Inactivation or removal of the budded particles of a nuclear polyhedrosis virus of a silkworm, Bombyx mori L. (Lep., Bombycidae)

Effective methods to inactivate or remove budded particles of a nuclear polyhedrosis virus of Bombyx mori (BmNPV) from a cell-cultured media or from host haemolymph that is infected by this virus have been developed. Two types of suspensions containing BmNPV budded virus particles, TC-100 media that cultured BmN4 cells infected by this virus and haemolymph of B. mori larvae infected by this virus, were treated by 6% (w/v) polyethylene glycol (PEG), 0.01% (w/v) chitosan, 0.05% (v/v) linoleic acid (an emulsion), and/or diethylether. Treatment by linoleic acid followed by PEG-precipitation and treatment by diethylether followed by PEG-precipitation were so effective that these treatments suppressed the viral titre of BmNPV-infected larval haemolymph from an original titre (> 10⁹ TCID₅₀ units/ml) to below a detectable limit. These methods are suggested as being potentially useful in an insect factory system; that is, a protein production system utilizing a baculovirus vector and its insect host or cultured cells on a large scale.     

THE STRUCTURE OF INSECT VIRUS PARTICLES

Thin sections have been cut of the virus particles from four types of insect virus diseases: cytoplasmic polyhedroses of lepidopterous larvae, a nuclear polyhedrosis of Tipula paludosa (Diptera), a granulosis from Melanchra persicariae (Lepidoptera), and a new virus disease without polyhedra from T. paludosa. The cytoplasmic polyhedral viruses are thought to have composite particles in some cases. The shape and enveloping membranes of the different virus particles are compared. In the new virus disease of T. paludosa some of the virus particles appear to be empty; inclusion bodies surrounded by complicated membranes are also demonstrated.     

Insect virus polyhedra, infectious protein crystals that contain virus particles

High-resolution atomic structures have been reported recently for two types of viral polyhedra, intracellular protein crystals produced by ubiquitous insect viruses. Polyhedra contain embedded virus particles and function as the main infectious form for baculoviruses and cypoviruses, two distinct classes of viruses that infect mainly Lepitoptera species (butterflies and moths). Polyhedra are extremely stable and protect the virus particles once released in the environment. The extensive crystal contacts observed in the structures explain the remarkable stability of viral polyhedra and provide hints about how these crystals dissolve in the alkaline midgut, releasing embedded virus particles to infect feeding larvae. The stage is now set to answer intriguing questions about the in vivo crystallization of polyhedra, how virus particles are incorporated into polyhedra, and what determines the size and shape of the crystals. Large quantities of polyhedra can be obtained from infected larvae and polyhedra can also be produced using insect cell expression systems. Modified polyhedra encapsulating other entities in place of virus particles have potential applications as a means to stabilize proteins such as enzymes or growth factors, and the extremely stable polyhedrin lattice may provide a framework for future engineered micro-crystal devices.     

Synthesis of bluetongue virus (BTV) corelike particles by a recombinant baculovirus expressing the two major structural core proteins of BTV.

The L3 and M7 genes of bluetongue virus (BTV), which encode the two major core proteins of the virus (VP3 and VP7, respectively), were inserted into a baculovirus dual-expression transfer vector and a recombinant baculovirus expressing both foreign genes isolated following in vivo recombination with wild-type Autographa californica nuclear polyhedrosis virus DNA. Spodoptera frugiperda insect cells infected with the recombinant synthesized large amounts of BTV corelike particles. These particles have been shown to be similar to authentic BTV cores in terms of size, appearance, stoichiometric arrangement of VP3 to VP7 (ratio, 2:15), and the predominance of VP7 on the surface of the particles. In infected insect cells, the corelike particles were observed in paracrystalline arrays. The formation of these structures indicates that neither the BTV double-stranded viral RNA species nor the associated minor core proteins are necessary for assembly of cores in insect cells. Furthermore, the three BTV nonstructural proteins NS1, NS2, and NS3, are not required to assist or direct the formation of empty corelike particles from VP3 and VP7.     

