Infection Of Chick Embryo Cells Research Articles

Persistence of Rous sarcoma virus in transformed non-permissive cells: characteristics of virus induction following Sendai virus-mediated fusion with permissive cells.

AbstractVirus induction in heterokaryons formed by non‐permissive cells transformed by Rous sarcoma virus (RSV) and permissive chick embryo (CE) cells, in the presence of inactivated Sendai virus, was studied with a clone of hamster BHK21 fibroblasts transformed by Schmidt‐Ruppin strain RSV (clone RS2) and subclones derived from this clone. The main results have been the following: (1) The number of heterokaryons giving rise to foci of Rous cells (focus‐forming infective centers, or FFICs) in mixed cultures is proportional to the number of RS2 cells when the initial CE:RS2 cell ratio attains 30, and almost all heterokaryons are then formed from single RS2 cells. (2) Recovery of FFICs also depends on the dose of Sendai virus. (3) Following fusion of RS2 cells with cells from different chick embryos the recovery of FFICs varies in parallel with the number of foci produced by free virus in cultures of these embryos. (4) The latent period of virus induction from the time of cell fusion is about 24 h, and somewhat longer than that of viral replication in newly infected CE cells. The growth of induced virus is similar to that of virus produced after de novo infection. (5) Virus induction requires no DNA synthesis but is inhibited, reversibly, by treatment for a few hours with inhibitors of DNA‐dependent synthesis (actinomycin D) and protein synthesis (puromycin and cycloheximide). Hence, non‐permissiveness of RS2 cells may be due to a partial block of transcription of the viral replicative DNA (provirus) present in the cells, and induction in heterokaryons to switching from partial to full transcription and translation of the provirus. However, it is also possible that some function, or factor (s) of the CE cells is required for virus production in heterokaryons. (6) The study of a subclone of RS2 cells 100‐ to 1000‐fold less inducible than other subclones suggesting that, in this subclone, non‐permissiveness may be due to a permanent block of synthesis of all viral proteins, at the level of transcription of the provirus.

Role of mitochondria in the production of RNA-containing tumor viruses.

The role of mitochondria in the reproduction of RNA-containing tumor viruses was examined by using ethidium bromide (EB) to induce degenerative effects in mitochondria. The effects of EB in murine and avian cells were monitored by electron microscopy. Chronically infected mouse (JLS-V5) cells, in which extensive mitochondrial changes were induced, continued to produce murine leukemia virus. Also, complete reproductive cycles of Rous sarcoma virus (RSV) occurred in newly infected chicken embryo cells exposed to EB. Morphological transformation characteristic of infection of chicken embryo cells by RSV occurred in cells which contained induced aberrant mitochondria. The results demonstrate that mitochondria play a relatively minor role, if any, in the reproduction of RNA-containing tumor viruses.

Ribonucleic acid species of intracellular nucleocapsids and released virions of vesicular stomatitis virus.

Infection of chicken embryo cells with vesicular stomatitis (VS) virus resulted in variable production of three classes of intracellular viral ribonucleocapsids with sedimentation coefficients of approximately 140S, 110S, and 80S, as well as three corresponding classes of released virions designated B, LT, and T. Intracellular nucleocapsids of each class contained three proteins of which the major N protein was firmly bound, and the minor L and NS1 proteins were readily dissociated with 0.5 m NaCl. The ribonucleic acid (RNA) species extracted from B, LT, and T virions, and from corresponding intracellular nucleocapsids, contained RNA species with approximate molecular weights of 3.2 x 10(6), 2.0 x 10(6), and 10(6), respectively, as determined by polyacrylamide gel electrophoresis. These values are roughly equivalent to sedimentation coefficients of 42S, 28S, and 23S for each of the virion and nucleocapsid RNA species. Cells infected at high multiplicity with undiluted passage VS virus gave rise primarily to virions and nucleocapsids containing 23S RNA, whereas cells productively infected with purified B virions produced predominantly B and LT virions and nucleocapsids. At late stages in the productive cycle of infection, more virions containing 42S RNA were produced, but the intracellular pool of nucleocapsids containing 28S and 23S RNA remained relatively constant. Additional studies by more refined techniques are required to test the hypothesis that nucleocapsids containing 28S and 23S RNA are precursors of the 42S RNA in infectious VS-B virions and that production of defective T and LT virions results from failure of ligation of the RNA precursors.

