Sorghum chlorotic spot virus binds to potyvirus cylindrical inclusions in tobacco leaf cells

Nicotiana benthamiana can be doubly infected with either potato virus Y or tobacco etch virus and sorghum chlorotic spot virus (SCSV). Immunogold labeling showed that cylindrical inclusions of either potyvirus bind virions of the unrelated rod-shaped furovirus SCSV. Not all cells in doubly infected N. benthamiana plants contained both viruses. In cells infected by the potyviruses but not by SCSV, cylindrical inclusions did not label with the antiserum to SCSV. Numbers of cells infected with SCSV did not increase in doubly infected plants compared to those in plants infected with SCSV alone. Systemic infection of N. benthamiana by either potyvirus was not prevented by SCSV infections. This provides further evidence that unrelated rod-shaped viruses can bind to potyvirus cylindrical inclusion bodies, and that this phenomenon is not limited to graminaceous hosts.     

Ultrastructural Studies of Barley Mesophyll Cella lnfected with Barley Yellow Mosaic Viruses

lexuous filamentous, rod-shaped particles, and laminated, pinwheel inclusions were observed in the mesophyll cells of the barley plants naturally infected with barley yellow mosaic viruses. These virus particles had a length of 480–920 nm and a width of 10–20 nm. In addition, bundles of filamentous structures which consisted of many particles with more 2000 nm in length were found in the leaves of the infected barley plants. The ultrastructural alterations of the infected mesophyll cells were rather conspicuous. The cytoplasmic matrix was lost seriously, and the chloroplast membrane system was destroyed. The cristae and matrix of the mitochondrium were decreased and some of them became vacuoles. The endoplasmic reticulum (ER) expanded teristic membranous network structures occurred in the cytoplasm of infected cells. The virus particles were often associated at one end with ER and with the membranes of network structures. The nucleus, membrane and wall of ceils also had somewhat variation.     

CYTOPLASMIC DIFFERENCES IN T-TYPE CYTOPLASMIC MALE-STERILE CORN AND ITS MAINTAINER

Edwardson , John R. (U. Florida, Gainesville.) Cytoplasmic differences in T-type cytoplasmic male-sterile corn and its maintainer. Amer. Jour. Bot. 49 (2) : 184–187. Illus. 1962.—Differences in inclusions in sections of T-type male-sterile and maintainer corn were found with electron microscopy. Some male-sterile root tip cells contain dense inclusions ca. 50–60 mμ dia. These inclusions are always associated with areas of dense cytoplasm, while no such inclusions or dense cytoplasmic areas occur in maintainer material. The majority of maintainer tapetal cells contain inclusions ca. 46–52 mμ dia.; the majority of male-sterile tapetal cells contain inclusions ca. 58–64 mμ dia. Well-defined membranes always enclose the inclusions in maintainer material; indistinct membranes are occasionally associated with inclusions in sterile material.     

Origins and formation of intracellular inclusions associated with two leguminous virus diseases

Summary Two hitherto undescribed viruses were isolated from naturally-infected white clover plants.One induced both cytoplasmic and intranuclear inclusions, and the other caused cytoplasmic inclusions of a new type, which we have called Corner inclusion bodies.All three kinds of inclusion were found in the same cells.Vital observations showed that the two kinds of cytoplasmic inclusion bodies developed principally from the plastids.The intranuclear inclusions were shown to develop from the nucleoli.Cytochemical tests showed that the cytoplasmic inclusions contained protein and ribonucleic acid.On the basis of these findings, the possibility of virus synthesis in plastids and nucleoli is discussed.     

Purification and Electron Microscopy Studies of a Probable Potyvirus from Polianthes tuberosa L.

A partial purification procedure is described for a flexuous filamentous virus from leaves of the ornamental crop tuberose (Polianthes tuberosa L.) exhibiting mottle symptoms. Particular problems associated with the purification included particle aggregation and the highly viscous nature of the plant sap. The former difficulty was partially alleviated by the addition of 1.0 M urea to the extraction buffer and the latter by treatment of the extract with cellulase. The mean virus particle length from negatively stained leaf dips was 742 nm (SD = 54) which is characteristic of viruses belonging to the potyvirus group. In addition, electron microscopy of thin sections of infected leaf tissue demonstrated the presence of several types of inclusion bodies similar in appearance to inclusions previously described in association with potyvirus infections in other plants.     