Intracellular localization of adeno-associated viral proteins expressed in insect cells

Production of vectors derived from adeno-associated virus (AAVv) in insect cells represents a feasible option for large-scale applications. However, transducing particles yields obtained in this system are low compared with total capsid yields, suggesting the presence of genome encapsidation bottlenecks. Three components are required for AAVv production: viral capsid proteins (VP), the recombinant AAV genome, and Rep proteins for AAV genome replication and encapsidation. Little is known about the interaction between the three components in insect cells, which have intracellular conditions different to those in mammalian cells. In this work, the localization of AAV proteins in insect cells was assessed for the first time with the purpose of finding potential limiting factors. Unassembled VP were located either in the cytoplasm or in the nucleus. Their transport into the nucleus was dependent on protein concentration. Empty capsids were located in defined subnuclear compartments. Rep proteins expressed individually were efficiently translocated into the nucleus. Their intranuclear distribution was not uniform and differed from VP distribution. While Rep52 distribution and expression levels were not affected by AAV genomes or VP, Rep78 distribution and stability changed during coexpression. Expression of all AAV components modified capsid intranuclear distribution, and assembled VP were found in vesicles located in the nuclear periphery. Such vesicles were related to baculovirus infection, highlighting its role in AAVv production in insect cells. The results obtained in this work suggest that the intracellular distribution of AAV proteins allows their interaction and does not limit vector production in insect cells.     

Quantification of rotavirus-like particles by gel permeation chromatography

There is a lack of accurate and practical methods that require only small amounts of sample for quantifying virus-like particles (VLP). In this work, gel permeation (GP) HPLC was used to quantify double-layered rotavirus-like particles (dlRLP) produced in insect cells. The proposed methodology utilized two columns in series (pore sizes of 200 and 50 nm) and had a high precision (relative standard deviation below 5%). GP-HPLC not only allowed the routine quantification of dlRLP, but also of assembly intermediaries and other viral structures present in the samples. For the first time, kinetics of dlRLP accumulation could be followed. This methodology is valuable for designing new production processes and for optimizing dlRLP monitoring.     

Structural Changes in Dengue Virus When Exposed to a Temperature of 37°C

Previous binding studies of antibodies that recognized a partially or fully hidden epitope suggest that insect cell-derived dengue virus undergoes structural changes at an elevated temperature. This was confirmed by our cryo-electron microscopy images of dengue virus incubated at 37°C, where viruses change their surface from smooth to rough. Here we present the cryo-electron microscopy structures of dengue virus at 37°C. Image analysis showed four classes of particles. The three-dimensional (3D) map of one of these classes, representing half of the imaged virus population, shows that the E protein shell has expanded and there is a hole at the 3-fold vertices. Fitting E protein structures into the map suggests that all of the interdimeric and some intradimeric E protein interactions are weakened. The accessibility of some previously found cryptic epitopes on this class of particles is discussed.     

同时表达蓝舌病毒四个主要结构蛋白可装配成病毒样颗粒

为研制蓝舌病毒（ｂｌｕｅｔｏｎｇｕｅ ｖｉｒｕｓ,ＢＴＶ）基因工程疫苗和进一步研究ＢＴＶ结构与功能的关系,对ＢＴＶ病毒样颗粒（ＶＬＰ）的装配进行了研究。同时在昆虫细胞中表达ＢＴＶ主要结构蛋白ＶＰ７、ＶＰ３、ＶＰ２与ＶＰ５,将细胞裂解液超速离心纯化后,发现主要存在两 形态的颗粒：一种与前文报道的病毒核心颗粒（ＣＬＰ）相同,直径约为６０ｎｍ￣７０ｎｍ,蛋白壳厚１０ｎｍ￣１５ｎｍ;另一种大小为７０ｎｍ￣     

Expression, self-assembly, and antigenicity of the Norwalk virus capsid protein.