Metabolic requirements for infection by rous sarcoma virus: III. The synthesis on viral DNA

Early stages in the infection of chick embryo cells with Rous sarcoma virus (RSV) were examined. Inhibition of protein synthesis with cycloheximide prior to and for a short interval after infection had little effect on subsequent transformation or virus yield, supporting an earlier conclusion ( Bader, 1966) that preexisting enzymes were used for the synthesis of viral DNA. Inhibition of virus reproduction after addition of 5-iododeoxyuridine (IdUrd) for various brief intervals after infection demonstrated that viral DNA synthesis is initiated within the first hour after exposure of cells to virus. Also, as the interval between infection and the addition of IdUrd increased, inhibition of progeny decreased, and the extent of inhibition suggested that viral DNAs accumulate logarithmically. A model is proposed for the residence and function of virus replicative genomes.

Membrane-bound proteins of Japanese encephalitis virus-infected chick embryo cells

The seven Japanese encephalitis virus specific polypeptides found in infected chick embryo cells were all bound to membranes. None were completely released from the membranes by treatment with neutral salt, alkaline salt, or dilute detergent, but two of them were partially released by both the neutral and alkaline salts. The polypeptides were released or attacked by trypsin at unequal rates and in the sequence: NV-5≥ NV-4 > V-3. NV-5 was released as a relatively undegraded soluble polypeptide, NV-4 was extensively degraded, and V-3 was degraded but part of its trypsinderived fragment (TF-2) remained membrane bound. We suggest that the three largest viral polypeptides are bound in such a manner that the larger the polypeptide, the more exposed and superficial it is. Treatment of virions with trypsin produced low molecular weight material and three discrete polypeptide fragments, probably all derived from the large virion envelope protein V-3; two (TF-1 and TF-3) had electrophoretic mobilities similar to the two naturally occurring nonvirion virus-specified polypeptides, NV-1 and NV-3.

Temperature-sensitive mutants isolated from L cells persistently infected with Newcastle disease virus.

Virus mutants (NDV(pi)) isolated from L cells persistently infected with the Herts strain of Newcastle disease virus have been previously reported by this laboratory to differ from the wild-type virus (NDV(o)) in several physical and biological properties. It has now been determined that, in addition to these differences, the NDV(pi) mutants are also spontaneously selected temperature-sensitive mutants. The temperature sensitivity of 10 NDV(pi) clones was confirmed by temperature inhibition, plaquing efficiency, and single-cycle yield experiments. The cut-off temperature, at which more than 90% of virus replication is inhibited was between 41 and 42 C. All 10 NDV(pi) clones were also found to be defective in virus-specific ribonucleic acid (RNA) synthesis in infected chick embryo cells at 42 C and are tentatively classified as RNA(-). The possible relationships of the temperature sensitivity, the other NDV(pi) properties, and the maintenance of the persistently infected state are discussed.

Characterization of the abortive infection of chick embryo cells by herpesvirus type I.

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Transmission of Avian Myeloblastosis by BAI Strain A Virus Ribonucleic Acid<xref ref-type="fn" rid="FN2">2</xref>

High-molecular-weight RNA was isolated from BAI strain A avian leukosis (myeloblastosis) virus by the sodium dodecyl sulfate dithiothreitol technique. Three of five preparations induced infection in synchronized chick embryo cells (CEC) in tissue cultures pretreated with diethylaminoethyl-dextran. In 2 series of studies, virus particles released by the CEC into the culture fluids caused neoplasms typical of the action of BAI strain A virus manifested by myeloblastosis and tumors of the kidney, liver, and ovary when inoculated into newly hatched chickens. Some of the cultures exhibited pronounced CEC morphologic alteration. Virus produced in the altered CEC cultures was infectious for, and caused morphologic changes on, serial passage in fresh CEC, but did not infect or alter bone marrow cells. A third RNA preparation infected CEC but did not alter the cells, though the virus induced tumors in the chickens. Agent liberated by the CEC cultures interfered with Rous sarcoma virus and MC29 virus superinfection and activated RSV nonproducer cells. RNA fractions inducing infection contained no virus particles that could be seen in the electron microscope, and infectivity was abolished by ribonuclease treatment. Protein content of the RNA preparations was less than 6% which was 406-fold less than that in intact virus particles as demonstrated by use of double labeling.

