Studies of a Defective Measles Virus

1968 ◽  
Vol 26 ◽  
pp. 208-209
Author(s):  
R. M. McCombs ◽  
M. Benyesh-Melnick ◽  
J. P. Brunschwig
Keyword(s):  
Inclusion Bodies ◽  
Measles Virus ◽  
Giant Cells ◽  
Helical Structures ◽  
Thin Sections ◽  
Virus Particles ◽  
Extracellular Virus ◽  
Culture Cells ◽  
Infected Cells ◽  
Eosinophilic Inclusions

Measles virus is an agent that is capable of replicating in a number of different culture cells and generally causes the formation of multinucleated giant cells. As a result of infection, virus is released from the cells into the culture fluids and reinfection can be initiated by this cell-free virus. The extracellular virus has been examined by negative staining with phosphotungstic acid and has been shown to be a rather pleomorphic particle with a diameter of about 140 mμ. However, no such virus particles have been detected in thin sections of the infected cells. Rather, the only virus-induced structures present in the giant cells are eosinophilic inclusions (intracytoplasmic or intranuclear). These inclusion bodies have been shown to contain helical structures, resembling the nucleocapsid observed in negatively stained preparations.

scholarly journals Observations of Measles Virus Infection of Cultured Human Cells

1960 ◽  
Vol 7 (1) ◽  
pp. 43-48 ◽  
Author(s):  
Fred Rapp ◽  
Irving Gordon ◽  
Richard F. Baker
Keyword(s):  
Electron Microscope ◽  
Latent Period ◽  
Measles Virus ◽  
Fluorescent Antibody ◽  
Giant Cells ◽  
Virus Antigen ◽  
Cell System ◽  
Extracellular Virus ◽  
Infected Cells ◽  
Spread Of Infection

The development of measles virus in cultures of both primary human amnion cells and H.Ep.-2 cells has been followed by means of the indirect fluorescent antibody technic and concurrent light and electron microscope observations. The immunofluorescence studies revealed that there is a latent period for development of demonstrable measles virus antigen. In amnion cells the latent period lasted for at least 3 days. In contrast, virus antigen could be detected in H.Ep.-2 cells as early as 12 hours following inoculation. In each cell system virus antigen was seen in either nucleus or cytoplasm of infected cells, or both. Early localization tended to be perinuclear. Intranuclear fluorescence was generally less bright and less widespread than cytoplasmic fluorescence. Giant cells and long cytoplasmic spindle-shaped processes appeared regularly in infected cultures. Infectious virus was liberated into the nutrient fluid but when extracellular virus was inhibited by antibody, spread of infection from cell to cell in the monolayer still continued. Results obtained in concurrent electron microscope studies will be presented separately. Correlation of the results of the immunofluorescence and electron microscope studies suggests the possibility that much of the immunofluorescence observed might be due to antigen in virus precursors or components.

scholarly journals AN ELECTRON MICROSCOPE STUDY OF THE DEVELOPMENT OF A MOUSE HEPATITIS VIRUS IN TISSUE CULTURE CELLS

1965 ◽  
Vol 24 (1) ◽  
pp. 57-78 ◽  
Author(s):  
J. F. David-Ferreira ◽  
R. A. Manaker
Keyword(s):  
Electron Microscope ◽  
Inclusion Bodies ◽  
Hepatitis Virus ◽  
Mouse Hepatitis Virus ◽  
Dense Material ◽  
Virus Particles ◽  
Culture Cells ◽  
Tissue Culture Cells ◽  
Tubular Body ◽  
Number Of Particles

