, Chrysosphaerella, Polylepidomonas and Paraphysomonas should no longer be classified within the family Mallomonadaceae. The new family Paraphysomonadaceae is established to include Chrysophyceae with an Ochromonas type of cell structure but which also produce silica scales.     

Virus-like particles from killer, neutral, and sensitive strains of Saccharomyces cerevisiae.

Procedures were developed for purification of virus-like particles (VLPs) from killer, neutral, and sensitive strains of Saccharomyces cerevisiae. Morphologically similar spherical VLPs measuring 40 nm in diameter were extracted from all three phenotypes. The particles were partially purified by high-speed centrifugation through a layer of CsCl (1.26 g/cm3) and sucrose density gradient centrifugation. Gradient-purified preparations contained three centrifugal species that sedimented at approximately 43, 102, and 162S. The 43S component is considered to be an artifact. Preparations from killer strains contained three double-stranded RNA (ds-RNA) components with molecular weights of 1.19 x 10(6), 1.29 x 10(6) and 2.54 x 10(6). VLPs from neutral and sensitive strains contained only the largest ds-RNA species. VLP preparations were subsequently separated into two major density components by CsCl equilibrium gradient centrifugation. The light component banding at 1.28 to 1.30 g/cm3 was void of nucleic acid, and the heavy component banding at 1.40 g/cm3 contained only the largest ds-RNA species.     

Characterization and replicase activity of double-layered and single-layered rotavirus-like particles expressed from baculovirus recombinants.

Rotavirus has a capsid composed of three concentric protein layers. We coexpressed various combinations of the rotavirus structural proteins of single-layered (core) and double-layered (single-shelled) capsids from baculovirus vectors in insect cells and determined the ability of the various combinations to assemble into viruslike particles (VLPs). VLPs were purified by centrifugation, their structure was examined by negative-stain electron microscopy, their protein content was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and GTP binding assays, and their ability to support synthesis of negative-strand RNAs on positive-sense template RNAs was determined in an in vitro replication system. Coexpression of all possible combinations of VP1, VP2, VP3, and VP6, the proteins of double-layered capsids, resulted in the formation of VP1/2/3/6, VP1/2/6, VP2/3/6, and VP2/6 double-layered VLPs. These VLPs had the structural characteristics of empty rotavirus double-layered particles and contained the indicated protein species. Only VPI/2/3/6 and VP1/2/6 particles supported RNA replication. Coexpression of all possible combinations of VPl, VP2, and VP3, the proteins of single-layered capsids, resulted in the formation of VP1/2/3, VP1/2, VP2/3, and VP2 single-layered VLPs. These VLPs had the structural characteristics of empty single-layered rotavirus particles and contained the indicated protein species. Only VP1/2/3 and VP1/2 VLPs supported RNA replication. We conclude that (i) the assembly of VP1 and VP3 into VLPs requires the presence of VP2, (ii) the role of VP2 in the assembly of VP1 and VP3 and in replicase activity is most likely structural, (iii) VP1 is required and VP3 is not required for replicase activity of VLPs, and (iv) VP1/2 VLPs constitute the minimal replicase particle in the in vitro replication system.     

共表达构建呈现人白细胞介素-13抗原肽的Qβ噬菌体病毒样颗粒

目的:构建呈现人白细胞介素-13(interleukin-13,IL-13)抗原肽的Qβ噬菌体病毒样颗粒(virus-like particles,VLPs)疫苗。方法:将人IL-13抗原肽经基因重组插入Qβ噬菌体衣壳蛋白(CP)的C端。在BL21细菌中,经IPTG诱导CP及C端接有IL-13抗原肽的CP(CP/IL-13)同时表达。以硫酸铵沉淀及蔗糖密度梯度离心进行VLPs纯化及分析嵌合VLPs的存在,以HPLC分析VLPs纯度,以电镜观察颗粒形态。小鼠经皮下免疫VLPs后采集血清,以ELISA检测人IL-13特异性Ig G抗体水平。结果:重组蛋白CP与CP/IL-13获得成功表达,两者在密度梯度离心中有一致的、与QβVLPs相同的沉降行为,而CP/IL-13单独无Qβ颗粒行为。经纯化获得了高纯度颗粒,嵌合颗粒与Qβ颗粒形态相似。此外,该VLPs疫苗诱导小鼠产生了IL-13特异的抗体应答。结论:利用共表达策略可成功构建呈现人IL-13抗原表位的嵌合VLPs,为以主动免疫方式调控IL-13在疾病中的病理作用,提供了具有临床应用潜能的疫苗形式。     

