Structure of an insect parvovirus (Junonia coenia Densovirus) determined by cryo-electron microscopy

Junonia coenia densovirus (JcDNV) belongs to the densovirus genus of the Parvoviridae family and infects the larvae of the Common Buckeye butterfly. Its capsid is icosahedral and consists of viral proteins VP1 (88 kDa), VP2 (58 kDa), VP3 (52 kDa) and VP4 (47 kDa). Each viral protein has the same C terminus but differs in the length of its N-terminal extension. Virus-like-particles (VLPs) assemble spontaneously when the individual viral proteins are expressed by a recombinant baculovirus. We present here the structure of native JcDNV at 8.7A resolution and of the two VLPs formed essentially from VP2 and VP4 at 17 A resolution, as determined by cryo-electron microscopy. The capsid displays a remarkably smooth surface, with only two very small spikes that define a pentagonal plateau on the 5-fold axes. JcDNV is very closely related to Galleria mellonella densovirus (GmDNV), whose structure is known (94% sequence identity with VP4 and 96% similarity). We compare these structures in order to locate the structural changes and mutations that may be involved in the species shift of these densoviruses. A single mutation at the tip of one of the two small spikes is a strong candidate as a species shift determinant. Difference imaging reveals that the 21 disordered amino acid residues at the N terminus of the capsid protein VP4 are located inside the capsid at the 5-fold axis, but the additional 94 amino acid residue extension of VP2 is not visible, suggesting that it is highly disordered. There is strong evidence of DNA ordering associated with the 3-fold axes of the capsid.     

Ultrastructural observations on fertilization and sporulation in Goussia iroquoina (Molnar and Fernando, 1974) in experimentally infected fathead minnows (Pimephales promelas, Cyprinidae)

The ultrastructural features of fertilization and sporogony of Eimeria iroquoina are described from the intestinal epithelium of experimentally infected fathead minnows (Pimephales promelas). Intact microgametes were observed in the cytoplasm of macrogametes. Within immature macrogametes the microgamete was segregated from the cytoplasm of the former by the plasma membrane of each cell plus additional membranes. Within mature macrogametes, only the plasma membranes separated the gametes. Fertilization by fusion of the limiting membrane of both gametes occurred after the entire microgamete lay within the cytoplasm of the macrogamete. The cytoplasm of the zygote cleaved into sporoblasts within cisternae of endoplasmic reticulum. The sporocystic wall was composed of an outer electron-lucent layer and an inner, thicker layer with periodic striations at right angles to the surface of the sporocyst. The sporocysts were bivalved and joined by a continuous suture. The sporozoites were morphologically similar to sporozoites and merozoites of other Coccidia. Due to the structure of the sporocyst, Eimeria iroquoina Molnar and Fernando, 1974 is amended to Goussia iroquoina (Molnar and Fernando, 1974).     

Assembly of double-shelled, virus-like particles in transgenic rice plants expressing two major structural proteins of rice dwarf virus

Rice dwarf virus (RDV) is a double-shelled particle that contains a major capsid protein (P8), a major core protein (P3), several minor core proteins, and viral genomic double-stranded RNA. Coexpression of P8 and P3 in transgenic rice plants resulted in formation of double-shelled, virus-like particles (VLPs) similar to the authentic RDV particles. The VLPs were not detected in transgenic rice plant cells expressing P8 alone. This in vivo result suggests that P8 interacted with P3 and that these two proteins provide the structural integrity required for the formation of VLPs in rice cells independently of other structural proteins, nonstructural proteins, or viral genomic double-stranded RNAs.     

Dissecting the role of protein-protein and protein-nucleic acid interactions in MS2 bacteriophage stability

To investigate the role of protein-protein and protein-nucleic acid interactions in virus assembly, we compared the stabilities of native bacteriophage MS2, virus-like particles (VLPs) containing nonviral RNAs, and an assembly-defective coat protein mutant (dlFG) and its single-chain variant (sc-dlFG). Physical (high pressure) and chemical (urea and guanidine hydrochloride) agents were used to promote virus disassembly and protein denaturation, and the changes in virus and protein structure were monitored by measuring tryptophan intrinsic fluorescence, bis-ANS probe fluorescence, and light scattering. We found that VLPs dissociate into capsid proteins that remain folded and more stable than the proteins dissociated from authentic particles. The proposed model is that the capsid disassembles but the protein remains bound to the heterologous RNA encased by VLPs. The dlFG dimerizes correctly, but fails to assemble into capsids, because it lacks the 15-amino acid FG loop involved in inter-dimer interactions at the viral fivefold and quasi-sixfold axes. This protein was very unstable and, when compared with the dissociation/denaturation of the VLPs and the wild-type virus, it was much more susceptible to chemical and physical perturbation. Genetic fusion of the two subunits of the dimer in the single-chain dimer sc-dlFG stabilized the protein, as did the presence of 34-bp poly(GC) DNA. These studies reveal mechanisms by which interactions in the capsid lattice can be sufficiently stable and specific to ensure assembly, and they shed light on the processes that lead to the formation of infectious viral particles.     

