Morphological Changes in Productive and Abortive Infection by Feline Herpesvirus

Feline herpesvirus produces characteristic morphological alterations in feline kidney cells. Nucleocapsid particles are formed in infected nuclei and are enveloped as they pass through the modified inner nuclear membrane. Aggregates of dense granular material and filamentous structures also regularly appear in infected nuclei. Infection of human embryonic lung cells by feline herpesvirus results in the appearance of intranuclear inclusion bodies, aggregates of dense granular material, and bundles of parallel filaments but no nucleocapsid particles.     

Histochemical evidence for lipoidal material in secretory granules of rat salivary glands

Synposis The granules of parotid acinar cells and submandibular granular tubule cells of rats contain one or more periodic acid-Schiff positive substances that are extracted during fixation with lipid solvents or acidic solutions or if frozen sections are stained in aqueous solutions. The granules in these cells can be stained by Schmorl's reaction, Luxol Fast Blue and a permanganate-Aldehyde Fuchsin sequence. The results obtained with these stains after a variety of fixation procedures strongly suggest that the secretory granules of these two cell types contain several components and that in parotid acinar and submandibular granular tubule cells, at least one of these components is a lipoidal substance.     

Morphogenesis of Type 2 Parainfluenza Virus Examined by Light and Electron Microscopy

The development of type 2 parainfluenza virus in HeLa and stable human amnion cells was examined by use of antisera labeled with fluorescein and ferritin. Serum containing antibody predominantly to soluble viral antigen gave specific fluorescence which was first detectable in small cytoplasmic foci 8 to 10 hr after initiation of infection. By 20 to 24 hr, when the production of infective virus and hemagglutinin was maximal, large perinuclear aggregates of fluorescence were observed which corresponded in distribution and time of appearance to the eosinophilic inclusions seen in similar preparations stained with azure eosin. The inclusions, examined by electron microscopy, were composed of fibrils, presumably viral ribonucleoprotein, which specifically bound the antibody labeled with ferritin. With antiserum to concentrated virus, on the other hand, specific fluorescence was most marked at the surface of infected cells. Foci of fluorescence at the surface represented segments of membrane which had become differentiated morphologically and antigenically to resemble the viral envelope. These were the sites where mature virions appeared. The latter exhibited marked pleomorphism; in some instances, particles were formed which lacked recognizable internal fibrils but which possessed an enclosing membrane bearing viral antigen. Filamentous forms showing an organized internal structure were also observed at the cell surface, but were never encountered in negatively stained preparations. No clear relationship between these filaments and the spherical or oval forms could be established. In negatively stained preparations, nucleocapsid released by rupture of viral particles was similar in appearance to that reported for other paramyxoviruses. It seems probable that this component has a helical configuration.     

A COMPARISON OF THE CHANGES IN FINE STRUCTURE OF L CELLS DURING SINGLE CYCLES OF VIRAL MULTIPLICATION, FOLLOWING THEIR INFECTION WITH THE VIRUSES OF MENGO AND ENCEPHALOMYOCARDITIS

The virus of encephalomyocarditis (EMC), examined by the negative-contrast method, is indistinguishable from the serologically related Mengovirus. The particles are 270 to 280 A in diameter. The surface of EMC is composed of an undetermined number of subunits. Frequent sampling of infected cells was carried out throughout one-step cycles of viral multiplication to observe cytopathic changes occurring in L cells infected by these two related RNA viruses. EMC and Mengovirus, which multiply at equal rates, in most respects elicit similar alterations in cell fine structure. Rearrangement and changes in nuclear material accompanied by formation of small vesicles in the centrosphere region commence at 4 to 6 hours after infection. Thereafter a progressive degeneration of the nucleus and vesiculation of the cytoplasm are observed up to 18 to 20 hours. Increased numbers of small dense granules, indistinguishable from ribonucleoprotein particles, appear in the cytoplasm between 8 and 14 hours after infection. L cells infected with Mengovirus become permeable to Erythrocin more slowly than those infected with EMC. Only in the case of Mengovirus infection are large aggregates of dense material first observed in the cytoplasm at 8 hours, followed by the appearance of crystals probably composed of Mengovirus particles, at 12 hours. Differences in the rates of cell permeability after infection with EMC and Mengovirus are discussed in relation to formation of virus crystals and plaque-type mutants.     