Samples taken at different intervals of time from suspension cultures of the NCTC 1469 line of mouse liver—derived (ML) cells infected with a mouse hepatitis virus have been studied with the electron microscope. The experiments revealed that the viruses are incorporated into the cells by viropexis within 1 hour after being added to the culture. An increasing number of particles are found later inside dense cytoplasmic corpuscles similar to lysosomes. In the cytoplasm of the cells from the samples taken 7 hours after inoculation, two organized structures generally associated and never seen in the controls are observed: one consists of dense material arranged in a reticular disposition (reticular inclusion); the other is formed by small tubules organized in a complex pattern (tubular body). No evidence has been found concerning their origin. Their significance is discussed. With the progression of the infection a system of membrane-bounded tubules and cisternae is differentiated in the cytoplasm of the ML cells. In the lumen of these tubules or cisternae, which are occupied by a dense material, numerous virus particles are observed. The virus particles which originate in association with the limiting membranes of tubules and cisternae are released into their lumen by a "budding" process. The virus particles are 75 mµ in diameter and possess a nucleoid constituted of dense particles or rods limiting an electron transparent core. The virus limiting membrane is sometimes covered by an outer layer of a dense material. In the cells from the samples taken 14 to 20 hours after inoculation, larger zones of the cell cytoplasm are occupied by inclusion bodies formed by channels or cisternae with their lumens containing numerous virus particles. In the samples taken 20 hours or more after the inoculation numerous cells show evident signs of degeneration.

scholarly journals Human Cytomegalovirus TR Strain Glycoprotein O Acts as a Chaperone Promoting gH/gL Incorporation into Virions but Is Not Present in Virions

2009 ◽  
Vol 84 (5) ◽  
pp. 2597-2609 ◽  
Author(s):  
Brent J. Ryckman ◽  
Marie C. Chase ◽  
David C. Johnson
Keyword(s):  
Endothelial Cells ◽  
Human Cytomegalovirus ◽  
Null Mutant ◽  
Virus Particles ◽  
Extracellular Virus ◽  
Biochemical Studies ◽  
Infected Cells ◽  
Low Passage ◽  
Er Export ◽  
Strain Ad169

ABSTRACT Human cytomegalovirus (HCMV) produces the following two gH/gL complexes: gH/gL/gO and gH/gL/UL128-131. Entry into epithelial and endothelial cells requires gH/gL/UL128-131, and we have provided evidence that gH/gL/UL128-131 binds saturable epithelial cell receptors to mediate entry. HCMV does not require gH/gL/UL128-131 to enter fibroblasts, and laboratory adaptation to fibroblasts results in mutations in the UL128-131 genes, abolishing infection of epithelial and endothelial cells. HCMV gO-null mutants produce very small plaques on fibroblasts yet can spread on endothelial cells. Thus, one prevailing model suggests that gH/gL/gO mediates infection of fibroblasts, while gH/gL/UL128-131 mediates entry into epithelial/endothelial cells. Most biochemical studies of gO have involved the HCMV lab strain AD169, which does not assemble gH/gL/UL128-131 complexes. We examined gO produced by the low-passage clinical HCMV strain TR. Surprisingly, TR gO was not detected in purified extracellular virus particles. In TR-infected cells, gO remained sensitive to endoglycosidase H, suggesting that the protein was not exported from the endoplasmic reticulum (ER). However, TR gO interacted with gH/gL in the ER and promoted export of gH/gL from the ER to the Golgi apparatus. Pulse-chase experiments showed that a fraction of gO remained bound to gH/gL for relatively long periods, but gO eventually dissociated or was degraded and was not found in extracellular virions or secreted from cells. The accompanying report by P. T. Wille et al. (J. Virol., 84:2585-2596, 2010) showed that a TR gO-null mutant failed to incorporate gH/gL into virions and that the mutant was unable to enter fibroblasts and epithelial and endothelial cells. We concluded that gO acts as a molecular chaperone, increasing gH/gL ER export and incorporation into virions. It appears that gO competes with UL128-131 for binding onto gH/gL but is released from gH/gL, so that gH/gL (lacking UL128-131) is incorporated into virions. Thus, our revised model suggests that both gH/gL and gH/gL/UL128-131 are required for entry into epithelial and endothelial cells.

scholarly journals ELECTRON MICROSCOPE OBSERVATIONS ON THE SURFACE ADENOSINE TRIPHOSPHATASE-LIKE ENZYMES OF HELA CELLS INFECTED WITH HERPES VIRUS

