A human virus protein, poliovirus protein 2BC, induces membrane proliferation and blocks the exocytic pathway in the yeast Saccharomyces cerevisiae.

Inducible synthesis of poliovirus protein 2BC in Saccharomyces cerevisiae arrests cell growth in the G2 phase of the cell cycle, while no effects are observed upon expression of poliovirus genes 2B or 2C, either individually or in combination. Expression of 2BC induces a number of morphological modifications in yeast cells, one of the most striking being the proliferation of small membranous vesicles that fill most of the cytoplasm. These vesicles are morphologically similar to the cytopathic vacuoles that proliferate during the infection of human cells by poliovirus. The transport and processing of several yeast proteins, including vacuolar carboxypeptidase Y, aminopeptidase I or yeast alpha-mating factor, is hampered upon expression of poliovirus 2BC, suggesting that transport of proteins through the Golgi apparatus is impaired by this viral protein. Finally, a number of 2BC variants were generated and the effects of their expression on yeast growth, cellular morphology and protein processing were analyzed. 2BC variants defective in the NTPase activity were still able to interfere with yeast growth and the exocytic system, while deletion of 30 amino acids at the N-terminus of 2BC impairs its function. These findings lend support to the idea that 2BC, but not 2B or 2C, is the protein responsible for vesicle proliferation in poliovirus-infected cells. In addition, the activity of a human virus protein in yeast cells opens new avenues to investigate the exact location at which poliovirus 2BC interferes with the vesicular system and to test the action of other animal virus proteins potentially involved in modifying the vesicular system in mammalian cells.     

Poliovirus protein 2BC increases cytosolic free calcium concentrations.

Poliovirus-infected cells undergo an increase in cytoplasmic calcium concentrations from the 4th h postinfection. The protein responsible for this effect was identified by the expression of different poliovirus nonstructural proteins in HeLa cells by using a recombinant vaccinia virus system. Synthesis of protein 2BC enhances cytoplasmic calcium concentrations in a manner similar to that observed in poliovirus-infected cells. To identify the regions in 2BC involved in modifying cytoplasmic calcium levels, several 2BC variants were generated. Regions present in both 2B and 2C are necessary to augment cellular free calcium levels. Therefore, in addition to inducing proliferation of membranous vesicles, poliovirus protein 2BC also alters cellular calcium homeostasis.     

应用免疫胶体金技术定位感病大麦细胞中的大麦和性花叶病毒

本文报道免疫胶体金标记技术的建立,并用此技术定位大麦叶和根组织超薄切片中大麦和性花叶病毒(BaMMV)。在感染病毒的大麦叶和根细胞中,病毒束、游离病毒颗粒和病毒外壳蛋白多分布于细胞质丰富的细胞中,且以液泡和叶绿体(仅叶组织)周围较多。在细胞器已解体的病根表皮细胞中,有时也可检测到大量游离病毒粒子。少数风轮体或板状集结体上也存在病毒或病毒外壳蛋白。细胞核、叶绿体、线粒体、细胞膜以及其他细胞器上都未见有特异性金颗粒标记。     

荞麦子叶发育过程中聚合的粗糙内质网

在荞麦（Fagopyrum esculentum Moench.）子叶的发育过程中观察到粗糙内质网的聚合现象,是一些粗糙内质网平行或成环形排列形成的。这些聚合的粗糙内质网附近通常有蛋白质团块存在,其形状不规则,无膜包被,与周围细胞质的界限不清。推测这些蛋白质团块不是在液泡或膨大的粗糙内质网囊泡中积累形成的,而是由聚合的粗糙内质网直接将合成的蛋白质分泌到细胞质中形成的。这种形式提高了子叶中贮存蛋白质的积累效率。     

A light and electron microscopic study of the preoptic nucleus of the grass frog (Rana pipiens), under normal conditions and following transection of the preoptico-neurohypophysial tract

Degenerative and regenerative processes occur in the preoptic neurons following transection of the preoptico-neurohypophysial tract. Three types of responses after transection were observed: affected, recovered, and degenerated neurons. However, transection of the tract did not stop the synthesis of neurosecretory granulated vesicles. The affected neurosecretory neurons showed nuclear changes, increased number of Golgi complexes, and dilated cisternae of rER, as well as, an increased number of dense bodies. The recovered neurosecretory neurons contained long non-dilated cisternae of rER which were organized in a concentric manner. Also seen were large nuclei with evenly distributed chromatin, less active Golgi complexes, and vesicles. The degenerated neurosecretory neurons exhibit pyknotic nuclei, a net of dilated cisternae of rER, dense bodies, and electron dense cytoplasm.     