A circumsporozoite-like protein is present in micronemes of mature blood stages of malaria parasites

We demonstrate for the first time the presence of a circumsporozoite (CS)-like protein in invasive blood stages of malaria parasites. Immunogold electron microscopy using antisporozoite monoclonal antibodies localized these antigens in the micronemes of merozoites. Western immunoblot and two-dimensional gel electrophoresis of mature blood stage extracts of Plasmodium falciparum, P. berghei, P. cynomolgi, and P. brasilianum identified polypeptides having the same apparent molecular mass and isoelectric points as the corresponding sporozoite (CS) proteins. The CS-like protein of merozoites is present in relatively minor amounts, compared to the CS protein of sporozoites. Mice with long-term P. berghei blood-induced infections develop antibodies which react with sporozoites.     

毁灭泰泽球虫Tyzzeria Perniciosa(Sporozoa:Eimeriidae)生活史各阶段的细胞化学研究:Ⅰ.多糖

本实验分别用过碘酸——雪夫氏剂染色方法(PAS)和乌洛托品——硝酸银染色方法在光镜和电镜下检验毁灭泰泽球虫生活史各时期体内的多糖及其分布。实验结果表明,子孢子内、各代裂殖体和裂殖子内都含有多糖。大配子和合子内除含有多糖外还含有成囊颗粒。成囊颗粒的成分是糖蛋白。无性世代的滋养体和多核体内未检出多糖。早期配子细胞,小配子体和小配子内也未检出多糖。本实验证明,毁灭泰泽球虫体内的多糖系由其自身合成,并在其发育过程中消耗。     

The maturation process of pVP2 requires assembly of infectious bursal disease virus capsids

Infectious bursal disease virus (IBDV) is a nonenveloped avian virus with a two-segment double-stranded RNA genome. Its T=13 icosahedral capsid is most probably assembled with 780 subunits of VP2 and 600 copies of VP3 and has a diameter of about 60 nm. VP1, the RNA-dependent RNA polymerase, resides inside the viral particle. Using a baculovirus expression system, we first observed that expression of the pVP2-VP4-VP3 polyprotein encoded by the genomic segment IBDA results mainly in the formation of tubules with a diameter of about 50 nm and composed of pVP2, the precursor of VP2. Very few virus-like particles (VLPs) and VP4 tubules with a diameter of about 25 nm were also identified. The inefficiency of VLP assembly was further investigated by expression of additional IBDA-derived constructs. Expression of pVP2 without any other polyprotein components results in the formation of isometric particles with a diameter of about 30 nm. VLPs were observed mainly when a large exogeneous polypeptide sequence (the green fluorescent protein sequence) was fused to the VP3 C-terminal domain. Large numbers of VLPs were visualized by electron microscopy, and single particles were shown to be fluorescent by standard and confocal microscopy analysis. Moreover, the final maturation process converting pVP2 into the VP2 mature form was observed on generated VLPs. We therefore conclude that the correct scaffolding of the VP3 can be artificially induced to promote the formation of VLPs and that the final processing of pVP2 to VP2 is controlled by this particular assembly. To our knowledge, this is the first report of the engineering of a morphogenesis switch to control a particular type of capsid protein assembly.     

Virus stability and protein-nucleic acid interaction as studied by high-pressure effects on nodaviruses.