1963 ◽  
Vol 19 (2) ◽  
pp. 337-347 ◽  
Author(s):  
M. A. Epstein ◽  
S. J. Holt
Keyword(s):  
Enzyme Activity ◽  
Hela Cells ◽  
Adenosine Triphosphatase ◽  
Plasma Membranes ◽  
Enzyme Reaction ◽  
Thin Sections ◽  
Virus Particles ◽  
Extracellular Virus ◽  
Simplex Virus ◽  
Vacuolar Membranes

HeLa cells infected with herpes simplex virus have been examined in thin sections by electron microscopy after cytochemical staining for the presence of surface enzymes splitting adenosine triphosphate. As with uninfected HeLa cultures (18), the opaque enzyme reaction product was localized at the plasma membranes of about half the cells, tending to be present where there were microvilli and absent on smooth surfaces. Where mature extracellular herpes particles were found in association with cell membranes showing the enzyme activity, they were invariably likewise stained, and conversely, those mature particles which lay close against cells without reaction product at the surface were themselves free of it. Particles found budding into cytoplasmic vacuoles were also always without opaque deposit since this was never seen at vacuolar membranes, even in cells having the activity at the surface. The enzyme reaction product thus provided a marker indicating the manner in which the particles escape from cells and mature by budding out through cellular membranes, carrying, in the process, a portion of the latter on to themselves to form the outer viral limiting membrane. In some instances, virus particles were observed with more opaque material covering them than was present at the cell membrane with which they were associated. This finding has been taken as evidence for a physiological waxing and waning of surface enzyme activity of adenosine triphosphatase type. The fine structure of the mature extracellular virus as prepared here, using glutaraldehyde fixation, is also recorded. The observations and interpretations are discussed in full.

scholarly journals Measles Virus Forms Inclusion Bodies with Properties of Liquid Organelles

2019 ◽  
Vol 93 (21) ◽  
Author(s):  
Yuqin Zhou ◽  
Justin M. Su ◽  
Charles E. Samuel ◽  
Dzwokai Ma
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Virus Replication ◽  
Inclusion Bodies ◽  
Measles Virus ◽  
Rna Replication ◽  
Infected Cells ◽  
Viral Inclusion ◽  
P Proteins ◽  
Liquid Liquid Phase Separation

ABSTRACT Nonsegmented negative-strand RNA viruses, including measles virus (MeV), a member of the Paramyxoviridae family, are assumed to replicate in cytoplasmic inclusion bodies. These cytoplasmic viral factories are not membrane bound, and they serve to concentrate the viral RNA replication machinery. Although inclusion bodies are a prominent feature in MeV-infected cells, their biogenesis and regulation are not well understood. Here, we show that infection with MeV triggers inclusion body formation via liquid-liquid phase separation (LLPS), a process underlying the formation of membraneless organelles. We find that the viral nucleoprotein (N) and phosphoprotein (P) are sufficient to trigger MeV phase separation, with the C-terminal domains of the viral N and P proteins playing a critical role in the phase transition. We provide evidence suggesting that the phosphorylation of P and dynein-mediated transport facilitate the growth of these organelles, implying that they may have key regulatory roles in the biophysical assembly process. In addition, our findings support the notion that these inclusions change from liquid to gel-like structures as a function of time after infection, leaving open the intriguing possibility that the dynamics of these organelles can be tuned during infection to optimally suit the changing needs during the viral replication cycle. Our study provides novel insight into the process of formation of viral inclusion factories, and taken together with earlier studies, suggests that Mononegavirales have broadly evolved to utilize LLPS as a common strategy to assemble cytoplasmic replication factories in infected cells. IMPORTANCE Measles virus remains a pathogen of significant global concern. Despite an effective vaccine, outbreaks continue to occur, and globally ∼100,000 measles-related deaths are seen annually. Understanding the molecular basis of virus-host interactions that impact the efficiency of virus replication is essential for the further development of prophylactic and therapeutic strategies. Measles virus replication occurs in the cytoplasm in association with discrete bodies, though little is known of the nature of the inclusion body structures. We recently established that the cellular protein WD repeat-containing protein 5 (WDR5) enhances MeV growth and is enriched in cytoplasmic viral inclusion bodies that include viral proteins responsible for RNA replication. Here, we show that MeV N and P proteins are sufficient to trigger the formation of WDR5-containing inclusion bodies, that these structures display properties characteristic of phase-separated liquid organelles, and that P phosphorylation together with the host dynein motor affect the efficiency of the liquid-liquid phase separation process.

