- Volume 33, Issue 2, 1976
Volume 33, Issue 2, 1976
- Articles
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Some Effects of Temperature on the Early Stages of Tobacco Necrosis Virus Multiplication
More LessSUMMARYThe number of infective centres which were established successfully following the manual inoculation of French bean leaves with tobacco necrosis virus strain D (TNV d ) or with TNV d RNA, decreased with increasing temperature between 13 and 30 °C. At 30 °C or above, primary and probably also secondary infections could not be established, though it is likely that a limited amount of virus RNA and nucleoprotein was produced at 30 °C in cells in which infection had been established previously at 23 °C. During the first day after inoculation, 23 °C was optimal for virus accumulation. Between 23 and 30 °C the rate at which lesions increased in diameter decreased with increasing temperature. The inhibitory effect of supraoptimal temperatures on the establishment of infection may be due to degradation of the infective entity by ribonuclease(s).
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Examination of the Polypeptides of Hepatitis B Surface Antigen
More LessSUMMARYWhen the polypeptides of hepatitis B surface antigen were examined by SDS-polyacrylamide gel electrophoresis under a variety of conditions, anomalous results were found to be due to (i) variable and at times incomplete dissociation of polypeptides after boiling with 1% SDS and reducing agent, (ii) reaggregation of solubilized material under certain electrophoretic conditions and during laboratory manipulations, and (iii) the variable presence of additional components in hepatitis B surface antigen prepared from certain individual donors. When these factors were taken into account, two major components were consistently identified by discontinuous buffer polyacrylamide gel electrophoresis, of apparent mol. wt. 60000 to 70000 and 12000 to 14000. However, in view of the demonstrated limitations of this technique in examining HBsAg polypeptides, alternative methods are necessary to confirm the true mol. wt. of the unique virus-specified amino acid sequence present.
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Long-Term Persistent Vesicular Stomatitis Virus and Rabies Virus Infection of Cells In Vitro
SUMMARYBHK 21 carrier cells persistently infected with VSV Indiana for over 2 years have been shedding generally very low levels of mature infectious virus or mature T particles (averaging less than one-hundredth p.f.u./cell/day) yet most cells are producing virus antigens and are resistant to homologous superinfection. However, large amounts of biologically active T particle RNP can be recovered from cytoplasmic extracts of these carrier cells even at times when they are shedding no detectable infectious virus. This recovered cytoplasmic RNP replicates (with helper B virions) to produce mature T particles, interferes strongly after DEAE dextranfacilitated uptake and, together with B virions, allows the establishment of a persistent carrier state in exposed cells.
No ‘provirus’ DNA copies of the VSV RNA genome are detectable (less than 1/40 copy/cell or 1 copy per 40 cells) in carrier cells after more than 2 years of persistent infection, and all transfection attempts have failed using DNA from these VSV carriers or DNA from carrier cells persistently infected with some other negative strand RNA viruses (measles, mumps, LCM, influenza, rabies).
Infectious viruses shed after more than 1 year from carrier cells originally infected with wild-type B virions are small plaque mutants showing a slight temperature sensitivity. Cured cell populations can be obtained from the long term VSV carrier culture by cloning in the presence or absence of antiviral antibody.
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RNA Synthesis in BHK 21 Cells Persistently Infected with Vesicular Stomatitis Virus and Rabies Virus
More LessSUMMARYVirus-induced RNA synthesis was studied in BHK 21 cells persistently infected with vesicular stomatitis virus (VSV) and rabies virus by labelling RNA synthesized in the presence of actinomycin D. During persistent infection the species of messenger RNA synthesized were similar in size and relative proportions to those seen during acute infection, but there were some minor differences. Full-sized B virion RNA was generally not detected during persistent infection, and new species (probably DI virion RNA) appeared.