Norwalk virus capsid protein was produced by expression of the second and third open reading frames of the Norwalk virus genome, using a cell-free translation system and baculovirus recombinants. Analysis of the expressed products showed that the second open reading frame encodes a protein with an apparent molecular weight of 58,000 (58K protein) and that this protein self-assembles to form empty viruslike particles similar to native capsids in size and appearance. The antigenicity of these particles was demonstrated by immunoprecipitation and enzyme-linked immunosorbent assays of paired serum samples from volunteers who developed illness following Norwalk virus challenge. These particles also induced high levels of Norwalk virus-specific serum antibody in laboratory animals following parenteral inoculation. A minor 34K protein was also found in infected insect cells. Amino acid sequence analysis of the N terminus of the 34K protein indicated that the 34K protein was a cleavage product of the 58K protein. The availability of large amounts of recombinant Norwalk virus particles will allow the development of rapid, sensitive, and reliable tests for the diagnosis of Norwalk virus infection as well as the implementation of structural studies.     

HCV结构蛋白在昆虫细胞中的表达及病毒样颗粒的组装

本文利用Bac-to-Bac杆状病毒表达系统构建了含有丙型肝炎病毒(Hepatitis C Virus,HCV)结构蛋白编码基因的重组杆状病毒vAcHCVspl,并获得了HCV结构蛋白在昆虫细胞Sf21中的表达。HCV mRNA转录和蛋白质表达时相分析表明,感染后16h HCV结构蛋白编码基因开始转录,72h达最高峰;蛋白质表达则是在感染后48h开始,72h达到高峰。电镜观察表明vAcHCVspl感染的Sf21细胞96h时在细胞质中可见很多空泡,空泡中可见50nm的球形颗粒,为HCV结构蛋白组装的病毒样颗粒。     

Virus-like particles of potato leafroll virus as potential carrier system for nucleic acids

Potato leafroll virus is a member of the polerovirus genus. The isometric virion is formed by a coat protein encapsidating single-stranded, positive-sense, mono-partite genomic RNA with covalently attached viral protein at the 5' end. The coat protein of the virus exists in two forms: i) a 23 kDa protein, the product of the coat protein gene, and ii) a 78 kDa protein, the product of the coat protein gene and an additional open reading frame expressed by read-through of the coat protein gene stop codon. The aim of this work was the expression of potato leafroll virus coat protein-based proteins that would be able to assemble into virus-like particles in insect cells. These modified particles were tested for their ability to encapsidate nucleic acids. Two types of N-terminally His-tagged coat protein constructs were used for the expression in insect cells: one, encoding a 23 kDa protein with the C-terminal amino-acid sequence corresponding to the wild type coat protein and the second with additional clathrin binding domain at the C-terminus. The expression of these two proteins by a recombinant baculovirus was characterized by Western immunoblotting with antibodies directed against potato leafroll virus. The protection or putative encapsidation of nucleic acids by these two coat protein derivatives was shown by DNase I and RNase A protection assays.     

Resolving structure and mechanical properties at the nanoscale of viruses with frequency modulation atomic force microscopy

Structural Biology (SB) techniques are particularly successful in solving virus structures. Taking advantage of the symmetries, a heavy averaging on the data of a large number of specimens, results in an accurate determination of the structure of the sample. However, these techniques do not provide true single molecule information of viruses in physiological conditions. To answer many fundamental questions about the quickly expanding physical virology it is important to develop techniques with the capability to reach nanometer scale resolution on both structure and physical properties of individual molecules in physiological conditions. Atomic force microscopy (AFM) fulfills these requirements providing images of individual virus particles under physiological conditions, along with the characterization of a variety of properties including local adhesion and elasticity. Using conventional AFM modes is easy to obtain molecular resolved images on flat samples, such as the purple membrane, or large viruses as the Giant Mimivirus. On the contrary, small virus particles (25-50 nm) cannot be easily imaged. In this work we present Frequency Modulation atomic force microscopy (FM-AFM) working in physiological conditions as an accurate and powerful technique to study virus particles. Our interpretation of the so called "dissipation channel" in terms of mechanical properties allows us to provide maps where the local stiffness of the virus particles are resolved with nanometer resolution. FM-AFM can be considered as a non invasive technique since, as we demonstrate in our experiments, we are able to sense forces down to 20 pN. The methodology reported here is of general interest since it can be applied to a large number of biological samples. In particular, the importance of mechanical interactions is a hot topic in different aspects of biotechnology ranging from protein folding to stem cells differentiation where conventional AFM modes are already being used.     