In this work, we evaluate the stability, dynamics and protein-nucleic acid interaction in Flock House virus (FHV). FHV is an RNA insect virus, non-enveloped, member of the family Nodaviridae. It is composed of a bipartite single-stranded RNA genome packaged in an icosahedral capsid of 180 copies of an identical protein (alpha protein). A fundamental property of many animal viruses is the post-assembly maturation required for infectivity. FHV is constructed as a provirion, which matures to an infectious virion by cleavage of alpha protein into beta and gamma subunits. We used high pressure, temperature and chemical denaturing agents to promote perturbation of the viral capsid. These effects were monitored by spectroscopy measurements (fluorescence, light scattering and CD) and size-exclusion chromatography. The data showed that FHV was stable to pressures up to 310 MPa at room temperature. The fluorescence emission and light scattering values showed small changes that were reversible after decompression. When we combined pressure and sub-denaturing urea concentrations (1 M), the changes were more drastic, suggesting dissociation of the capsid. However, these changes were reversible after pressure release. The complete dissociation of FHV could be observed only under high urea concentrations (10 M). There were no significant changes in emission spectra up to 5 M urea. FHV also was stable when we used temperature treatments (high and low). We also compared the effects of urea and pressure on FHV wild type and cleavage-defective mutant VLPs (virus-like particles). The VLPs and authentic particles are distinguishable by protein-RNA interactions, since VLPs pack cellular RNA and native particles contain viral RNA. Our results demonstrated that native particles are more stable than VLPs to physical and chemical treatments. Our data point to the specificity of the interaction between the capsid protein and the viral RNA. This specificity is crucial to the stability of the particle, which makes this interaction an excellent target for drug development.     

Development of Caryospora simplex (Apicomplexa: Eimeriidae) from sporozoites to oocysts in human embryonic lung cell culture

Leighton tubes containing monolayers of human embryonic lung cells were inoculated with 70,000 or 30,000 sporozoites of the viperid coccidium Caryospora simplex and examined at 1, 2, 4, 6, 8, 10, 12, 14, 16, and 18 days post-inoculation (PI). By day 1 PI, sporozoites had penetrated cells and were within parasitophorous vacuoles. Most sporozoites became spherical and then underwent karyokinesis several times between days 2 and 6 PI. Mature Type I meronts were found on days 6-16 PI and contained 8 to 22 short, stout merozoites. Mature Type II meronts were present on days 10-18 PI and contained 8 to 22 long, slender merozoites. Developing gamonts (undifferentiated sexual stages) were observed on days 14 and 16 PI. Mature micro- and macrogametes and thin-walled unsporulated oocysts were present on days 16 and 18 PI. Attempts to sporulate oocysts in tissue culture medium or in a 2.5% (w/v) aqueous solution of K2Cr2O7 at 25 degrees C and 37 degrees C were unsuccessful; only a few oocysts developed to the contracted sporont stage. Four Swiss-Webster mice injected intraperitoneally with merozoites obtained from Leighton tubes on day 10 PI did not acquire infections. This is the second coccidium reported to complete its entire development, from sporozoite to oocyst, in cell culture.     

Production of porcine parvovirus virus-like particles using silkworm larvae

Porcine parvovirus (PPV) is a significant causative agent of porcine reproductive failure, causing serious economic losses in the swine industry. PPV is a nonenveloped virus, and its capsid is assembled from three viral proteins (VP1, VP2, and VP3). The major capsid protein, VP2, is the main target for PPV neutralizing antibodies and vaccine development. In this study, PPV-VP2 protein was expressed in silkworm larvae, and its antigenicity and production were compared with those in B. mori cells (Bm5). The recombinant VP2 protein was expressed successfully in silkworm larvae and Bm5 cells with a size of approximately 64 kDa. The formation of virus-like particles (VLPs) by recombinant PPV-VP2 was confirmed through transmission electron microscopy. The recombinant PPV-VP2 protein assembled into spherical particles with diameters ranging from 20 to 22 nm. The antigenicity of PPV-VLPs was comparatively analyzed between Bm5 cells and silkworm larvae by ELISA, hemagglutination and hemagglutination inhibition assays. Consequently, it was confirmed that the PPV-VLPs produced in the silkworm larvae were more antigenic than VLPs produced in Bm5 cells. Therefore, it is expected that economical and effective vaccine development will be possible by mass production of PPV-VLPs in silkworm larvae.     

Biochemical characterization of a smaller form of recombinant Norwalk virus capsids assembled in insect cells.