Combined Fluorescent-Antibody and Electron Microscopy Study of Marek''s Disease Virus-Infected Cell Culture

Duck embryo fibroblast (DEF) and chicken embryo fibroblast (CEF) cultures infected with Marek's disease virus were studied by combined fluorescent antibody and electron microscopy techniques. In both DEF and CEF cultures, cells containing immunofluorescent (IF) antigen also contained herpesvirus particles; conversely, cells lacking this antigen lacked herpesvirus particles. Two morphologically distinct IF antigens were detected in the cytoplasm. (i) A granular antigen in the perinuclear region was brightly stained with the conjugated antibody. This antigen was composed of a granular mass of osmiophilic material and did not contain virions. (ii) A diffuse antigen, present throughout the cytoplasm of infected cells, was less brightly stained. The area of the cell with the highest concentration of this antigen contained small vesicles, folded membranes, and fine electron-dense granules. Naked virions were occasionally seen in these areas. A diffuse nuclear IF antigen was occasionally seen in infected cells. This antigen was often separated from the nuclear membrane and the nucleolus by a clear margin. The intranuclear IF antigen was composed of a fine granular aggregate and naked herpesvirus particles which were randomly distributed throughout the nucleus. Viral capsids in antibody-treated cells were coated with fine filamentous material.     

Cellular Compartmentalization of Herpesvirus Antigens During Viral Replication

HEp-2 cells infected with herpes simplex virus develop five distinct immunofluorescent elements. Three (small nuclear granules, large nuclear granules, and an amorphous mass filling the nucleus) contain antigens which react with a rabbit serum prepared against boiled infected cell debris. A labeled pool of human antibody revealed antigens making up cytoplasmic granules and those responsible for a diffuse cytoplasmic fluorescence. All five immunofluorescent elements are demonstrable with a rabbit serum prepared against unheated infected cell debris. Viral antigens are segregated in the nucleus or in the cytoplasm; within the limits of detection, each antigen accumulates in one compartment only. The antigens responsible for the diffuse cytoplasmic fluorescence and for the amorphous nuclear mass are synthesized early in infection; they are formed in arginine-deprived cells and exist in a form which does not sediment on centrifugation at 79,000 x g for 2 hr. The antigens comprising the nuclear and cytoplasmic granules arise relatively late in infection; they are not formed in arginine-deprived cells, and they are readily sedimented on centrifugation at 79,000 x g for 2 hr. Heating (60 C for 2 hr) confers on one or more cytoplasmic viral antigens a new specificity; the altered antigens are demonstrable with labeled rabbit anti-boiled infected cell serum which normally does not combine with cytoplasmic antigens.     

The effects of colchicine on the ultrastructure of the dental epithelium and odontoblasts of teleost tooth buds

Secretory granule ultrastructure of teleost inner dental epithelial (IDE) cells has been reported to be similar to procollagen granules of other cells synthesizing collagen. This study describes the ultrastructure of secretory products in odontogenic cells during enameloid matrix formation in cichlids after inhibition of granule secretion with colchicine. Thirty-six fish were injected with 0.1 mg colchicine, then three were killed first at 2-hr intervals for 12 hr, then daily for 5 days. Tooth buds were processed for transmission electron microscopy, and ultrastructural alterations were assessed for each post-injection interval. Four hours post-injection, IDE cells contained increased numbers of secretory granules, lightly stained granules, dilated cisternae of the granular endoplasmic reticulum, and intercellular amorphous material. After 6 hr, the IDE intercellular amorphous material additionally contained electron dense deposits, and after 8 hr, the intercellular material had fibers similar in appearance to enameloid collagen. No ultrastructural changes were detected in odontoblasts that were in close proximity to the enameloid matrix. Only odontoblasts synthesizing predentin were affected by colchicine, and the observed alterations were similar to those seen in IDE cells. It is concluded that IDE cells synthesize and secrete ectodermal enameloid matrix collagen.     