Effects of foot-and-mouth disease virus nonstructural proteins on the structure and function of the early secretory pathway: 2BC but not 3A blocks endoplasmic reticulum-to-Golgi transport

Infection of cells by picornaviruses leads to the generation of intracellular membrane vesicles. The expression of poliovirus (PV) 3A protein causes swelling of the endoplasmic reticulum (ER) and inhibition of protein trafficking between the ER and the Golgi apparatus. Here, we report that the nonstructural proteins of a second picornavirus, foot-and-mouth disease virus (FMDV), also perturb the secretory pathway. FMDV proteins 3A, 2B, 2C, and 2BC expressed alone in cells were recovered from crude membrane fractions, indicating membrane association. Immunofluorescence microscopy showed that 3A was located in a reticular structure and 2B was located in the ER, while 2C was located in both the ER and the bright punctate structures within the Golgi apparatus. 2BC gave punctate cytoplasmic staining and also caused accumulation of ER proteins in large vesicular structures located around the nuclei. The effect of the FMDV proteins on the trafficking of the vesicular stomatitis virus glycoprotein (G protein) from the ER to the cell surface was determined. Unlike its PV counterpart, the 3A protein of FMDV did not prevent trafficking of the G protein to the cell surface. Instead, surface expression of the G protein was blocked by 2BC, with retention of the G protein in a modified ER compartment staining for 2BC. The results suggest that the nonstructural proteins of different picornaviruses may vary in their ability to perturb the secretory pathway. Since FMDV 2BC can block the delivery of proteins to the cell surface, it may, as shown for PV 3A, play a role in immune evasion and contribute to the persistent infections observed in ruminants.     

Origin and development of protein bodies in cotyledons of Vicia faba

Storage proteins of the field bean (Vicia faba L., var. minor, cv. “Fribo”) are synthesized and accumulated in the cotyledons during stage 2 of seed development. Deposition of protein reserves takes place in the protein bodies. The generation of protein bodies was investigated electronmicroscopically using ultra-thin sections as well as the freeze-fracturing technique. During the initial period of storage protein formation, globulins are deposited in large vacuoles which later are transformed to give increasing numbers of small vacuoles with decreasing size. The vacuoles disappear early during the stage of storage protein formation and generate the first protein bodies. During the subsequent period of maximum storage protein formation, which takes place at the rough endoplasmic reticulum (rER), swollen ER strands appear which seem to be entirely filled with protein, and these generate ER-produced protein vacuoles (ERPVAC). The vesicles are transformed in a manner comparable to the vacuoles in the initial period of developmental stage 2 and thus generate the major quantity of protein bodies. Both processes seem to represent only two variants of an uniform mechanism of protein body generation.     

Retention of cartilage oligomeric matrix protein (COMP) and cell death in redifferentiated pseudoachondroplasia chondrocytes

Cartilage oligomeric matrix protein (COMP) is a large extracellular glycoprotein that is found in the territorial matrix surrounding chondrocytes. Two skeletal dysplasias, pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (EDM1) are caused by mutations in the calcium binding domains of COMP. In this study, we identified two PSACH mutations and assessed the effect of these mutations on redifferentiated chondrocyte structure and function. We confirmed, in vitro, that COMP is retained in enormous cisternae of the rough endoplasmic reticulum (rER) and relatively absent in the PSACH matrix. The rER accumulation may compromise chondrocyte function, leading to chondrocyte death. Moreover, while COMP appears to be deficient in the PSACH matrix, the matrix appeared to be normal but the over-all quantity was reduced. These results suggest that the abnormality in linear growth in PSACH may result from decreased chondrocyte numbers which would also affect the amount of matrix produced.     