MORPHOLOGICAL DEVELOPMENT OF CHIKUNGUNYA VIRUS

1966 ◽  
Vol 12 (5) ◽  
pp. 895-900 ◽  
Author(s):  
Marybelle M. T. Chain ◽  
Frances W. Doane ◽  
D. M. McLean
Keyword(s):  
Chikungunya Virus ◽  
Phosphotungstic Acid ◽  
Morphological Development ◽  
Infective Virus ◽  
Chick Embryo Fibroblast ◽  
Virus Particles ◽  
Outer Membranes ◽  
Extracellular Virus ◽  
Dense Cores ◽  
Infected Cells

Chikungunya virus was first detected by electron microscopy of primary chick embryo fibroblast cultures 5 hours after inoculation. Two types of particles were observed in the cytoplasm of infected cells, but not in uninoculated cells. The smaller, presumably precursor particles, measured 260 to 280 Å. The larger particles contained dense cores 260 to 280 Å in diameter and outer shells 500 to 560 Å in diameter. In cell lysates stained negatively with phosphotungstic acid 12 and 24 hours after inoculation, virus particles surrounded by fine outer membranes showed diameters of 540 to 580 Å. Infective virus was first detected 5 hours after inoculation and maximum yields of cell-associated and extracellular virus were attained at 8 to 10 hours.

Maize Necrotic Lesion Virus Particles and Associated Cellular Inclusions

1988 ◽  
Vol 46 ◽  
pp. 296-297
Author(s):  
O. E. Bradfute ◽  
Raymond Louie
Keyword(s):  
Zea Mays L ◽  
Necrotic Lesion ◽  
Mesophyll Cells ◽  
Thin Sections ◽  
Virus Particles ◽  
Cellular Inclusions ◽  
Maize Roots ◽  
Close Proximity ◽  
Infested Field ◽  
Infected Cells

Maize necrotic lesion virus (MNLV), a newly found soil-borne virus, is apparently one of a complex of viruses infecting roots of maize (Zea mays L.) in northern Ohio (1). Maize roots become infected when plants are grown in infested, field soil that has undergone air-dry storage or in autoclaved, greenhouse soil infested with diseased roots. Symptoms on leaves of rub-inoculated maize and other monocot seedlings first appear as chlorotic local lesions that become necrotic after 24-36 hr.Numerous isometric virus particles of two sizes, ca. 17 and 29 nm in diameter, were observed in crude extracts from MNLV lesions negatively stained in phosphotungstic acid, pH 4.8 (Fig. 1). At pH 6.9 the larger virus particles were frequently stain-penetrated and clumped together or embedded in an amorphous matrix (Fig. 2). MNLV-infected cells were also examined in thin sections cut from fixed and embedded chlorotic lesions (Fig. 3-4). In the cytoplasm of some mesophyll cells, numerous isometric virus particles of both sizes were clearly recognized in close proximity to each other and to masses of electron-dense, amorphous inclusions.

scholarly journals ELECTRON MICROSCOPE OBSERVATIONS ON INTRACELLULAR VIRUS-LIKE PARTICLES ASSOCIATED WITH THE CELLS OF THE LUCKÉ RENAL ADENOCARCINOMA

1956 ◽  
Vol 2 (6) ◽  
pp. 725-742 ◽  
Author(s):  
Don W. Fawcett
Keyword(s):  
Electron Microscope ◽  
Tumor Cells ◽  
Inclusion Bodies ◽  
Hollow Spheres ◽  
Virus Multiplication ◽  
Leopard Frog ◽  
Uniform Size ◽  
Thin Sections ◽  
Virus Particles ◽  
Renal Adenocarcinoma