Light and electron microscopy of virus inclusions inAmaranthus lividus cells

Summary Amaranthus plants infected with a virus of rod-shaped particles showed under the light microscope intracytoplasmic amorphous and crystalline inclusions.The submicroscopic organization of mesophyll cells from infectedAmaranthus leaves by electron microscopy is described. Besides big crystalline inclusions, long dark inclusions correspondent to needle-like inclusions observed by light microscopy are definable in the cytoplasm. The amorphous inclusion bodies were formed by an overgrown protrusion of vacuolate cytoplasm containing virus particles, long very dark stained inclusions forming dense bands and rings, normal elements of the cytoplasm such as mitochondria, endoplasmic reticulum and ribosomes, and some spherosomes. Inclusions and virus particles were not found in chloroplasts, mitochondria or nuclei of infected cells.     

Reovirus core protein mu2 determines the filamentous morphology of viral inclusion bodies by interacting with and stabilizing microtubules

Cells infected with mammalian reoviruses often contain large perinuclear inclusion bodies, or "factories," where viral replication and assembly are thought to occur. Here, we report a viral strain difference in the morphology of these inclusions: filamentous inclusions formed in cells infected with reovirus type 1 Lang (T1L), whereas globular inclusions formed in cells infected with our laboratory's isolate of reovirus type 3 Dearing (T3D). Examination by immunofluorescence microscopy revealed the filamentous inclusions to be colinear with microtubules (MTs). The filamentous distribution was dependent on an intact MT network, as depolymerization of MTs early after infection caused globular inclusions to form. The inclusion phenotypes of T1L x T3D reassortant viruses identified the viral M1 genome segment as the primary genetic determinant of the strain difference in inclusion morphology. Filamentous inclusions were seen with 21 of 22 other reovirus strains, including an isolate of T3D obtained from another laboratory. When the mu2 proteins derived from T1L and the other laboratory's T3D isolate were expressed after transfection of their cloned M1 genes, they associated with filamentous structures that colocalized with MTs, whereas the mu2 protein derived from our laboratory's T3D isolate did not. MTs were stabilized in cells infected with the viruses that induced filamentous inclusions and after transfection with the M1 genes derived from those viruses. Evidence for MT stabilization included bundling and hyperacetylation of alpha-tubulin, changes characteristically seen when MT-associated proteins (MAPs) are overexpressed. Sequencing of the M1 segments from the different T1L and T3D isolates revealed that a single-amino-acid difference at position 208 correlated with the inclusion morphology. Two mutant forms of mu2 with the changes Pro-208 to Ser in a background of T1L mu2 and Ser-208 to Pro in a background of T3D mu2 had MT association phenotypes opposite to those of the respective wild-type proteins. We conclude that the mu2 protein of most reovirus strains is a viral MAP and that it plays a key role in the formation and structural organization of reovirus inclusion bodies.     

大麦黄花叶病毒侵染的大麦叶肉细胞超微结构研究

在自然感染大麦黄花叶病毒的大麦叶肉细胞中可见线条状和杆状的病毒粒体以及风轮状内含体。这些病毒的长度一般为480—920nm,宽为lo—20nm。此外,还观察到一种由许多病毒组成的堆束状结构,这种病毒的直径为13nm 左右,长度可达2000nm 以上。感病叶肉细胞的超微结构变化是相当明显的。在病害严重的细胞中,细胞基质丧失严重;叶绿体膜系统破坏;线粒体的嵴和基质减少;内质网膨大或断裂,小泡大量出现,病毒粒体的一端往往与内质网联结在一起,特征性膜性网络结构在感染的细胞质中形成。细胞核和细胞膜也发生了变化。     

Morphogenesis of Respiratory Syncytial Virus in a Green Monkey Kidney Cell Line (Vero)

The structure and morphogenesis of respiratory syncytial (RS) virus particles in a green monkey kidney cell line (Vero) were examined. Infected cells contained dense intracytoplasmic inclusions composed of filamentous structures. In places where inclusion material was associated with membranes, structural modifications were induced. There was a thickening of the membrane and an addition of projections 12 to 15 nm in length. The same changes were most frequently observed after association of isolated filamentous structures with the cytoplasmic membrane. The budding-off process was clearly visualized. The diameter of mature virus particles varied between 90 and 130 nm and that of the internal component varied between 11 and 15 nm. The similarities between ultrastructural features of cells infected with RS virus and pneumonia virus of mice are pointed out. It is proposed that these two viruses should be classified together in a third subgroup of myxoviruses.     