Potential subversion of autophagosomal pathway by picornaviruses

The RNA replication complexes of small positive-strand RNA viruses such as poliovirus are known to form on the surfaces of membranous vesicles in the cytoplasm of infected mammalian cells. These membranes resemble cellular autophagosomes in their double-membraned morphology, cytoplasmic lumen, lipid-rich composition and the presence of cellular proteins LAMP 1 and LC3. Furthermore, LC3 protein is covalently modified during poliovirus infection in a manner indistinguishable from that observed during bona fide autophagy. This covalent modification can also be induced by the expression of viral protein 2BC in isolation. However, differences between poliovirus-induced vesicles and autophagosomes also exist: the viral-induced membranes are smaller, at 200-400 nm in diameter, and can be induced by the combination of two viral proteins, termed 2BC and 3A. Experimental suppression of expression of proteins in the autophagy pathway was found to reduce viral yield, arguing that this pathway facilitates viral infection, rather than clearing it. We have hypothesized that, in addition to providing membranous surfaces for assembly of viral RNA replication complexes, double-membraned vesicles provide a topological mechanism to deliver cytoplasmic contents, including mature virus, to the extracellular milieu without lysing the cell.     

Potential subversion of autophagosomal pathway by picornaviruses

The RNA replication complexes of small positive-strand RNA viruses such as poliovirus are known to form on the surfaces of membranous vesicles in the cytoplasm of infected mammalian cells. These membranes resemble cellular autophagosomes in their double-membraned morphology, cytoplasmic lumen, lipid-rich composition and the presence of cellular proteins LAMP 1 and LC3. Furthermore, LC3 protein is covalently modified during poliovirus infection in a manner indistinguishable from that observed during bona fide autophagy. This covalent modification can also be induced by the expression of viral protein 2BC in isolation.However, differences between poliovirus-induced vesicles and autophagosomes also exist: the viral-induced membranes are smaller, at 200- 400 nm in diameter, and can be induced by the combination of two viral proteins, termed 2BC and 3A. Experimental suppression of expression of proteins in the autophagy pathway was found to viral yield, arguing that this pathway facilitates viral infection, rather than clearing it. We have hypothesized that, in addition to providing membranous surfaces for assembly of viral RNA replication complexes, double-membraned vesicles provide a topological mechanism to deliver cytoplasmic contents, including mature virus, to the extracellular milieu without lysing the cell.     

The fine structure of the dorsal column nucleus and the nucleus of bischoff of the python (Python reticulatus)

Three types of neuronal perikaryal profiles were identified in the dorsal column nucleus and the nucleus of Bischoff of the python (Python reticulatus). Type I neuronal profiles are large (diameters 12–20 μm) with a deeply indented uncleus. The cisterns of rough endoplasmic reticulum (rER) are mostly randomly dispersed. Axosomatic synapses are few. Type II neuronal profiles (9–11 μm) have a smooth, round, or slightly oval nucleus. Several small stacks of rER are present. Type III neuronal profiles (8–10 μm) have little cytoplasm. The nuclear margin is irregular but not deeply infolded. The rER usually consists of a single long perinuclear ribosome-studded cistern. Two types of astrocytic profiles have been identified. Both types contain abundant filaments. Type I astrocytes are large cells, and the nucleus is very irregular in shape. Type II astrocytes are smaller and are found among the myelinated axons in the dorsal funiculus. Two classes of axon terminals have been identified. One class contains round synaptic vesicles (R profiles) and the other flattened vesicles (F profiles). Some R profiles are small (SR profiles), others are large (LR profiles). Some R profiles also contain a few large, dense-cored vesicles. The R and F profiles establish axodendritic and axoaxonal synapses, some of which are located in the synaptic glomeruli and others in the extraglomerular neuropil. In most of the axoaxonal synapses, the presynaptic element is an F profile and the post synaptic element an LR profile. Occasionally, LR profiles are presynaptic to F profiles. The findings in the python are compared with those of the dorsal column nuclei of the rat, cat, and monkey.     