The expression of the single capsid protein of Norwalk virus (NV) in Spodoptera frugiperda (Sf9) insect cells infected with recombinant baculovirus results in the assembly of virus-like particles (VLPs) of two sizes, the predominant 38-nm, or virion-size VLPs, and smaller, 23-nm VLPs. Here we describe the purification and biochemical characterization of the 23-nm VLPs. The 23-nm VLPs were purified to 95% homogeneity from the medium of Sf9 cultures by isopycnic CsCl gradient centrifugation followed by rate-zonal centrifugation in sucrose gradients. The compositions of the purified 23- and 38-nm VLPs were compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and protein immunoblots. VLPs of both sizes showed a doublet at 58 kDa, the size of the full-length capsid protein. Upon alkaline treatment, the 23-nm VLPs underwent dissociation into soluble intermediates that were able to reassemble into 23- and 38-nm VLPs upon dialysis, suggesting that the assembly of both types of structures has a common pathway. Antigenic and biochemical properties of the 38- and 23-nm VLPs were examined and found to be conserved. Immunoprecipitation assays using polyclonal and monoclonal antibodies indicated that immunodominant epitopes on the capsid protein as well as conformational epitopes are conserved in the two types of particles. The trypsin cleavage site at residue 227 was protected in the assembled particles of both sizes but exposed after alkaline dissociation. These results, and the conservation of the binding activity of both forms of recombinant NV VLPs to cultured cells (L. J. White, J. M. Ball, M. E. Hardy, T. N. Tanaka, N. Kitamoto, and M. K. Estes, J. Virol. 70:6589-6597, 1996), suggest that the tertiary folding of the capsid protein responsible for these properties is conserved in the two structures. We hypothesize that the 23-nm VLPs are formed when 60 units of the NV capsid protein assembles into a structure with T=1 symmetry.     

Display of whole proteins on inner and outer surfaces of grapevine fanleaf virus‐like particles

Virus‐like particles (VLPs) derived from nonenveloped viruses result from the self‐assembly of capsid proteins (CPs). They generally show similar structural features to viral particles but are noninfectious and their inner cavity and outer surface can potentially be adapted to serve as nanocarriers of great biotechnological interest. While a VLP outer surface is generally amenable to chemical or genetic modifications, encaging a cargo within particles can be more complex and is often limited to small molecules or peptides. Examples where both inner cavity and outer surface have been used to simultaneously encapsulate and expose entire proteins remain scarce. Here, we describe the production of spherical VLPs exposing fluorescent proteins at either their outer surface or inner cavity as a result of the self‐assembly of a single genetically modified viral structural protein, the CP of grapevine fanleaf virus (GFLV). We found that the N‐ and C‐terminal ends of the GFLV CP allow the genetic fusion of proteins as large as 27 kDa and the plant‐based production of nucleic acid‐free VLPs. Remarkably, expression of N‐ or C‐terminal CP fusions resulted in the production of VLPs with recombinant proteins exposed to either the inner cavity or the outer surface, respectively, while coexpression of both fusion proteins led to the formation hybrid VLP, although rather inefficiently. Such properties are rather unique for a single viral structural protein and open new potential avenues for the design of safe and versatile nanocarriers, particularly for the targeted delivery of bioactive molecules.     

Development of Caryospora simplex (Apicomplexa: Eimeriidae) from Sporozoites to Oocysts in Human Embryonic Lung Cell Culture1

Leighton tubes containing monolayers of human embryonic lung cells were inoculated with 70,000 or 30,000 sporozoites of the viperid coccidium Caryospora simplex and examined at 1, 2, 4, 6, 8, 10, 12, 14, 16, and 18 days post-inoculation (PI). By day 1 PI, sporozoites had penetrated cells and were within parasitophorous vacuoles. Most sporozoites became spherical and then underwent karyokinesis several times between days 2 and 6 PI. Mature Type I meronts were found on days 6–16 PI and contained 8 to 22 short, stout merozoites. Mature Type II meronts were present on days 10–18 PI and contained 8 to 22 long, slender merozoites. Developing gamonts (undifferentiated sexual stages) were observed on days 14 and 16 PI. Mature micro- and macrogametes and thin-walled unsporulated oocysts were present on days 16 and 18 PI. Attempts to sporulate oocysts in tissue culture medium or in a 2.5% (w/v) aqueous solution of K₂Cr₂O₇ at 25/°C and 37°C were unsuccessful; only a few oocysts developed to the contracted sporont stage. Four Swiss-Webster mice injected intraperitoneally with merozoites obtained from Leighton tubes on day 10 PI did not acquire infections. This is the second coccidium reported to complete its entire development, from sporozoite to oocyst, in cell culture.     