Comparison of Subacute Sclerosing Panencephalitis and Measles Viruses: an Electron Microscope Study

The ultrastructure of CV-1 cells infected with subacute sclerosing panencephalitis (SSPE) viruses was compared with that of CV-1 cells infected with the wild or Edmonston strain of measles virus. Both SSPE viruses and the measles viruses produced two types of nucleocapsid structures: smooth filaments, 15 to 17 nm in diameter, and granular filaments, 22 to 25 nm. The smooth and granular filaments produced by SSPE and measles virus did not differ in appearance. In CV-1 cells infected with SSPE viruses, smooth filaments formed large intranuclear inclusions and granular filaments occupied a large area of the cytoplasm, but always spared the area under the cell membrane. Particles budding from the surface of these cells contained no nucleocapsids. In CV-1 cells infected with measles virus, only small aggregates of smooth filaments were seen in the nuclei. Granular filaments in the cytoplasm predominantly occupied the area under the cell membrane, and were aligned beneath the cell membrane in a parallel fashion and assembled into budding particles. These differences between SSPE and measles virus may be regarded as quantitative, but they do distinguish SSPE viruses from measles virus. Moreover, the formation of large nuclear inclusions filled with smooth filaments appears to be a characteristic process of SSPE, but not of measles, since this type of inclusion is invariably seen in SSPE brain tissues, brain cultures derived from them, and CV-1 cells infected with SSPE viruses.     

FINE STRUCTURE OF NUCLEAR CRYSTALS IN LEAVES OF TWO DIANTHUS SPECIES

Examination of leaf cells of Dianthus barbatus with the election microscope revealed the presence of crystals in the nuclei, but not in the cytoplasm or in other organelles. They were at first thought to be viral crystalline inclusions, but no evidence could be adduced to support this possibility. The crystals were found in the nuclei of cells from all parts of the leaf, including parenchyma cells of the vascular elements. They were usually 0.5–2 μ long, and occasionally up to 5 μ. In longitudinal section they appeared as electron-dense striations, about 10–15 mμ wide, alternating with electron-transparent bands, 6–8 mμ wide. In cross section they had a honeycomb appearance, resulting from closely packed hexagonal elements, with electron-transparent centers. Similar crystals were found in nuclei of D. chinensis leaf cells, but not in other members of the Caryophyllaceae, including two varieties of D. caryophyllus, D. deltoides, D. arenarius, Silene acaulis, Lychnis chalcedonica, Saponaria calibrica, and Stellaria media. This appears be the first fine structural report of a lattice inclusion in nuclei of cells of healthy higher plants.     

Characterization of intracytoplasmic prokaryote infections in Dreissena sp. (Bivalvia: Dreissenidae)

This study characterizes intracytoplasmic infections with prokaryote microorganisms in Dreissena sp. (near Dreissena polymorpha) from northeastern Greece and represents the first report of such infections in freshwater bivalves. Light microscope observations of stained tissues revealed basophilic, cytoplasmic inclusion bodies in 87.5% (28/32) of the mussels sectioned. Inclusions in epithelial cells and connective tissues were noted, respectively, in 34.4 and 71.9% of the sample, with 5 mussels (15.6%) having both tissue types infected. Epithelial cell infections were observed in histological sections only in digestive gland tubules and ducts; within tubules, inclusions were present more often in secretory than digestive cells. Connective tissue infections, however, were systemic; among the 32 mussels sectioned, inclusions were found in the gills (65.6%), foot (12.5%), mantle (9.4%), labial palps (6.3%), digestive gland (6.3%), stomach (6.3%), and gonads (3.1%). Cytoplasmic inclusions (maximum dimension, 138 microm) were prominent enough in the gills to be visible in 17.0% of the 247 mussels dissected. Ultrastructurally, prokaryote cells in gill connective tissues were clearly characteristic of Chlamydiales-like organisms, with each intracytoplasmic inclusion containing a loosely packed mixture of elementary, reticulate, intermediate bodies, and blebs. Prokaryote colonies in digestive gland epithelial cells exclusively contained 1 of 4 morphological cell types and were considered Rickettsiales-like. Hexagonal, virus-like particles were present in the cytoplasm of the largest of these Rickettsiales-like prokaryotes. Although host stress was evident from localized cell necrosis and dense hemocyte infiltration, overall infection was fairly benign, with no major, adverse impact on body condition evident among sectioned or dissected mussels. A possible negative effect was partial constriction of gill water tubes, but at the infection intensity observed (typical range 1 to 7 inclusion bodies per section), significant interference with respiration and other metabolic functions of the gills was highly unlikely.     