Number of Virus Particles in Insects and Plants Infected with Wound Tumor Virus

A new procedure for counting virus particles was employed to measure the concentration of wound tumor virus in purified virus preparations, in plant tumors, and in the insect vector. Partially purified wound tumor virus was used to establish the quantitative features of the method. A 1-g amount of plant tumor tissue contained an average of 5 x 10(10) virus particles and 1 g of insect tissue contained 2 x 10(10) particles.     

Hybrid human immunodeficiency virus Gag particles as an antigen carrier system: induction of cytotoxic T-cell and humoral responses by a Gag:V3 fusion.

In attempts to increase the immunogenicity of recombinant antigens, a number of particulate antigen presentation systems have been developed. In this study, we used human immunodeficiency virus Gag particles as carriers for the human immunodeficiency virus envelope V3 region. Gag:V3 fusion proteins were expressed from baculovirus expression vectors; they migrated to the insect cell membrane and budded from the cells as hybrid particles. An immunization study carried out with rats showed that the particles elicited a strong anti-Gag antibody response and a weak antibody response to the V3 region. A strong anti-V3 cytolytic T-cell response was elicited in immunized mice. These data show that retroviral Gag particles can be used as antigen presentation vehicles.     

Release of Dengue Virus Genome Induced by a Peptide Inhibitor

Dengue virus infects approximately 100 million people annually, but there is no available therapeutic treatment. The mimetic peptide, DN59, consists of residues corresponding to the membrane interacting, amphipathic stem region of the dengue virus envelope (E) glycoprotein. This peptide is inhibitory to all four serotypes of dengue virus, as well as other flaviviruses. Cryo-electron microscopy image reconstruction of dengue virus particles incubated with DN59 showed that the virus particles were largely empty, concurrent with the formation of holes at the five-fold vertices. The release of RNA from the viral particle following incubation with DN59 was confirmed by increased sensitivity of the RNA genome to exogenous RNase and separation of the genome from the E protein in a tartrate density gradient. DN59 interacted strongly with synthetic lipid vesicles and caused membrane disruptions, but was found to be non-toxic to mammalian and insect cells. Thus DN59 inhibits flavivirus infectivity by interacting directly with virus particles resulting in release of the genomic RNA.     

The composite insect trap: an innovative combination trap for biologically diverse sampling

Documentation of insect diversity is an important component of the study of biodiversity, community dynamics, and global change. Accurate identification of insects usually requires catching individuals for close inspection. However, because insects are so diverse, most trapping methods are specifically tailored to a particular taxonomic group. For scientists interested in the broadest possible spectrum of insect taxa, whether for long term monitoring of an ecosystem or for a species inventory, the use of several different trapping methods is usually necessary. We describe a novel composite method for capturing a diverse spectrum of insect taxa. The Composite Insect Trap incorporates elements from four different existing trapping methods: the cone trap, malaise trap, pan trap, and flight intercept trap. It is affordable, resistant, easy to assemble and disassemble, and collects a wide variety of insect taxa. Here we describe the design, construction, and effectiveness of the Composite Insect Trap tested during a study of insect diversity. The trap catches a broad array of insects and can eliminate the need to use multiple trap types in biodiversity studies. We propose that the Composite Insect Trap is a useful addition to the trapping methods currently available to ecologists and will be extremely effective for monitoring community level dynamics, biodiversity assessment, and conservation and restoration work. In addition, the Composite Insect Trap will be of use to other insect specialists, such as taxonomists, that are interested in describing the insect taxa in a given area.     