Function of dynein and dynactin in herpes simplex virus capsid transport

After fusion of the viral envelope with the plasma membrane, herpes simplex virus type 1 (HSV1) capsids are transported along microtubules (MTs) from the cell periphery to the nucleus. The motor ATPase cytoplasmic dynein and its multisubunit cofactor dynactin mediate most transport processes directed toward the minus-ends of MTs. Immunofluorescence microscopy experiments demonstrated that HSV1 capsids colocalized with cytoplasmic dynein and dynactin. We blocked the function of dynein by overexpressing the dynactin subunit dynamitin, which leads to the disruption of the dynactin complex. We then infected such cells with HSV1 and measured the efficiency of particle binding, virus entry, capsid transport to the nucleus, and the expression of immediate-early viral genes. High concentrations of dynamitin and dynamitin-GFP reduced the number of viral capsids transported to the nucleus. Moreover, viral protein synthesis was inhibited, whereas virus binding to the plasma membrane, its internalization, and the organization of the MT network were not affected. We concluded that incoming HSV1 capsids are propelled along MTs by dynein and that dynein and dynactin are required for efficient viral capsid transport to the nucleus.     

Ultrastructure of the liver cells in Salamandra salamandra (L.) in the end of Winter

Following hibernation, most salamander liver cells undergo alterations. The liver cells containing the structure of their active period present a normal nucleus, glycogen granules disseminated throughout the entire cytoplasm and granular mitochondria with several peripheral cristae. Most cells, however, possess altered mitochondria, transformed into electron-dense masses, or clear vesicles with the possibility of digesting the ambient organelles. Other cells have clear vacuoles with similar digestive possibilities. The endoplasmic reticulum is poorly represented in the liver cells that have suffered advanced alteration. The nucleus of these cells is homogeneously granular. The sytoplasm is populated by electron-dense bodies and lipid vacuoles. The nucleolemma and plasmalemma are thickened. The pigmented cells, altered concomitantly with the liver cells, contain granular aggregates. Some of the pigmented cells exhibit electron-clear formations similar to the reflecting platelets of the iridocytes of the integument of lower vertebrates.     

The leader protein of cardioviruses inhibits stress granule assembly

Stress granules (SG) are cytoplasmic aggregates of stalled translation preinitiation complexes that form in cells exposed to various environmental stresses. Here, we show that stress granules assemble in cells infected with Theiler's murine encephalomyelitis virus (TMEV) mutants carrying alterations in the leader (L) protein, but not in cells infected with wild-type TMEV. Stress granules also formed in STAT1-deficient cells, suggesting that SG formation was not a consequence of increased type I interferon (IFN) production when cells were infected with the mutant virus. Ectopic expression of the wild-type L protein was sufficient to inhibit stress granule formation induced by sodium arsenite or thapsigargin treatment. In conclusion, TMEV infection induces stress granule assembly, but this process is inhibited by the L protein. Unlike poliovirus-induced stress granules, TMEV-induced stress granules did not contain the nuclear protein Sam68 but contained polypyrimidine tract binding protein (PTB), an internal ribosome entry site (IRES)-interacting protein. Moreover, G3BP was not degraded and was found in SG after TMEV infection, suggesting that SG content could be virus specific. Despite the colocalization of PTB with SG and the known interaction of PTB with viral RNA, in situ hybridization and immunofluorescence assays failed to detect viral RNA trapped in infection-induced SG. Recombinant Theiler's viruses expressing the L protein of Saffold virus 2 (SAFV-2), a closely related human theilovirus, or the L protein of mengovirus, an encephalomyocarditis virus (EMCV) strain, also inhibited infection-induced stress granule assembly, suggesting that stress granule antagonism is a common feature of cardiovirus L proteins.     