Characterization of Peanut Stripe Virus Isolates from Soybean in Taiwan

Potyvirus isolates were obtained in Taiwan from soybean showing crinkle, mottle, mosaic or blistering. They were identified as peanut stripe virus (PStV) on the basis of host range, serology, molecular weight of the capsid proteins and morphology of cytoplasmic cylindrical inclusions. PStV was found to be closely related serologically to adzuki bean mosaic virus (AzMV), blackeye cowpea mosaic virus (BICMV), and the bean common mosaic virus (BCMV) strain NY 15. A clear differentiation of PStV from these related viruses was possible on the basis of the cylindrical inclusion morphology. Only the peanut isolate of PStV from the USA and the three soybean isolates of PStV from Taiwan produced pinwheels, scrolls and curved laminated aggregates whereas the other serologically related viruses produced scrolls only. Whilst the peanut isolate of PStV infected all nine peanut cvs tested, the soybean isolate PN of PStV infected two peanut cvs only. AzMV, BICMV and two strains of soybean mosaic virus did not infect any of the peanut cultivars tested. On the other hand, nineteen and three of the 27 soybean cvs were susceptible to the soybean isolate PN and the peanut isolate of PStV, respectively. The capsid proteins of the peanut and the three soybean isolates of PStV and of AzMV appeared to be proteolytically undegraded and to have nearly identical molecular weights of 35 kD. Based upon results of virus surveys in soybean plantings in Taiwan, the incidence of soybean isolates of PStV in soybean is similar to that of soybean mosaic virus, suggesting that these PStV strains might be economically significant to soybean production m Taiwan.     

Formation of virion-occluding proteinic spindles in a Baculovirus disease of Aedes triseriatus

An unusual process of inclusion formation was studied in Aedes triseriatus larvae infected with a Baculovirus (BV) similar to the nuclear polyhedrosis virus (NPV) type. In this disease virion-occluding proteinic inclusions initially developed individually. However, as the disease progressed the proteinic inclusions gradually coalesced eventually forming large rugose ellipsoids and finally, large smooth-surfaced spindles. Nuclei in late stages of infection usually contained two to five rugose ellipsoidal inclusions, frequently measuring 5 μm to 7 μm in diameter by 10 μm to 15 μm in length. The ellipsoidal forms exhibited different chemical behavior from the spindles.     

Circular dichroic spectrum of poly-L-tyrosine in aqueous solution

The UV and CD spectra of poly-L-tyrosine were investigated at pH 10.6 and pH 11.2. At pH 10.6 (μ=0.1), the CD spectrum exhibits a medium positive band at 230mμ, an extremely small negative band at 217mμ, and a large positive band at 200mμ. At pH 11.2 (μ=0.1), a new positive CD band appears at 277mμ while the bands at 230mμ and 217mμ are shifted to longer wavelengths by 15 and 10mμ respectively. These results, together with UV spectral data and a specific rotation- pH profile, suggest that at pH 10.6, poly-L-tyrosine exists in the helical conformation with only a small fraction of its side chains ionized; at pH 11.2, the polypeptide retains its helical structure but with a considerable increase in ionization.     

Ultrastructure of Inclusions Formed by Ribgrass Mosaic Virus in Leaf Cells

Diffrent types of cytoplasmic inclusions were observed in ultrathin sections of plants systemically infected with three different strains of ribgrass mosaic virus (RMV) (tobamovirus group). Tissue from uninoculated plants did not contain such inclusions. Most common were “rounded plates” consisting of layers of aligned virus particles 300 nm long. RMV also induced angled layer aggregates in Capsicum annuum plants. A novel type of inclusion for the tobamovirus group were the abundant spiral aggregates found in Digitalis purpurea, systemically infected with strain D of RMV. In these aggregates the virions become circularly arranged around a center. The orientation of the particles changes in such a way that virions being 300 nm apartare cut in the longitudinal and in the transverse direction respectively.     

Narcissus latent, a virus with filamentous particles and a novel combination of properties

As previously reported, narcissus latent virus (NLV) has flexuous filamentous particles measuring c. 650 nm × 13 nm, is manually transmissible to Nicotiana clevelandii and Tetragonia expansa, and is transmitted by the aphid Myzus persicae following brief acquisition access periods. In contrast to previous reports the virus particle protein has an apparent mol. wt of c. 45 kD. Moreover, infected cells in N. clevelandii leaves contain cytoplasmic inclusion bodies resembling those of potyviruses. In vitro translation of NLV RNA produced only one major product (mol. wt c. 25 kD) which was not precipitated by antisera to virus particle protein or to cytoplasmic inclusion protein. Antisera to 12 potyviruses and nine carlaviruses failed to react with sap containing NLV particles. Similarly antiserum to NLV particles did not react with particles of seven potyviruses or four carlaviruses. A weak reaction was detected between NLV particles and antiserum to particles of maclura mosaic virus (MMV), a virus which resembles NLV in particle morphology and particle-protein size, and in inducing pinwheel inclusions. The cytoplasmic inclusion proteins (CIPs) of NLV, MMV and from narcissus plants with yellow stripe symptoms were serologically inter-related. These proteins were also serologically related to, and had mol. wt similar to, the CIP of members of the potyvirus group. Particles with the size and antigenic specificity of those of NLV were found consistently in narcissus plants with yellow stripe disease. Narcissus latent and narcissus yellow stripe viruses therefore seem to be synonymous and, together with MMV, have properties distinct from those of any previously described virus group.     