Enzyme studies (phosphorylation) during Newcastle disease virus infection of chick embryo cells.

The infection of chicken embryo cells with Newcastle disease virus (NDV) caused the increased incorporation of 3H-uridine into viral ribonucleic acid (RNA). However, when uridine kinase activity was assayed, it was found that infected cells demonstrated the identical activity as uninfected chicken embryo cells. Hexokinase and adenosine triphosphatase showed the same trend as uridine kinase. However, carbamyl phosphate synthetase was found to increase in activity during NDV infection.

Purification and properties of Semliki Forest virus nucleocapsids

A method is described for the purification of Semliki Forest virus nucleocapsids from infected chick embryo cells. Cell extracts are sedimented through a 5–20% sucrose gradient, and the peak of nucleocapsids at 145 S is recovered and dialyzed against a buffer lacking magnesium, to dissociate contaminating cellular ribosomes and ribosome oligomers. The dialyzed material is sedimented twice in succession into sucrose gradients of high viscosity, giving rise to a monodisperse band of nucleocapsids free of other ultraviolet-absorbing components or of particulate contamination. Purified nucleocapsids are spherical particles 40 nm in diameter which are frequently penetrated by negative stains. They have a mottled surface appearance suggestive of a subunit structure. They contain predominantly 45 S viral RNA and a single protein of molecular weight approximately 32,000. Purified nucleocapsid preparations are essentially free of contaminating cellular RNA, but 25% of the the protein in these preparations is derived from the host cells. This contaminating protein cosediments with nucleocapsids and is of a variety of molecular weights; it may be adsorbed to the viral RNA in the nucleocapsids. Nucleocapsids have a 260 nm:280 nm ultraviolet absorbance ratio of 1.75–1.80, and buoyant densities in potassium tartrate and cesium chloride of approximately 1.34 and 1.47 g/ml, respectively. These figures suggest that the RNA content of nucleocapsids may be substantially higher than 30% of their total mass.

Altered viral ribonucleic acid synthesis by a temperature-sensitive mutant of eastern equine encephalitis virus

A temperature-sensitive mutant (Ets-4) derived from eastern equine encephalitis virus induced the synthesis of unusually large amounts of viral RNA in infected chick embryo cells. Ets-4 produced two to four times more RNA at 37 °C; however, its yield of mature virus was about 10 times less than the parent virus. Viral RNA extracted from infected chick embryo cells was fractionated on sucrose gradients. Three types of viral RNA were synthesized by both viruses: the infectious 45 s RNA, 27 s “interjacent” RNA and a 20 s ribonuclease resistant double-stranded RNA.Ets-4 produced greater amounts of each RNA species than did encephalitis virus. Production of the 45 s infectious RNA by Ets-4 as measured by plaque assay was less than that of the parent virus, but the incorporation of radioactive uridine into this RNA species was greater, suggesting that the 45 s RNA was biologically defective. On a specific infectivity basis (infectious 45 s RNA/radioactive 45 s RNA) the 45 s RNA of Eta-4 possessed only 2% of the infectivity observed for its parent. Viral protein synthesis, measured by the complement fixation and hemagglutinin tests, induced by Ets-4 was depressed below that of the parent virus indicating that the increased RNA synthesis of the mutant did not result in correspondingly increased amounts of virus protein. The apparent defect in viral protein synthesis may be the result of a defect in viral RNA made by Eta-4.On the basis of the information available, it appeared that viral RNA synthesis in Eta-4 infected cells was out of control. Control seemed to be restored to the level and pattern of the parent by superinfection with the parent encephalitis virus or Venezuelan equine encephalitis virus but not with other viruses.

Overgrowth stimulating factor released from Rous sarcoma cells.