Cleavage of Poliovirus-Specific Polypeptide Aggregates

Zonal electrophoresis resolves two aggregates of poliovirus type 2 cytoplasmic polypeptides. The more negatively charged aggregate contains mainly noncapsid viral-specific polypeptides (NCVP) 2 and x, whereas the other consists of the capsid polypeptides (VP) 0, 1, 2, and 3 (VP0, VP1, VP2, VP3). After treatment with sodium deoxycholate (DOC), the aggregates sediment at 5 to 6S. Their electrophoretic mobilities are unaffected by DOC or RNase. The capsid polypeptide aggregate is similar in mobility to virions but can be converted to a faster electrophoretic form, resembling empty capsids, by heating. If infected HeLa cells are allowed to synthesize poliovirus polypeptides in the presence of iodoacetamide, no capsid polypeptides are produced, but rather NCVP1a (the precursor to capsid polypeptides) is accumulated, along with NCVP2 and NCVPx. When analyzed by electrophoresis and centrifugation, uncleaved NCVP1a migrates with the NCVP2-x aggregate. NCVP1a can be cleaved to capsid-like polypeptides in vitro by using extracts of infected cells, but not uninfected cells, indicating either a virus-specified protease or a cellular enzyme activated during infection. After cleavage of NCVP1a by infected cell extracts, the capsid polypeptides which are produced dissociate from the NCVP2-x complex.     

Fine structure of epithelial cells of Lieberkühn's crypts in feline panleukopenia.

Electron microscopic observation was carried out on epithelial cells of Lieberkühn's crypts of cats naturally affected with feline panleukopenia. The most important change was the replication of feline panleukopenia. The most important change was the replication of feline panleukopenia virus in the nucleus with associated alterations in the lining epithelial cells of the crypts. In these cells in the early stage of infection, virus particles 20 nm in average diameter were found either singly or in small regularly arrayed clusters everywhere in the markedly swollen nucleus. In the course of infection, the nucleus of infected cells became rather atrophic with a marked margination of chromatin granules. Its major portion was occupied with masses of fine fibrillar substance. It was a "viral matrix area" in which appeared a large compact aggregate of virus particles showing a crystalline array. At the same time, the outer membrane of the nuclear envelope partially extended and disrupted. Membranous elements related to it in the cytoplasm were regularly distributed almost always with particles indistinguishable from the virus particles in the nucleus. From these results it was suggested that the major portion of the infected nucleus, or the site of viral replication, might correspond to the amphophilic intranuclear inclusion body revealed by light microscopy.     

Ultrastructure of alarm substance cells in the epidermis of the channel catfish, Ictalurus punctatus (Rafinesque)

The cytoplasm of the alarm substance cells in the epidermis of an ostariophysian fish, channel catfish, contained fine homogeneously dispersed fibrillar material. Most cyto-plasmic organelles were concentrated perinuclearly. The mitochondria were of the tubular type and had matrix granules 30-50 nm in diameter. The saccules of the Golgi bodies did not form a parallel array and were comparatively widely separated. The nucleus was irregular in shape and had a distinct nucleolus made up of deeply stained granules meshed with areas of less granulation. The nuclear envelope was in the form of a double membrane having a wavy appearance and occasionally it showed invaginations penetrating deeply into the nucleoplasm. Lysosomes and lipid inclusions were encountered in the vicinity of the nucleus. Proteins were demonstrated histochemically, but carbohydrates were not detected. Differences between the alarm substance cells of channel catfish and those of other ostariophysian fish were described and discussed.     

STRUCTURE AND DEVELOPMENT OF VIRUSES OBSERVED IN THE ELECTRON MICROSCOPE : IV. VIRUSES OF THE RI-APC GROUP

Representative viruses of the RI-APC group were observed with the electron microscope in thin sections of infected HeLa cells. The viral particles varied in density, were approximately 60 mµ in diameter and had a center to center spacing when close packed of about 65 mµ. Many of the less dense particles exhibited an internal body averaging 24 mµ in diameter. It was suggested that within the nucleus the virus differentiated from dense granular and reticular material and formed crystals. Disintegration of the crystals and disruption of the nuclear membrane with release of virus into the cytoplasm appeared to occur at any stage. No evidence to suggest development of the virus in the cytoplasm was obtained. It was possible to deduce the structure of the viral crystal from the electron micrographs. The viral particles are packed in a cubic body—centered lattice. Correlative histochemical observations in the light microscope which are now in progress revealed that the crystals and non-crystalline aggregates of virus were strongly Feulgen-positive.     