Purification and partial characterization of virus-like particles from Schneider line 2 Drosophila cells

A procedure has been developed for the purification of virus-like particles (VLPs) from Schneider line 2 Drosophila cells. The VLPs were precipitated with polyethylene glycol from the cytoplasmic fraction of lysed cells and further purified by equilibrium centrifugation in CsCl density gradients, in which they band at a density of 1.366 g/ml. Electron micrographs of these preparations revealed polyhedral particles with a diameter of 310–330 Å. We have also found particles of this size in thin sections of the intact cells. Sedimentation of the VLPs through 10–70% sucrose gradients yields a sedimentation coefficient of 235 S. Preliminary studies show that the VLPs contain double-stranded RNA species of 10 S, 14.5 S, 16 S, and 18 S.     

Isolation and characterization of a new dsRNA virus fromWickerhamia fluorescens

Virus-like particles (VLPs) were isolated from the yeastWickerhamia fluorescens strain CCY61-1-1. The VLPs are approximately 42 nm in diameter and contain only one species of dsRNA molecule. The apparent length of the dsRNA determined by native agarose gel electrophoresis was 4.6 kbp. Analysis of protein content of the VLPs showed them to contain one major capsid protein with an apparent molar mass of 74.5 kDa.     

Prevalence of virus-like particles within a staghorn scleractinian coral (<Emphasis Type="Italic">Acropora muricata</Emphasis>) from the Great Barrier Reef

Transmission electron microscopy (TEM) was used to determine whether Acropora muricata coral colonies from the Great Barrier Reef (GBR), Australia, harboured virus-like particles (VLPs). VLPs were present in all coral colonies sampled at Heron Island (southern GBR) and in tagged coral colonies sampled in at least two of the three sampling periods at Lizard Island (northern GBR). VLPs were observed within gastrodermal and epidermal tissues, and on rarer occasions, within the mesoglea. These VLPs had similar morphologies to known prokaryotic and eukaryotic viruses in other systems. Icosahedral VLPs were observed most frequently, however, filamentous VLPs (FVLPs) and phage were also noted. There were no clear differences in VLP size, morphology or location within the tissues with respect to sample date, coral health status or site. The most common VLP morphotype exhibited icosahedral symmetry, 120–150 nm in diameter, with an electron-dense core and an electronlucent membrane. Larger VLPs of similar morphology were also common. VLPs occurred as single entities, in groups, or in dense clusters, either as free particles within coral tissues, or within membrane-bound vacuoles. VLPs were commonly observed within the perinuclear region, with mitochondria, golgi apparatus and crescent-shaped particles frequently observed within close proximity. The host(s) of these observed VLPs was not clear; however, the different sizes and morphologies of VLPs observed within A. muricata tissues suggest that viruses are infecting either the coral animal, zooxanthellae, intracellular bacteria and/or other coral-associated microbiota, or that the one host is susceptible to infection from more than one type of virus. These results add to the limited but emerging body of evidence that viruses represent another potentially important component of the coral holobiont.     