An assay is described for a nonzviral factor from the medium of Rous sarcoma cells which stimulates rapid and sustained cellular overgrawth in crowded chick embryo cultures. The factor is nondialyzable and thermnolabile and is released in large amounts several days after the first visible transformation of newly infected chick embryo cells.

Virus nucleic acids formed in chick embryo cells infected with Semliki Forest virus.

Summary The three species of virus RNA (45 s, 26 s and 20 s) formed during infection of chick embryo cells with Semliki Forest virus were isolated by sequential extraction of the cells with phenol and then with phenol + sodium dodecyl sulphate. They were then separated either by sucrose density gradient centrifugation, or by polyacrylamide gel electrophoresis. The 20 s RNA was relatively resistant to the action of ribonuclease, in contrast to the single-stranded 26 s and 45 s species. The 20 s RNA was the first species to be formed in infected cells followed by 26s and then by 45s RNA. An attempt to demonstrate the intermediate role of 20s RNA in virus RNA synthesis by the displacement of radioactive by non-radioactive uridine (‘pulse-chase’ experiment) was unsuccessful, but both 26 s and 45 s RNA displaced radioactivity from 20 s RNA on heating above the thermal transition temperature and slow cooling. Comparison of the sedimentation velocities and electrophoretic mobilities of the 26s and 45s RNA species, together with the sharp thermal transition at 65° of 45s RNA to 26s RNA suggested that 45s RNA consisted of two 26s RNA species, and that the different sedimentation characteristics did not indicate different conformations of the same molecule.

Specific interference among strains of Newcastle disease virus: II. Comparison of interference by active and inactive virus

High concentrations of NDV-B1, NDV-B1-UV, and NDV-L-UV interfere with the infection of chick embryo cells by NDV-L when added prior to or simultaneously with this virus. When time is allowed to elapse between infection by interfering virus and NDV-L there is a temperature-dependent increase in interference lasting about 90 minutes. The interference induced by ultraviolet (UV)-irradiated virus then begins to disappear immediately, while that induced by active virus remains constant for about 4 hours before increasing again. The second increase in interference exhibited by cells infected with active virus has been correlated with the multiplication of the interfering virus. The interfering capacity of a virus inoculum has been shown to be associated with the virus particle, and the degree of interference is proportional to the number of virus particles in the inoculum. UV irradiation initially reduces both infectivity and the simultaneous and early interfering capacities of the virus with the same single-hit kinetics. UV irradiation in excess of about three hits to the infectious capacity of the virus continues to inactivate infectivity but has no further effect on interfering capacity, and no effect at all on the viral hemagglutinin and RDE. NDV-B1-UV, NDV-L-UV, and heat-inactivated NDV-L all induce similar levels of simultaneous and early interference which are 5- to 50-fold lower than the levels of interference induced by active NDV-B1. Preliminary evidence suggests that different mechanisms may be responsible for the interference induced by active and inactive virus.

The Nucleic Acid of Sendai Virus and Ribonucleic Acid Synthesis in Cells Infected by Sendai Virus

Summary RNA was extracted from purified Sendai virus, labelled with 32P, and sedimented in sucrose density gradients. Under conditions of high ionic strength (0·1 m-NaCl) the sedimentation coefficient was 57 S. In the presence of 0·005 m-EDTA, the sedimentation coefficient was reduced to 40 S, indicating that the RNA of the virus exists as a single-stranded molecule. The intracellular synthesis of virus-directed RNA during the latent period of infection of chick embryo cells by Sendai virus in the presence of actino- mycin was also studied. Cells were extracted by the phenol + sodium dodecyl sulphate method at 2,4, 6 and 7 hr after infection, having previously been exposed to [3H]uridine for at least 2 hr. It was found that the first newly formed RNA had a sedimentation coefficient of 18 S' and could be detected between 2 and 4 hr after infection. From 4 hr onwards a 57 S component was found in increasing amounts until 7 hr after infection. Substantially similar results were obtained when infected cells were treated with detergent alone, and the mixture immediately centrifuged on density gradients except that the principal RNA components now had sedimentation values of 57 S and 23 S.