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Purification of Two Components of Mouse L Cell Interferon: Electrophoretic Demonstration of Interferon Proteins
More LessSUMMARYMouse L cell interferon induced by Newcastle disease virus was purified by the procedure described previously (Yamamoto et al. 1974) followed by gel filtration. The two fractions obtained containing interferon species S (36000 daltons) and F (24000 daltons), respectively, were analysed electrophoretically at pH 4.3. or in the presence of sodium dodecyl sulphate (SDS) at pH 7.2. In both fractions, interferon activity was invariably associated with distinct protein bands. In the F-containing fraction there were essentially no other proteins, and in the S-containing fraction, impurity proteins were well separated from the interferon activity. The apparent mol. wt. determined by SDS-gel electrophoresis showed little or no dependence on gel concentration, suggesting that the interferons had low carbohydrate contents, and did not change after reduction with thiol reagents in SDS and urea.
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The Multiplication Cycle of Tobacco Rattle Virus in Tobacco Mesophyll Protoplasts
More LessSUMMARYWhen protoplasts inoculated with tobacco rattle virus (TRV) were sampled after successive intervals at 22 or 25 °C in light, the following sequence of events was detected. Infective TRV-RNA appeared at 7 h and approached its maximum concentration by 12 h. Antigen detected by staining with fluorescent antibody to TRV particles, infective TRV nucleoprotein, and both long and short TRV particles were produced by 9 h, and increased up to about 40 h. Infective RNA synthesized at about 9 h was apparently incorporated into nucleoprotein particles about 4 to 5 h later. Up to 16 h, long virus particles outnumbered short, but their proportion steadily decreased until 40 h, when it stabilized at about 20%. Half the final yield of long particles was produced by 22 h, and half that of short particles by 30 h. Nearly all the long virus particles were associated with mitochondria at all times, but after the first few hours the short ones were predominantly free in the cytoplasm. Inoculating short virus particles up to 8 h before, or 4 h after long particles instead of simultaneously, did not greatly affect the number of antigen-producing infections produced, but somewhat decreased the proportion of infective RNA incorporated into nucleoprotein. These results provide support for the idea that separation in time or place within the cell of the synthesis, translation or assembly into nucleoprotein of the different genome parts may be one of the advantages of multipartite genomes as a modus vivendi for plant viruses.
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Inhibitory Effect of Herpes Simplex Virus Type 1 on Type 2 Virus Replication
Dana Zelená, J. Roubal and V. VonkaSUMMARYSimultaneous infection with herpes simplex type 1 and type 2 viruses of chick embryo fibroblasts (CEF), which are only permissive for type 2 virus, or rabbit embryo fibroblasts (REF), which are permissive for both virus types, resulted in a marked reduction of type 2 virus production. This effect was dependent on the m.o.i. of type 1, being expressed at a high rather than a low m.o.i. The rate of interference decreased with the prolongation of the interval between infection with type 2 and type 1 viruses. No evidence suggestive of interferon involvement was obtained. Partial inactivation of type 2 virus by ultraviolet irradiation enhanced the inhibitory effect of type 1 virus. On the other hand, u.v. irradiation of type 1 virus resulted in a progressive loss of inhibitory activity. The results of the present experiments suggest that a type 1 genome function is responsible for the interfering effect, and that an early step in the growth of type 2 virus is sensitive to the particular type 1 virus product involved.
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Enhancement by Phytohaemagglutinin of Inactivation of Herpes Simplex Virus by Concanavalin A
M. Ito and A. L. BarronSUMMARYInactivation of herpes simplex virus (HSV) by concanavalin A (Con A) was enhanced by treatment with phytohaemagglutinin (PHA)-P using a two-stage reaction procedure. Treatment with PHA alone failed to inactivate HSV. Enhancement of inactivation was also effective when HSV was exposed to PHA first. Our results suggest that the envelope of HSV contains receptor sites for Con A which are important in the infectious process, as well as receptor sites for PHA which are not critical for infectivity. Direct interaction of Con A with PHA was demonstrated and the reaction was reversed by α-methyl-d-glucoside. The data indicate that PHA stabilized the binding of Con A to the virus since reversal of inactivation by α-methyl-d-glucoside was minimal following treatment with Con A and PHA. Con A inactivated only enveloped virions and enhancement by PHA was a general phenomenon.