Assembly of the capsid protein of red-spotted grouper nervous necrosis virus during purification,and role of calcium ions in chromatography

Currently virus-like particles (VLPs) are receiving much attention as platforms for next generation vaccines. However, chromatography-based methods for purifying VLPs remain challenging. Unlike traditional methods using density gradient for purifying VLPs, there have been few advances in explaining how assembled particles can be obtained by chromatography. Nervous necrosis virus (NNV) infects over 30 species of fish and leads to large economic losses in the farmed fish industry. Previously we developed a heparin chromatography-based method for purifying red-spotted grouper NNV (RGNNV) VLPs. However it is unclear how the assembled RGNNV VLPs are obtained by this method. It is known that assembly of NNV capsid proteins depends on calcium ions. In the present study, we found that the yield of purified RGNNV capsid protein in heparin chromatography was enhanced when calcium ions were present during binding. Also, it appears that the capsid protein of RGNNV undergoes partial disassembly and reassembly during sample preparation prior to heparin chromatography and the protein finally undergoes assembly during the chromatography. Therefore, our results indicated that heparin-binding affinity of RGNNV capsid protein is linked to its ability for VLP formation. The assembly of RGNNV capsid proteins recombinantly produced is a good model for explaining VLP formation during chromatography-based purification processes.     

Regeneration of Phosphorus and Nitrogen by Four Species of Heterotrophic Nanoflagellates Feeding on Three Nutritional States of a Single Bacterial Strain

Three physiological states of a single bacterial strain, namely, balanced, phosphorus-rich, and nitrogen-rich bacteria, were obtained by culturing a bacterial strain in chemostats under three different nutrient regimens. Each was shown to be distinctly different in elemental composition with respect to C/N/P ratio. These bacteria were fed to four species of heterotrophic nanoflagellates in batch culture grazing experiments, and the percent regeneration efficiencies of bacterium-bound nitrogen and phosphorus by the flagellates were compared. All flagellate species regenerated comparable amounts of nitrogen, which was thought to be due to their similar internal C/N ratios. There was, however, interspecies variation with regard to phosphorus regeneration: the two faster-growing species (Paraphysomonas imperforata and Bodo designis) released significantly more phosphorus than the two slower-growing species (Stephanoeca diplocostata and Jakoba libera). The observed differences were thought to have been influenced by a combination of life cycle strategies and internal C/P ratios.     

Isometric Virus-like particles in Abies alba Mill. and Other Abies Species: Partial Purification and Improved Detection by Means of Immunoelectron Microscopy

Abstract Two types of isometric virus-like particles (VLPs) with diameters of c. 20 and 35 nm were detected in partially purified extracts from Abies alba Mill., A. homolepis Sieb. et Zucc., and five other Abies species. An immunoelectron microscopical test was developed by means of which the large VLPs could often be detected even in crude sap preparations. They were to be widespread in A. alba and A. homolepis in the Federal Republic of Germany and Southern France. Theirpresence in glasshouse-grown seedlings of A. alba and A. homolepis shows that they are seed-transmissible. The small particles were detected only in partically purified preparations. Neither type of VLP was found in coniferous genera otherthan Abies.     

Scale–bearing Chrysophyceae (Mallomonadaceae) from Central and Northern Canada

In this study, scale–bearing Chrysophyceae (Mallomonadaceae) have been examined by means of transmission and scanning electron microscopy. Lakes in four areas in central and northern Canada, viz. Experimental Lakes Area (ELA), in northwestern Ontario, Whiteshell Provincial Park and Southern Indian Lake in eastern and northern Manitoba, respectively, and Saqvaqjuac on the west coast of Hudson Bay in the Northwest Territories have been investigated. Forty–three species of the genera Mallomonas, Paraphysomonas, Spiniferomonas and Synura have been identified in addition to three species of the genus Chrysochromulina (Prymnesiophyceae). Ten species are new to Canada; five of these have not previously been recorded from North America. Paraphysomonas elegantissima sp. nov. is described. The composition of the Canadian chrysophycean flora is compared with the chrysophycean flora of North America as a whole.     