Assembly of severe acute respiratory syndrome coronavirus RNA packaging signal into virus-like particles is nucleocapsid dependent

The severe acute respiratory syndrome coronavirus (SARS-CoV) was recently identified as the etiology of SARS. The virus particle consists of four structural proteins: spike (S), small envelope (E), membrane (M), and nucleocapsid (N). Recognition of a specific sequence, termed the packaging signal (PS), by a virus N protein is often the first step in the assembly of viral RNA, but the molecular mechanisms involved in the assembly of SARS-CoV RNA are not clear. In this study, Vero E6 cells were cotransfected with plasmids encoding the four structural proteins of SARS-CoV. This generated virus-like particles (VLPs) of SARS-CoV that can be partially purified on a discontinuous sucrose gradient from the culture medium. The VLPs bearing all four of the structural proteins have a density of about 1.132 g/cm(3). Western blot analysis of the culture medium from transfection experiments revealed that both E and M expressed alone could be released in sedimentable particles and that E and M proteins are likely to form VLPs when they are coexpressed. To examine the assembly of the viral genomic RNA, a plasmid representing the GFP-PS580 cDNA fragment encompassing the viral genomic RNA from nucleotides 19715 to 20294 inserted into the 3' noncoding region of the green fluorescent protein (GFP) gene was constructed and applied to the cotransfection experiments with the four structural proteins. The SARS-CoV VLPs thus produced were designated VLP(GFP-PS580). Expression of GFP was detected in Vero E6 cells infected with the VLP(GFP-PS580), indicating that GFP-PS580 RNA can be assembled into the VLPs. Nevertheless, when Vero E6 cells were infected with VLPs produced in the absence of the viral N protein, no green fluorescence was visualized. These results indicate that N protein has an essential role in the packaging of SARS-CoV RNA. A filter binding assay and competition analysis further demonstrated that the N-terminal and C-terminal regions of the SARS-CoV N protein each contain a binding activity specific to the viral RNA. Deletions that presumably disrupt the structure of the N-terminal domain diminished its RNA-binding activity. The GFP-PS-containing SARS-CoV VLPs are powerful tools for investigating the tissue tropism and pathogenesis of SARS-CoV.     

Three-dimensional structure of HIV-1 virus-like particles by electron cryotomography

While the structures of nearly every HIV-1 protein are known in atomic detail from X-ray crystallography and NMR spectroscopy, many questions remain about how the individual proteins are arranged in the mature infectious viral particle. Here, we report the three-dimensional structures of individual HIV-1 virus-like particles (VLPs) as obtained by electron cryotomography. These reconstructions revealed that while the structures and positions of the conical cores within each VLP were unique, they exhibited several surprisingly consistent features, including similarities in the size and shape of the wide end of the capsid (the "base"), uniform positioning of the base and other regions of the capsid 11nm away from the envelope/MA layer, a cone angle that typically varied from 24 degrees to 18 degrees around the long axis of the cone, and an internal density (presumably part of the NC/RNA complex) cupped within the base. Multiple and nested capsids were observed. These results support the fullerene cone model for the viral capsid, indicate that viral maturation involves a free re-organization of the capsid shell rather than a continuous condensation, imply that capsid assembly is both concentration-driven and template-driven, suggest that specific interactions exist between the capsid and the adjacent envelope/MA and NC/RNA layers, and show that a particular capsid shape is favored strongly in-vivo.     

The Living, Endogenous Stages of the Rat Coccidium, Eimeria nieschulzi

SYNOPSIS. The living, endogenous stages of Eimeria nieschulzi Dieben, 1924 (Landers isolate) were studied under the phase contrast microscope. Active sporozoites were found as early as 2.5 hours after exposure and as late as 48 hours after exposure. The first generation schizont was recognized by the presence of a refractile globule remaining from the sporozoite. First and second generation merozoites were only weakly motile and had small paired organelles. Third generation merozoites were seen 48–120 hours after exposure and were strongly motile from 72 hours after exposure onward. The paired organelle consisted of 2 intertwining portions, one 5.5 μ long, the other tapering to a slender filament and continuing to about the posterior quarter of the parasite. The fourth generation merozoites were short, curved, and weakly motile. A paired organelle about 3 μ long was seen. Gametocytes and gametes were seen 144–192 hours after exposure. Macrogametes appeared to elaborate refractile granules in the vicinity of the nucleus. No motility of any type was seen in the macrogametes. Microgametocytes were recognized when nuclear material moved to the periphery of the parasite for the formation of microgametes. Observations on living organisms agreed generally with those made on fixed and stained organisms with the exception that the living merozoites were about 20% larger.