Immunoenzyme localization of cathepsins in the Golgi region of rat hepatocytes and renal tubule cells

Localization of carboxyl proteinase (cathepsin D) and cysteine proteinases (cathepsins B, H, and L) in Golgi region was studied using an immunoenzyme technique. Rat livers and kidneys were used. The results obtained from the livers were similar to those from the kidneys. All cathepsins were detected in lysosomal compartments such as secondary lysosomes, multivesicular bodies (endosomes), and autophagosomes. Rough endoplasmic reticulum (rER), including nuclear envelope was focally stained. Most of Golgi cisternae were negative, but sometimes only one cisterna or the terminal portion of the cisterna were stained focally. Rarely, the trans Golgi network (TGN) was positive for the proteinases. Among numerous Golgi vesicles, only a few of them were stained. The positive vesicles were divided into two groups, one had a bristle coat and heavily stained, and other were smaller than 40 nm in diameter and weakly stained. The small vesicles seemed to bud from the ER and to fuse with the Golgi cisternae, while the large clathrin-coated vesicles seem to bud from the TGN. The results suggests that cathepsins are transported by vesicular system from the rER to lysosomes via Golgi apparatus. In addition, it is suggested that the small vesicles transport the proteinases from the ER to the Golgi cisternae and the large clathrin-coated vesicles from the Golgi cisternae to the lysosomes.     

Immunoenzyme localization of cathepsins in the Golgi region of rat hepatocytes and renal tubule cells

Summary Localization of carboxyl proteinase (cathepsin D) and cysteine proteinases (cathepsins B, H, and L) in Golgi region was studied using an immunoenzyme technique. Rat livers and kidneys were used. The results obtained from the livers were similar to those from the kidneys. All cathepsins were detected in lysosomal compartments such as secondary lysosomes, multivesicular bodies (endosomes), and autophagosomes. Rough endoplasmic reticulum (rER), including nuclear envelope was focally stained. Most of Golgi cisternae were negative, but sometimes only one cisterna or the terminal portion of the cisterna were stained focally. Rarely, the trans Golgi network (TGN) was positive for the proteinases. Among numerous Golgi vesicles, only a few of them were stained. The positive vesicles were divided into two groups, one had a bristle coat and heavily stained, and other were smaller than 40 nm in diameter and weakly stained. The small vesicles seemed to bud from the ER and to fuse with the Golgi cisternae, while the large clathrin-coated vesicles seem to bud from the TGN. The results suggests that cathepsins are transported by vesicular system from the rER to lysosomes via Golgi apparatus. In addition, it is suggested that the small vesicles transport the proteinases from the ER to the Golgi cisternae and the large clathrin-coated vesicles from the Golgi cisternae to the lysosomes.     

Modification of cellular autophagy protein LC3 by poliovirus

Poliovirus infection remodels intracellular membranes, creating a large number of membranous vesicles on which viral RNA replication occurs. Poliovirus-induced vesicles display hallmarks of cellular autophagosomes, including delimiting double membranes surrounding the cytosolic lumen, acquisition of the endosomal marker LAMP-1, and recruitment of the 18-kDa host protein LC3. Autophagy results in the covalent lipidation of LC3, conferring the property of membrane association to this previously microtubule-associated protein and providing a biochemical marker for the induction of autophagy. Here, we report that a similar modification of LC3 occurs both during poliovirus infection and following expression of a single viral protein, a stable precursor termed 2BC. Therefore, one of the early steps in cellular autophagy, LC3 modification, can be genetically separated from the induction of double-membraned vesicles that contain the modified LC3, which requires both viral proteins 2BC and 3A. The existence of viral inducers that promote a distinct aspect of the formation of autophagosome-like membranes both facilitates the dissection of this cellular process and supports the hypothesis that this branch of the innate immune response is directly subverted by poliovirus.     