The common renal adenocarcinoma of the leopard frog was studied in thin sections with the electron microscope. Approximately a third of the tumors examined were found to contain spheroidal bodies of uniform size and distinctive morphology that are believed to be virus particles. These consist of hollow spheres (90 to 100 mµ) having a thick capsule and a dense inner body (35 to 40 mµ) that is eccentrically placed within the central cavity (70 to 80 mµ). Virus particles of this kind occur principally in the cytoplasm but occasionally they are also found in the nucleus and in the extracellular spaces of the tumor. The intranuclear inclusion bodies that are visible with the light microscope are largely comprised of hollow, spherical vesicles with thin limiting membranes. These are embedded in a finely granular matrix. A few of the thin walled vesicles contain a dense inner body like that of the cytoplasmic virus particles. This suggests that they may be immature virus particles. The inclusion bodies are believed to be formed in the course of virus multiplication but they usually contain very few mature virus particles. Bundles of dense filaments and peculiar vacuolar inclusions also occur in the cytoplasm of the tumor cells. These seem to be related in some way to the presence of virus but their origin and significance remain obscure. These findings are discussed in relation to previous work suggesting that the Lucké adenocarcinoma is caused by an organ-specific filtrable agent. It is concluded that the "virus particles" found in electron micrographs of the tumor cells may be the postulated tumor agent. On the other hand, the possibility remains that the particles described here are not those that are causally related to the tumors.

scholarly journals THE STRUCTURE OF INSECT VIRUS PARTICLES

1956 ◽  
Vol 2 (3) ◽  
pp. 301-306 ◽  
Author(s):  
Kenneth M. Smith
Keyword(s):  
Inclusion Bodies ◽  
Virus Disease ◽  
Composite Particles ◽  
Insect Virus ◽  
Virus Diseases ◽  
Thin Sections ◽  
Virus Particles ◽  
Nuclear Polyhedrosis

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

scholarly journals Upon Infection, Cellular WD Repeat-Containing Protein 5 (WDR5) Localizes to Cytoplasmic Inclusion Bodies and Enhances Measles Virus Replication

2017 ◽  
Vol 92 (5) ◽  
Author(s):  
Dzwokai Ma ◽  
Cyril X. George ◽  
Jason L. Nomburg ◽  
Christian K. Pfaller ◽  
Roberto Cattaneo ◽  
...  
Keyword(s):  
Viral Replication ◽  
Inclusion Body ◽  
Virus Replication ◽  
Inclusion Bodies ◽  
Measles Virus ◽  
Rna Replication ◽  
Viral Proteins ◽  
Cellular Protein ◽  
Infected Cells ◽  
Wd Repeat

ABSTRACTReplication of negative-strand RNA viruses occurs in association with discrete cytoplasmic foci called inclusion bodies. Whereas inclusion bodies represent a prominent subcellular structure induced by viral infection, our knowledge of the cellular protein components involved in inclusion body formation and function is limited. Using measles virus-infected HeLa cells, we found that the WD repeat-containing protein 5 (WDR5), a subunit of histone H3 lysine 4 methyltransferases, was selectively recruited to virus-induced inclusion bodies. Furthermore, WDR5 was found in complexes containing viral proteins associated with RNA replication. WDR5 was not detected with mitochondria, stress granules, or other known secretory or endocytic compartments of infected cells. WDR5 deficiency decreased both viral protein production and infectious virus yields. Interferon production was modestly increased in WDR5-deficient cells. Thus, our study identifies WDR5 as a novel viral inclusion body-associated cellular protein and suggests a role for WDR5 in promoting viral replication.IMPORTANCEMeasles virus is a human pathogen that remains a global concern, with more than 100,000 measles-related deaths annually despite the availability of an effective vaccine. As measles continues to cause significant morbidity and mortality, understanding the virus-host interactions at the molecular level that affect virus replication efficiency is important for development and optimization of treatment procedures. Measles virus is an RNA virus that encodes six genes and replicates in the cytoplasm of infected cells in discrete cytoplasmic replication bodies, though little is known of the biochemical nature of these structures. Here, we show that the cellular protein WDR5 is enriched in the cytoplasmic viral replication factories and enhances virus growth. WDR5-containing protein complex includes viral proteins responsible for viral RNA replication. Thus, we have identified WDR5 as a host factor that enhances the replication of measles virus.

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