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Pathogenesis of Cytomegalovirus Infection. Distribution of Viral Products, Immune Complexes and Autoimmunity During Latent Murine Infection
More LessSUMMARYDuring studies on the mechanisms of virus latency, reactivation and resultant tissue injury in mice infected with murine cytomegalovirus (MCMV) in utero or at birth, we found the occurrence of three distinct pathological groups. In the first group, mice died within 4 weeks of exposure to virus and showed evidence of tissue injury due to MCMV in multiple tissues and organs of the body. The second group consisted of mice which survived the initial infection and was composed of a minority (about 25%) which shed virus (chronically infected). The third group (about 75%) consisted of mice in which shedding of virus could not be detected (latently infected). Study of the latter group indicated that virus was not detected in brain, thymus, liver, kidneys, urine or serum by co-cultivation techniques or by cellular DNA-MCMV DNA hybridization. In contrast, virus could be activated from spleen cells by co-cultivation with allogenic but not syngeneic feeder cells and MCMV-DNA was detected in amounts equivalent to 3 to 4 virus genomes per 100 spleen cells. In both the latently infected and chronically infected mice, in all strains studied evidence of virus-antivirus immune complex deposits in the renal glomeruli occurred. Only one of the six infected strains (C57 Br/cdJ) studied showed manifestations of autoimmune disease with the formation of antibodies to nuclear antigens, DNA and soluble nucleoprotein.
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Molluscum Contagiosum — A Defective Poxvirus?
More LessSUMMARYPurified preparations of molluscum contagiosum virus contain a DNA-dependent RNA polymerase (EC 2.7.7.6) with similar but not identical properties to those of the enzyme found in vaccinia virions. The ultraviolet inactivation kinetics of the RNA polymerase from both viruses were similar, displaying fast and slow components. Ultraviolet irradiation destroyed the interfering capacities of molluscum and inactivated vaccinia virions, and the interferon-inducing capacity of molluscum virus slowly and with first-order kinetics. Inactivation studies of the interferon-inducing capacity of vaccinia virus were complicated by cytotoxic effects. Electron microscopical studies showed all stages of virus growth in vaccinia-infected mouse embryo cells; molluscum virus appeared to be degraded in lysosome-like bodies. In preliminary studies, marked changes in cytoplasmic RNA synthesis and in patterns of polypeptide synthesis were found in vaccinia-infected but not in molluscum-infected mouse embryo cells.
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Different States of Aggregation of Tobacco Rattle Virus Coat Protein
More LessSUMMARYIt was shown by gel chromatography, sucrose gradient centrifugation and immunodiffusion that tobacco rattle virus (TRV) coat protein can exist in various discrete states of aggregation. Mol. wt. values of about 20000, 75000 and 210000 were estimated for the coat protein subunit and two discrete oligomeric aggregates, and a sedimentation coefficient of about 35S for the characteristic disc-like polymeric aggregate. Some serological differences between the 35S-aggregate and a mixture of oligomers and monomers of the coat protein subunit were demonstrated by agar gel immunodiffusion and immunoelectrophoresis.
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Polypeptides Specified by Bacteriophage T1
More LessSUMMARYThe proteins synthesized during the replication of phage T1 in u.v.-irradiated Escherichia coli strain B have been examined by polyacrylamide gel electrophoresis of polypeptides pulse-labelled with 14C-amino acids. Up to 50 discrete bands were identified of which about 30 were sufficiently distinct to be classified in terms of time of synthesis. Three polypeptides were synthesized only during the first 6 to 8 min post infection (Class I, Early); 16 or 17 were synthesized predominantly during the later stages of replication starting from 6 to 8 min after infection (Class III, Late); three classes of proteins were made continuously, two at constant rate (Class II, Continuous), five at decreasing rate (Class IV, Early-continuous) and five at increasing rate (Class V, Late-continuous). Of the 14 polypeptides identified as structural components of the virion, three (P7, P10 and P11) account for about 85% of the particle weight with P7 comprising 50% of the particle. P7 and P10 appear to result from the cleavage of larger polypeptides. Preliminary studies with amber mutants suggest that normal levels of T1 DNA synthesis are not required for the manufacture of late proteins and that a phage-controlled function may control the switch-off of proteins made early and the switch-on of proteins made late.