Ultrastructure of neuromuscular contacts in the embryonic pond snail Lymnaea stagnalis L

Ultrastructural characteristics of muscle fibers and neuromuscular contacts were investigated during two stages of embryogenesis of the pulmonate snail Lymnaea stagnalis. The first muscle cells appear as early as during metamorphosis (50-55% of embryonic development), whereas previously, in the trochophore/veliger stages (25-45%), muscular elements cannot be detected at all. The first muscle fibers contain large amounts of free numbers, a well-developed rER system and only a few irregularly arranged contractile elements. The nucleus is densely packed with heterochromatine material. At 75% adult-like postmetamorphic stage, the frequency of muscle fibers increases significantly, but, bundles of muscle fibers cannot yet be observed. Furthermore the muscle cells are characterized by large numbers of free ribosomes and numerous rER elements. Fine axon bundles and single axon processes, both accompanied by glial elements, can already be found at this time. Axon varicosities with different vesicle and/or granule contents form membrane contacts with muscle fibers, but without revealing membrane specialization on the pre- or postsynaptic side. The late development of the muscle system and neuromuscular contacts during Lymnaea embryogenesis correlates well with the maturation of different forms of behavior of adult, free-living life, and also with the peripheral appearance of chemically identified components of the embryonic nervous system of central origin.     

Improved distribution of antigenic site specificity of poliovirus-neutralizing antibodies induced by a protease-cleaved immunogen in mice.

Previous studies showed that the distribution of antigenic site specificity of neutralizing antibodies to type 3 poliovirus obtained with the inactivated poliovirus vaccine can be deficient as compared with that obtained following poliovirus infection. This observation was shown by the relatively low capacity of sera from inactivated-poliovirus-vaccine-immunized persons to neutralize poliovirus cleaved at antigenic site 1. We investigated possibilities for improving the situation in a mouse model. Balb/c mice were immunized with intact or trypsin-cleaved type 3 poliovirus (Saukett strain). Sera from mice immunized with the intact virus readily neutralized the intact virus but neutralized the cleaved virus only rarely. In contrast, cleaved-virus-immunized mice produced antibodies that were able to neutralize the cleaved virus as well as the intact one. Mice immunized with a 100-fold-higher dose of the intact virus produced significant levels of antibodies to the cleaved virus, too. Somewhat surprisingly, mice immunized with high doses of the cleaved virus produced antibodies specific for the intact loop between beta sheets B and C of VP1 (virion protein 1), which should be cleaved in the immunogen. This was shown by a higher titer of antibodies to intact Saukett virus than to the corresponding cleaved virus, as well as to a type 1/type 3 hybrid poliovirus in which only the BC loop amino acids were derived from type 3 poliovirus. The cleavage-induced enhanced availability of antigenic determinants residing outside the BC loop was also shown by increased neutralization titers of monoclonal antibodies specific for some of these other determinants. These results indicate that by using a trypsin-cleaved type 3 poliovirus as a parenteral immunogen, it is possible to change the distribution of antigenic site specificities of neutralizing antibodies to resemble that following poliovirus infection.     

Remodeling the endoplasmic reticulum by poliovirus infection and by individual viral proteins: an autophagy-like origin for virus-induced vesicles

All positive-strand RNA viruses of eukaryotes studied assemble RNA replication complexes on the surfaces of cytoplasmic membranes. Infection of mammalian cells with poliovirus and other picornaviruses results in the accumulation of dramatically rearranged and vesiculated membranes. Poliovirus-induced membranes did not cofractionate with endoplasmic reticulum (ER), lysosomes, mitochondria, or the majority of Golgi-derived or endosomal membranes in buoyant density gradients, although changes in ionic strength affected ER and virus-induced vesicles, but not other cellular organelles, similarly. When expressed in isolation, two viral proteins of the poliovirus RNA replication complex, 3A and 2C, cofractionated with ER membranes. However, in cells that expressed 2BC, a proteolytic precursor of the 2B and 2C proteins, membranes identical in buoyant density to those observed during poliovirus infection were formed. When coexpressed with 2BC, viral protein 3A was quantitatively incorporated into these fractions, and the membranes formed were ultrastructurally similar to those in poliovirus-infected cells. These data argue that poliovirus-induced vesicles derive from the ER by the action of viral proteins 2BC and 3A by a mechanism that excludes resident host proteins. The double-membraned morphology, cytosolic content, and apparent ER origin of poliovirus-induced membranes are all consistent with an autophagic origin for these membranes.     