The immunolocalization of small nuclear ribonucleoprotein particles in testicular cells during the cycle of the seminiferous epithelium of the adult rat

The objective of this study was to determine the cellular and subcellular distribution of small nuclear ribonucleoprotein particles (snRNPs) in the adult rat testis in relation to the different cell types at the various stages of the cycle of the seminiferous epithelium. The distribution of snRNPs in the nucleus and cytoplasm of germ cells was quantitated in an attempt to correlate RNA processing with morphological and functional changes occurring during the development of these cells. Light-microscopic immunoperoxidase staining of rat testes with polyclonal anti-Sm and monoclonal anti-Y12 antibodies localized spliceosome snRNPs in the nuclei and cytoplasm of germ cells up to step 10 spermatids. Nuclear staining was intense in Sertoli cells, spermatogonia, spermatocytes, and in the early steps of round spermatid development. Although comparatively weaker, cytoplasmic staining for snRNPs was strongest in mid and late pachytene spermatocytes and early round spermatids. Quantitative electron-microscopic immunogold labeling of Lowicryl embedded testicular sections confirmed the light-microscopic observations but additionally showed that the snRNP content peaked in the cytoplasm of midpachytene spermatocytes and in the nuclei of late pachytene spermatocytes. The immunogold label tended to aggregate into distinct loci over the nuclear chromatin. The chromatoid body of spermatids and spermatocytes and the finely granular material in the interstices of mitochondrial aggregates of spermatocytes were found to be additional sites of snRNP localization and were intensely labeled. This colocalization suggests that these dense cytoplasmic structures may be functionally related. Anti-U1 snRNP antibodies applied to frozen sections showed the same LM localization pattern as spliceosome snRNPs. Anti-U3 snRNP antibodies applied to frozen sections stained nucleoli of germ cells where pre-rRNA is spliced.     

LIPOFUSCIN (AGING) PIGMENT GRANULES OF THE NEWBORN HUMAN LIVER

We have observed pigmented cytoplasmic granules, with the characteristic staining properties of lipofuscin (ceroid, "wear-and-tear") pigment, in newborn human liver. The pigment is found at the periphery of the lobule in hepatocytes and some bile ductular cells. It is acid-fast, PAS-positive after diastase digestion, slightly argyophilic and sudanophilic, and markedly Schmorl's- and peroxidase positive in paraffin sections. Difficult to see in sections stained with hematoxylin and eosin, the pigment can be detected in unstained sections. The granules also resemble lipofuscin found in adult tissues, in their ultra-structural and enzymatic properties. They are polymorphic, contain granular material of moderate and high electron opacity, and are delimited by a single membrane. Acid phosphatase and β-glucuronidase activities are visualized in the newborn granules, identifying them as lysosomes. The granules also contain copper and, to a much lesser extent, iron. The accumulation of lipofuscin pigment in lysosomes in many tissues correlates well with aging, and this process has been interpreted as a reflection of cellular degeneration or wear-and-tear. However, the presence of lipofuscin granules as a constant component of neonatal liver suggests that they are not a measure of cellular senescence.     

Evidence that ADH-stimulated intramembrane particle aggregates are transferred from cytoplasmic to luminal membranes in toad bladder epithelial cells

In freeze-fracture (FF) preparations of ADH-stimulated toad urinary bladder, characteristic intramembrane particle (IMP) aggregates are seen on the protoplasmic (P) face of the luminal membrane of granular cells while complementary parallel grooves are found on the exoplasmic (E) face. These IMP aggregates specifically correlate with ADH-induced changes in water permeability. Tubular cytoplasmic structures whose membranes contain IMP aggregates which look identical to the IMP aggregates in the luminal membrane have also been described in granular cells from unstimulated and ADH-stimulated bladders. The diameter of these cytoplasmic structures (0.11 +/- 0.004 micrometers) corresponds to that of tubular invaginations of the luminal membrane seen in thin sections of ADH-treated bladders (0.13 +/- 0.005 micrometers). Continuity between the membranes of these cytoplasmic structures (which are not granules) and the luminal membrane has been directly observed in favorable cross-fractures. In FF preparations of the luminal membrane, these apparent fusion events are seen as round, ice-filled invaginations (0.13 +/- 0.01 micrometer Diam), of which about half have the characteristic ADH-associated aggregates near the point of membrane fusion. They are less numerous than, but linearly related to, the number of aggregates counted in the same preparations (n = 78, r = 0.71, P less than 0.01). These observations suggest that the IMP aggregates seen in luminal membrane after ADH stimulation are transferred preformed by fusion of cytoplasmic with luminal membrane.     