Apparent global ubiquity of species in the protist genus Paraphysomonas

Evidence is presented for the ubiquity of protist species. Using the example of protists that leave traces (siliceous scales) of their recent population growth, we show that most - perhaps all species in the genus Paraphysomonas, are ubiquitous. Of the species recorded in surveys carried out worldwide, we have identified 78% of their number in 0.1 cm2 of sediment collected from a freshwater pond (total area 10(8) cm2) in England. Moreover, the pond appears to act like a microcosm of aquatic environments in general, for species that are globally rare or abundant, are likewise rare or abundant in the pond. We assume that the rate of neutral migration to the pond is greatest for the globally abundant species. As these species are probably capable of growth in a broad range of conditions, they will more frequently encounter the environment they require for population growth. Thus globally abundant species are also locally abundant in the pond - a pattern that will be amplified by periodic cyst production. Ubiquitous dispersal is probably driven by very high absolute abundance of individuals, and the water column of the pond was estimated to support >10(14) Paraphysomonas individuals. Ubiquity will dampen rates of speciation, and the evidence presented here indicates that global species richness of Paraphysomonas is indeed modest - perhaps close to what is already known.     

Binding and internalization of human papillomavirus type 33 virus-like particles by eukaryotic cells.

Infection of cells by human papillomaviruses (HPVs) associated with malignant genital lesions has not been studied because of the lack of an in vitro system and the unavailability of virions. We have now used virus-like particles (VLPs) of HPV type 33 to analyze the initial events in the interaction of the HPV capsid with cell lines. Binding of VLPs to HeLa cells was observed in biochemical assays and by immunofluorescence. VLP binding was inhibited by antisera raised against VLPs but not by monoclonal antibodies recognizing either L1 or L2 epitopes accessible on VLPs. Under saturating conditions, approximately 2 x 10(4) VLPs were bound per cell, with a dissociation constant of about 100 pM. VLPs composed of L1 alone bound as well as VLPs composed of both capsid proteins, indicating that L2 is not required for initial binding. VLPs dissociated into capsomers did not bind, demonstrating that intercapsomer contacts are required. Neither capsomers nor simian virus 40 virions competed with VLP binding. Uptake of VLPs by small and smooth endocytic vesicles was demonstrated by immunoelectron microscopy. Cellular binding of VLPs was sensitive to trypsin but not to sialidase, N-glycosidase, or octyl-beta-D-glycopyranoside treatment, suggesting that a cell surface protein is involved in the VLP binding. Cell lines originating from a variety of tissues and organisms as distantly related as insects and humans bound VLPs with similar efficiency and specificity. Therefore, the putative receptor mediating VLP attachment should be highly conserved and cannot be responsible for the species and tissue specificity of HPVs.     

Preclinical model to test human papillomavirus virus (HPV) capsid vaccines in vivo using infectious HPV/cottontail rabbit papillomavirus chimeric papillomavirus particles

A human papillomavirus (HPV) vaccine consisting of virus-like particles (VLPs) was recently approved for human use. It is generally assumed that VLP vaccines protect by inducing type-specific neutralizing antibodies. Preclinical animal models cannot be used to test for protection against HPV infections due to species restriction. We developed a model using chimeric HPV capsid/cottontail rabbit papillomavirus (CRPV) genome particles to permit the direct testing of HPV VLP vaccines in rabbits. Animals vaccinated with CRPV, HPV type 16 (HPV-16), or HPV-11 VLPs were challenged with both homologous (CRPV capsid) and chimeric (HPV-16 capsid) particles. Strong type-specific protection was observed, demonstrating the potential application of this approach.     

Isolation and characterization of the dsRNA virus from the yeast Endomyces magnusii

Abstract Virus-like particles (VLPs) have been isolated from the yeast Endomyces magnusii . The VLPs measure 43 nm in diameter and contain six species of dsRNA (0.78, 0.83, 1.77, 1.84, 2.64, 4.30 kb respectively). E. magnusii produces a 'toxic' protein, which reduces the growth, and changes the colony morphology, of sensitive strains of Hansenula sp. growing on solid media. All strains of E. magnusii tested produced the 'toxin' and contained the VLPs. Current procedures of curing failed to destroy the ability to produce the 'toxin'.     