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Antibody-induced Redistribution of Virus Antigens on the Surface of Influenza Virus-infected Cells
More LessSUMMARYThe behaviour of virus-specific antigen-antibody complexes at the plasma membrane of HeLa cells infected with influenza virus (A0PR8) was studied by immunofluorescence- and immunoelectron-microscopy. As early as 7 h after infection the virus-induced plasma membrane antigens are evenly distributed over the surface of the infected cells. The addition of antiviral antibodies interferes with this distribution. The appearance of a subsequent patchlike and ‘capping’ distribution, followed by the disappearance of virus antigen-antibody complexes from the cell surface, is described. These findings demonstrate the potential mobility of virus-induced antigens in the plane of the cell surface.
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Homologous Interference Induced by a Temperature-sensitive Mutant Derived from an HVJ (Sendai Virus) Carrier Culture
More LessSUMMARYHomologous interference between a temperature-sensitive small plaque mutant (HVJ-pB) derived from an HVJ (haemagglutinating virus of Japan — the Sendai strain of parainfluenza 1 virus) carrier culture of BHK cells and the original wild-type virus (HVJ-W) has been investigated. Prior infection of LLCMK2, HeLa, BHK or mouse L cells with HVJ-pB, both at permissive and non-permissive temperatures, for 24 h resulted in a reduced yield of superinfecting HVJ-W, reflecting a smaller number of cells capable of producing the superinfecting virus. However, HVJ-pB did not interfere with the replication of vesicular stomatitis virus, Sindbis virus or Newcastle disease virus. Interference in this system seems to be due to inhibition of the attachment of superinfecting HVJ-W as a result of intracellular mechanisms operating at a late stage in the replication of the interfering virus. There is also blocking or destruction of cellular receptors by extracellular particles of the interfering virus. Protein synthesis coded for by the complete virus genome is required to establish and maintain the interference, and treatment with actinomycin D has no effect on the interference phenomenon.
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Gel Filtration of Hepatitis B Surface Antigen: Increased size of the native particle
More LessSUMMARYThe Stokes radius of unpurified hepatitis B antigen (HBsAg) was determined by chromatography on a carefully calibrated Sepharose 4B column. A value of 14.2 nm was found by this procedure, contrasting with a published value of 11 nm for purified, pepsin-treated HBsAg. Chromatography at pH 3 appeared to reduce the Stokes radius of HBsAg to 11 nm. Evidence is presented to show that serum proteins adsorbed to HBsAg can be removed with pepsin or acid.
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The Production and Action of Interferon in Chinese Hamster Cells
More LessSUMMARYThe interferon system has been investigated in primary cell cultures established from Chinese hamster embryos and new born pups. Interferon synthesis was induced with Sindbis virus, ultraviolet irradiated Newcastle disease virus (u.v.-NDV) and with polyriboinosinic acid-polyribocytidylic acid complex [poly (rI). poly (rC)]. Only u.v.-NDV induced significant production of interferon, maximum amounts being produced in ‘aged’ cells. Its apparent mol. wt. was 25000. CHO-K1 cells, an established line of Chinese hamster cells, did not synthesize interferon in response to viruses, but were sensitive to its action.
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Expression of Virus Structural Proteins on Murine Cell Surfaces in Association with the Production of Murine Leukaemia Virus
More LessSUMMARYWe have used a quantitative radiolabelled antibody procedure to measure the amount of certain virus structural antigens on the surface of BALB/c RAG cells producing endogenous B-tropic type C virus. RAG cells expressed group specificities of MuLV p30 on their cell surface but did not express gp70 group specificities. However, type specificities of gp70 were expressed on BALB/c cell lines infected with Moloney leukaemia virus. The majority of p30 antigens detected on the RAG cell surface were removed by trypsin and their reappearance was prevented by cycloheximide, even in the presence of ‘conditioned medium’ containing MuLV. Passive adsorption of exogenous MuLV p30 to the surface of virus negative BALB/c fibroblasts reached a maximum of 20% of the protein detectable on virus producing RAG cells. These data support the hypothesis that much, but not all, of the surface p30 is expressed de novo on the cell membrane and not derived from passive adsorption of p30 released from shed virus or as a by-product of virus infection of a cell.
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