Nuclear localization of herpesvirus proteins: potential role for the cellular framework.

Two herpes simplex virus proteins, the major capsid protein and the major DNA binding protein, are specifically localized to the nucleus of infected cells. We have found that the major proportion of these proteins is associated with the detergent-insoluble matrix or cytoskeletal framework of the infected cell from the time of their synthesis until they have matured to their final binding site in the cell nucleus. These results suggest that these two proteins may interact with or bind to the cellular cytoskeleton during or soon after their synthesis and throughout transport into the cell nucleus. In addition, the DNA binding protein remains associated with the nuclear skeleton at times when it is bound to viral DNA. Thus, viral DNA may also be attached to the nuclear framework. We have demonstrated that the DNA binding protein and the capsid protein exchange from the cytoplasmic framework to the nuclear framework, suggesting the direct movement of the proteins from one structure to the other. Inhibition of viral DNA replication enhanced the binding of the DNA binding protein to the cytoskeleton and increased the rate of exchange from the cytoplasmic framework to the nuclear framework, suggesting a functional relationship between these events. Inhibition of viral DNA replication resulted in decreased synthesis and transport of the capsid protein. We have been unable to detect any artificial binding of these proteins to the cytoskeleton when solubilized viral proteins were mixed with a cytoskeletal fraction or a cell monolayer. This suggested that the attachment of these proteins to the cytoskeleton represents the actual state of these proteins within the cell.     

Control of membrane morphogenesis in bacteriophage PM2

The regulation of membrane formation in bacteriophage PM2 serves as a simple model for changes in membrane structure in eukaryotic cells. Prior to Pseudomonas host lysis, wild-type virions mature to an icosahedral morphology at the inner face of the cytoplasmic membrane. The proliminary charcterization of two temperature-sensitive mutants of PM2 is described. In cells infected at the restrictive temperature with ts 1, an abundance of “empty” virus-size membrane vesicles are seen. Synthesis of DNA is also reduced in ts 1 infected cells. The preponderance of vesicles is not sen in cells infected with wil-type virus or with ts 1 at the permissive temperature. The “empty” appearance of the viral membranes suggests that viral DNA is not encapsulated. The major viral capsid protein (MW 26,000) is located just out side the viral membrane and normallyl sediments with host and virus membranes; insted, large amounts of capsid protein can be precipitated from the supernatant with TCA. Compared to cells infected with wild type virus, cells infected with is 5 at th restrictive temperature produce inside the cell an aboundance of virus-soze membrane vesicles. Taken Together, These results with viral mutants suggest that formation of a viral membrane of the proper size does not require a DNA core around which to form, or an outer scaffolding of coat protein against which to form a spherical bilayer.     

Development and fine structure of the yolk nucleus of previtellogenic oocytes in the medaka Oryzias latipes

The development and fine structure of yolk nuclei in the cytoplasm of previtellogenic oocytes were examined by electron microscopy during several stages of oogenesis in the medaka, Oryzias latipes. Shortly after oogenesis starts, oocytes 20-30 microm in diameter have much electron-dense (basophilic) cytoplasm, within which a continuous or discontinuous, irregular ring-shaped lower electron-dense area of flocculent appearance (LF) begins to emerge around the nucleus. The yolk nucleus is first recognized within an LF area as a few fragments of dense granular thread measuring 20-25 nm in width. The threads consist of two rows of very dense granules resembling ribosomes or ribonucleoprotein (RNP)-like particles in size and electron density. These thread-like fragments gradually increase in number and length until they assemble into a compact, spherical mass of complicated networks. Analysis of serial sections suggests that the yolk nucleus is a complicated mass of numerous, small deformed vacuoles composed of a single lamella with double layers of ribosomes or RNP-like granules, rather than a mass of granular threads. When oocytes develop to greater than 100 microm in diameter, the yolk nucleus begins to fragment before dispersing throughout the surrounding cytoplasm, concomitantly with the disappearance of LF areas. At this stage of oogenesis, a restricted region of the granulosa cell layer adjacent to the yolk nucleus becomes somewhat columnar in morphology, fixing the vegetal pole region of the oocyte.