The mechanism of genetic resistance of chick embryo cells to infection by Rous sarcoma virus-Bryan strain (BS-RSV)

The early steps of Rous sarcoma virus-Bryan strain (BS-RSV) infection of genetically resistant and susceptible chick embryo cells were investigated. The methods used were the uptake of virus by cells grown in monolayer, and the ability of infected cells to form foci of transformed cells after plating on resistant and susceptible assay plates. It was found that the initial attachment of virus to either genetic type of cells did not differ appreciably. Attachment on susceptible cells was immediately followed by penetration, which was 70% complete after 1 hour. Attachment on resistant cells was not followed by penetration of the cell membrane. It was concluded that the genetic block of resistance to infection of chick embryo cells by BS-RSV occurs at the step of viral penetration of the cell membrane.

Fragmentation of the nucleus in Rous sarcoma virus-infected chick embryo cells.

Abstract Infection of chick embryo cells with Rous sarcoma virus causes fragmentation of the nucleus as early as 36 hours after infection. Neither Rous-associated virus nor Newcastle disease virus causes nuclear fragmentation. The presence of both tryptose phosphate broth and calf serum in the medium are required for fragmentation to occur. An assay procedure for Rous sarcoma virus based upon nuclear fragmentation is described.

Failure of Function of the “Early Protein” induced by an Influenza Virus in Cells infected by Newcastle Disease Virus

AFTER infection of chick embryo cells with fowl plague virus (FPV), an influenza A virus, or with Newcastle disease virus (NDV), a para-influenza virus, “early proteins” are synthesized before viral RNA-synthesis starts1–4. These “early proteins” might function as an RNA-dependent RNA polymerase as has been demonstrated for picornaviruses5. In contrast to the picornaviruses, the “early proteins” of the myxoviruses are stable1–4. The purpose of this investigation was to determine whether or not the “early protein” induced by FPV is able to function for the multiplication of NDV.

Defective T particles of vesicular stomatitis virus: I. Preparation, morphology, and some biologic properties

Infection of chick embryo cells with vesicular stomatitis virus under conditions of high multiplicity and serial undiluted passage (VSV up) results in two kinds of progeny: a low yield of infectious bullet-shaped (B) particles and a high yield of noninfectious (T) particles. The smaller T particles can be partially purified by rate zonal centrifugation in sucrose gradients and readily identified by electron microscopy. T particles appear almost spherical with a diameter of ~65 mμ compared with the typically cylindrical B particles with dimensions of ~65 × 180 mμ. Despite this difference in size, T is strikingly similar to B in its ultrastructure, buoyant density, antigenicity, incorporation of uridine- 3H during growth, and capacity to inhibit cellular RNA and interferon synthesis. The data strongly suggest that T is a distinct, truncated form of B which contains only a portion of the VSV genome. The accompanying paper provides proof that T is the physical equivalent of the transmissible interfering component of Cooper and Bellett (1959).

The mechanism of interference between an avion leukosis virus and rous sarcoma virus: II. Early steps of infection by RSV of cells under conditions of interference

Infection of chick embryo cells by Rous sarcoma virus RSV (RAV-1) can be halted immediately after adsorption of the virus by adding antiviral antibody. Within 5 minutes at 37 ° the virus becomes insusceptible to inactivation by antibody. It can, however, still be inactivated by lowering the pH of the medium to 2.2. The rate at which adsorbed virus becomes insusceptible to antibody inactivation is about 8 times slower at 8 ° than at 37 °. The rate at which it becomes insusceptible to low pH treatment is very low at 8 °, so that it can all be inactivated by this treatment even after 5 hours. When chick embryo cells are infected with RAV-1, they become relatively resistant to infection by RSV (RAV-1). In such resistant cells, some of the superinfecting RSV (RAV-1) particles which ultimately initiate infection can be inactivated by antiserum long after they would have fully penetrated into normal cells; i.e., the attainment of insensitivity to antibody requires many hours instead of a few minutes. The subsequent step which leads to insensitivity to inactivation at pH 2.2 proceeds after a normal interval once the virus has become insusceptible to antibody. Therefore it is concluded that RAV-1 particles block the sites at which the superinfecting RSV (RAV-1) particles become insusceptible to antibody.