The ND10 component promyelocytic leukemia protein relocates to human papillomavirus type 1 E4 intranuclear inclusion bodies in cultured keratinocytes and in warts

Human papillomavirus type 1 (HPV1) E4 protein is associated with cytoplasmic and nuclear inclusions in productively infected keratinocytes. Here we have used transient expression of HPV1 E4 (also known as E1E4) protein in keratinocytes to reproduce formation of E4 inclusions. Immunofluorescence analysis showed that progressive formation of inclusions correlated with diminished colocalization between E4 and keratin intermediate filaments (IFs). Our results support a model in which the HPV1 E4-keratin IF association is transient, occurring only at an early stage of inclusion formation. We also demonstrate that E4 induces relocation of the promyelocytic leukemia protein (PML) from multiple intranuclear speckles (ND10 bodies) to the periphery of nuclear E4 inclusions and that this activity is specific to full-length E4 protein. Analysis of HPV1-induced warts demonstrated that nuclear PML-E4 inclusions were present in productively infected keratinocytes, indicating that reorganization of PML occurs during the virus's replication cycle. It has been suggested that ND10 bodies are the sites for papillomavirus genome replication and virion assembly. Our finding that E4 induces reorganization of ND10 bodies in vitro and in vivo is further strong evidence that these domains play an important role in the papillomavirus life cycle. This study indicates that HPV1 is analogous to other DNA viruses that disrupt or reorganize ND10 domains, possibly to increase efficiency of virus infection. We hypothesize that HPV1 E4-induced reorganization of PML is necessary for efficient replication of the virus during the virus-producing phase.     

Interactions between pepper ringspot virus and cylindrical inclusions of two potyviruses

Immunoelectron microscopy showed that cylindrical inclusions (CI) of the potyvirus potato virus Y (PVY) bound in addition to their homologous virions those of a co-infecting rod-shaped virus, pepper ringspot virus (PRV), in infected Nicotiana benthamiana leaf cells. The latter virus does not code for cylindrical inclusions and is cl assified as a Tobravirus. Virions of PRV were scattered throughout the cell cytoplasm and not associated with mitochondria in PVY + PRV double infections. Binding of PRV to mitochondria was disrupted in PVY + PRV infected cells. In double infections with a second potyvirus, tobacco etch virus (TEV), and PRV in N. benthamiana cells, TEV-CI bound homologous TEV virions but did not bind PRV. In contrast to PVY + PRV infections, virions of PRV attached end-on to mitochondrial limiting membranes in PRV-only and in TEV + PRV double infections. The results are interpreted to mean that there are differences in the PRV virion binding sites of PVY-CI and TEV-CI. In previous reports, potyviral CI have been nondiscriminating in binding virions or capsid proteins of other co-infecting rod-shaped viruses.     

Chloroplast microtubules in young leaves ofSedum rotundifolium

Well-defined tubular inclusions were detected in mesophyll chloroplasts of youngSedum rotundifolium leaves. The size and distinctly uniform arrangement of tubular inclusions were the most noticeable features. The chloroplast usually contained a large inclusion, sometimes extending almost as long as the chloroplast. Such inclusions were built up from microtubules exhibiting aggregates of either large plate-like or paracrystalline structures depending on the section angles. These inclusions were often quite huge, measuring as much as 7.1 μm in length and 2.6 μm in width. The diameter of the microtubule was approximately 25 nm. The microtubule aggregates were non-membrane bounded structures enclosed partly by the thylakoids. The microtubules in the aggregate were all displaying the same definite orientation. Cross-sectional views clearly demonstrated the characteristic hexagonal arrangement within paracrystalline structures. Longitudinal sections of the chloroplast microtubules showed that they were in perpendicular orientation to the chloroplast envelope. They were not connected to these membranes in any case. In general, one microtubular aggregate was seen in each chloroplast section. The role of tubular inclusions in the chloroplasts related to the photosynthetic mode was discussed.