Secretory production system of bionanocapsules using a stably transfected insect cell line

Bionanocapsules (BNCs) are hollow nanoscale particles composed of L protein of the hepatitis B virus surface antigen that represent specific affinity for human hepatocytes. BNCs can transfer genes and drugs into human hepatocytes efficiently and specifically. BNC can be expressed in yeast cells. In this study, we developed a new L particle production system using a stably transfected insect cell line. For this purpose, we established a host–vector system using the Trichoplusia ni insect cell line. L particles were efficiently secreted by the overexpression of the L protein, which was fused to the secretion signal peptide. The concentration of L particles was reached approximately 1.7 μg/ml in 5 days during cultivation in a serum-free medium without antibiotic selective pressure. The production of L particles was maintained for at least 75 days. The secretory production of L particles facilitated their easy purification by chromatography. Furthermore, it was demonstrated that purified L particles can transfect only human hepatocytes. Therefore, an insect cell expression system is an attractive tool for the production of BNC.     

Intracellular distribution of rotavirus structural proteins and virus-like particles expressed in the insect cell-baculovirus system

The production of virus-like particles (VLP) is of interest to several fields. However, little is known about their assembly when they are expressed in insect cells, as it occurs in conditions different to those of native virus. Knowledge of the localization of recombinant proteins and of the site of accumulation of VLP can increase the understanding of VLP assembly and be useful for proposing production strategies. In this work, the rotavirus proteins VP6 and the fusion protein GFPVP2 were expressed in High Five insect cells. Recombinant proteins and rotavirus-like particles (RLP) were located and visualized by confocal, epifluorescence and electron microscopy. Single-layered (sl) RLP (conformed by GFPVP2) accumulated in the cytoplasm as highly ordered aggregates. In contrast, VP6 formed fibrillar structures composed of various tubes of VP6 that were not associated to microtubules. Coexpression of GFPVP2 and VP6 altered the distribution of both proteins. VP6 formed aggregates, even when all other conditions of individual protein expression remained unchanged. Double-layered (dl) RLP were observed in dense zones of the cytoplasm, but were not in ordered aggregates. It was determined that the assembly of both slRLP and dlRLP occurs intracellularly. Accordingly, strategies for the optimum assembly of dlRLP should guarantee that each cell produces both recombinant proteins.     

应用HA、NA、M1和M2蛋白制备H5N1高致病性禽流感病毒样颗粒

目的:应用重组杆状病毒表达系统制备由HA、NA、M1和M2蛋白组成的H5N1高致病性禽流感病毒样颗粒,为研究H5N1高致病性禽流感疫苗奠定基础。方法:构建能共表达A/chicken/Jilin/2003(H5N1)禽流感病毒血凝素(HA)和神经氨酸酶(NA)、A/PR/8/34(H1N1)流感病毒基质蛋白(M1)和离子通道蛋白(M2)的2个二元重组杆状病毒,共同感染HighFive细胞,同时表达HA、NA、M1和M2蛋白,使这4种蛋白在感染的细胞内自主组装成病毒样颗粒。经差速离心和蔗糖密度梯度超速离心收获病毒样颗粒,通过Western印迹鉴定病毒样颗粒的组成,透射电镜观察病毒样颗粒形态,血凝试验测定病毒样颗粒的活性。结果:HA、NA、M1、M2蛋白在昆虫细胞中共表达,并组装成病毒样颗粒;电镜观察到病毒样颗粒的形态与流感病毒一致,直径约80 nm;血凝试验显示该病毒样颗粒具有凝集鸡红细胞的活性。结论:应用该方法可以制备流感病毒样颗粒,为H5N1流感疫苗研究提供了可行方案。