Immunochemical studies on the role of the Golgi complex in protein-body formation in rice seeds

Antibodies raised against purified glutelins and prolamines were employed as probes to study the cellular routes by which these proteins are deposited into protein bodies of rice (Oryza sativa L.) endosperm. Three morphologically distinct protein bodies, large spherical, small spherical, and irregularly-shaped, were observed, in agreement with existing reports. Immunocytochemical studies showed the presence of glutelins in the irregularly-shaped protein bodies while the prolamines were found in both the large and small spherical protein bodies. Both the large and small spherical protein bodies, distinguishable by electron density and gold-labeling patterns, appear to be formed by direct deposition of the newly formed proteins into the lumen of the rough endoplasmic reticulum (ER). In contrast, glutelin protein bodies are formed via the Golgi apparatus. Small electron-lucent vesicles are often found at one side of the Golgi. Electron-dense vesicles, whose contents are labeled by glutelin antibody-gold particles, are commonly observed at the distal side of the Golgi apparatus and fuse to form the irregularly shaped protein bodies in endosperm cells. These observations indicate that the transport of rice glutelins from their site of synthesis, the ER, to the site of deposition, the protein bodies, is mediated by the Golgi apparatus.Abbreviations BSA bovine serum albumin - Da dalton - DAF days after flowering - ER endoplasmic reticulum - GL irregularly shaped - L large spherical - S small spherical (protein bodies) - PBS phosphate-buffered saline - PTA phosphotungstic acid     

Desiccation Alters the Stability and Distribution of Pea Seed-borne Mosaic Virus in Pea (Pisum sativum) Cotyledon Cells

As a seed transmitted pathogen, pea seed-borne mosaic vires (PSbMV) not only replicates in embryonic cells but can also withstand seed desiccation. To understand the mechanism of PSbMV tolerance to seed desiccation, the authors compared the stability of viral coat protein (CP) and the distribution of viral particles in the cotyledon cells of pea ( Pisum sativum L. ) embryos collected before and after the dehydration process. Before dehydration, when the embryo was fresh and immature, degradation of CP was observed and a predominantly perinuclear distribution of viral particles in the cotyledon cells was evident. After dehydration, when the embryo was dry and mature, degradation of CP did not occur and the perinuclear viral distribution disappeared. Instead, aggregates containing PSbMV CP were found in the cytoplasm. Electron microscopy showed that these aggregates were composed of PSbMV particles. The formation of PSbMV particle aggregates is apparently triggered by seed dehydration and may be favorable to the virus survival in the desiccated embryonic cells.     

Cotton embryogenesis: The pollen tube in the stigma and style

Summary The ultrastructure and composition of the pollen tube of cotton (Gossypium hirsutum) growing in the tissues of the stigma and style of the flower were examined. The distal portion of the tube is densely cytoplasmic and contains the vegetative nucleus and the two sperms. The vegetative nucleus is highly convoluted and the membrane contains many pores and connections with the ER. No organized nucleolus is present but 4–6 membrane-bound, protein containing bodies are found in the nucleus. Mitochondria containing numerous cristae are abundant in the cytoplasm. Dictyosomes are also plentiful and are engaged in the production of many large vesicles. Rough ER is conspicuous and polysomes are found in the cytoplasm. Plastids are few in number, poorly developed, and contain little starch. Many uniform, small vesicles are found throughout the cytoplasm. Lipid bodies frequently with small vesicles associated with them are found in the tube. In the proximal region vacuoles form and the cytoplasm becomes pressed against the wall. In the transition zone the ER frequently becomes distended and filled with protein. The wall has two distinct layers: one strongly PAS positive, the other faintly PAS positive. The inner wall is apparently formed by the deposition of large dictyosome vesicles. Plug structure and development were studied.