Processing of infectious bursal disease virus (IBDV) polyprotein and self-assembly of IBDV-like particles in Hi-5 cells

The capsid of infectious bursal disease virus (IBDV), with a size of 60-65 nm, is formed by an initial processing of polyprotein (pVP2-VP4-VP3) by VP4, subsequent assemblage of pVP2 and VP3, and the maturation of VP2. In Sf9 cells, the processing of polyprotein expressed was restrained in the stage of VP2 maturation, leading to a limited production of capsid, i.e., IBDV-like particles (VLPs). In the present study, another insect cell line, High-Five (Hi-5) cells, was demonstrated to efficiently produce VLPs. Meanwhile, in this system, polyprotein was processed to pVP2 and VP3 protein and pVP2 was further processed to the matured form of VP2. Consequently, Hi-5 cells are better in terms of polyprotein processing and formation of VLPs than Sf9. In addition to the processing of pVP2, VP3 was also degraded. With insufficient intact VP3 protein present for the formation of VLPs, the excessive VP2 form subviral particles (SVPs) with a size of about 25 nm. The ratio of VLPs to SVPs is dependent on the multiplicity of infections (MOIs) used, and an optimal MOI is found for the production of both particles. VLPs were separated from SVPs with a combination of ultracentrifugation and gel-filtration chromatography, and a large number of purified particles of both were obtained. In conclusion, the insect cell lines and MOIs were optimized for the production of VLPs, and pure VLPs with morphology similar to that of the wild-type viruses can be effectively prepared. The efficient production and purification of VLPs benefits not only the development of an antiviral vaccine against IBDV but also the understanding of the structure of this avian virus that is economically important.     

Impact of internal RNA on aggregation and electrokinetics of viruses: comparison between MS2 phage and corresponding virus-like particles

We compare for the first time the electrokinetic and aggregation properties of MS2 phage (pH 2.5 to 7, 1 to 100 mM NaNO(3) electrolyte concentration) with those of the corresponding virus-like particles (VLPs), which lack entirely the inner viral RNA component. In line with our previous work (J. Langlet, F. Gaboriaud, C. Gantzer, and J. F. L. Duval, Biophys. J. 94:3293-3312, 2008), it is found that modifying the content of RNA within the virus leads to very distinct electrohydrodynamic and aggregation profiles for MS2 and MS2 VLPs. Under the given pH and concentration conditions, MS2 VLPs exhibit electrophoretic mobility larger in magnitude than that of MS2, and both have similar isoelectric point (IEP) values (～4). The electrokinetic results reflect a greater permeability of MS2 VLPs to electroosmotic flow, developed within/around these soft particles during their migration under the action of the applied electrical field. Results also support the presence of some remaining negatively charged component within the VLPs. In addition, MS2 phage systematically forms aggregates at pH values below the IEP, regardless of the magnitude of the solution ionic strength, whereas MS2 VLPs aggregate under the strict condition where the pH is relatively equal to the IEP at sufficiently low salt concentrations (<10 mM). It is argued that the stability of VLPs against aggregation and the differences between electrokinetics of MS2 and corresponding VLPs conform to recently developed formalisms for the stability and electrohydrodynamics of soft multilayered particles. The differences between the surface properties of these two kinds of particles reported here suggest that VLPs may not be appropriate for predicting the behavior of pathogenic viruses in aqueous media.     

Suitability and perspectives on using recombinant insect cells for the production of virus-like particles

Virus-like particles (VLPs) can be produced in recombinant protein production systems by expressing viral surface proteins that spontaneously assemble into particulate structures similar to authentic viral or subviral particles. VLPs serve as excellent platforms for the development of safe and effective vaccines and diagnostic antigens. Among various recombinant protein production systems, the baculovirus–insect cell system has been used extensively for the production of a wide variety of VLPs. This system is already employed for the manufacture of a licensed human papillomavirus-like particle vaccine. However, the baculovirus–insect cell system has several inherent limitations including contamination of VLPs with progeny baculovirus particles. Stably transformed insect cells have emerged as attractive alternatives to the baculovirus–insect cell system. Different types of VLPs, with or without an envelope and composed of either single or multiple structural proteins, have been produced in stably transformed insect cells. VLPs produced by stably transformed insect cells have successfully elicited immune responses in vivo. In some cases, the yield of VLPs attained with recombinant insect cells was comparable to, or higher than, that obtained by baculovirus-infected insect cells. Recombinant insect cells offer a promising approach to the development and production of VLPs.