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Volume 65,
Issue 2,
1984
Volume 65, Issue 2, 1984
- Review Article
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The Current Picture of the Structure and Assembly of Tobacco Mosaic Virus
More LessIntroduction. Tobacco mosaic virus (TMV) has long been a favourite object for studies on both the structure and assembly of rod-shaped viruses. Soon after its initial purification by Stanley (1935) investigations of its structure were begun using both chemical and X-ray diffraction techniques (Bawden & Pirie, 1937; Bernal & Fankuchen, 1941). Moreover, the problems in solving such a large structure led to many developments in techniques. Since the virus has a helical structure (Watson, 1954) and forms an extremely well oriented gel rather than crystals, the three-dimensional structural information is convoluted into two dimensions because of the azimuthal disorder of the particles in the gel. Despite this difficulty, techniques have been evolved allowing the virus structure to be solved to a resolution approaching 0.4 nm (Stubbs et al., 1977). The protein alone will form true crystals as one of its aggregates (the disk; see below) the structure of which has been solved to a resolution of 0.3 nm (Champness et al., 1976; Bloomer et al., 1978), allowing a detailed atomic model to be built.
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- Animal
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Ultrastructural and Biochemical Evidence that the L929 Cell Retrovirus Lacks the env Gene Translation Product
More LessSUMMARYLCV, a murine retrovirus released by L929 mouse cell fibroblasts, is non-infectious when inoculated into SC-1, mink, D-17 or Vero cells. Ultrastructural examination by thin sectioning, freeze-etching or negative staining revealed the absence, on the viral envelope, of the radially disposed spikes. Polyacrylamide gel electrophoresis of radiolabelled viral components showed the absence of the glycosylated protein gp70 as well as of the p15E cleavage product of the polyprotein precursor gPr90env. The premature loss of the gp70 molecule from LCV to the culture medium was ruled out since no peak of d-[14C]glucosamine-labelled glycoprotein was detected by affinity chromatography or immunoprecipitation of concentrated medium. The ultrastructural and biochemical results all supported the hypothesis that the absence of infectivity was due to the lack of gp70 glycoprotein in the envelope of LCV. A possible block at a translational or post-translational level was also investigated by immunofluorescence studies with antisera directed against ecotropic or xenotropic gp70; Moloney murine leukaemia virus-infected or NZB cells were used as positive controls for eco- or xenotropic viruses respectively. The absence of fluorescent stain in L929 cells further supported these results and suggested that LCV and the L929 parental cell line lack the uncleaved precursor and the final product of the env gene translation process.
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Integration of Proviral DNA in Kirsten Murine Sarcoma Virus-infected Mouse Fibroblasts
More LessSUMMARYThe structure and sites of integration of proviral DNA were studied in 19 clonally related Kirsten murine sarcoma virus-transformed non-producer NIH/3T3 cell lines. The majority of these cell lines contained a single provirus, inserted colinearly with respect to unintegrated linear viral DNA, and lacking detectable methylation at MspI/HpaII sites. Although all proviruses were located at distinct integration sites in the host cell genome, the possible existence of similarities between some adjacent host flanking sequences, suggested from restriction mapping data, could not be ruled out. In three phenotypically reverted cell lines no change in either proviral DNA or adjacent host flanking sequences was detectable. In addition, the revertant proviruses lacked detectable methylation at MspI/HpaII sites. These findings suggest that changes in cellular function(s) may be responsible for loss of transformed phenotype in these cells.
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Phenotypic Mixing of Retroviruses in Mitogen-stimulated Lymphocytes: Analysis of Xenotropic and Defective Endogenous Mouse Viruses
More LessSUMMARYIn addition to the known induction of xenotropic endogenous virus in B-mitogen-stimulated murine lymphocyte cultures, distinguishable defective viruses were also induced in different mouse strains (NFS/N, 129, BALB/c). AKR cells produced xenotropic virus and also, in contrast to BALB/c, ecotropic virus. The drug bromodeoxyuridine appeared to have differential effects on virus expression, amplifying xenotropic virus induction but inhibiting the spontaneous production of the ecotropic virus in AKR cultures and of the defective virus in NFS/N cells. Infecting stimulated BALB/c or AKR cultures with Friend leukaemia virus resulted in the production of ecotropic-xenotropic pseudotype viruses, indicating that the infecting ecotropic virus replicates in the cells in which xenotropic virus is induced. No pseudotypes or recombinants were observed following infection of spleen cells releasing defective viruses. Friend leukaemia virus and xenotropic virus with an ecotropic envelope replicated equally well in stimulated lymphocytes from the different strains examined. Taken together, these findings indicate that the non-infectious viruses are encoded by defective proviruses, rather than resulting from faulty, host cell-controlled, virus maturation.
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Replication of Transmissible Gastroenteritis Coronavirus (TGEV) in Swine Alveolar Macrophages
H. Laude, B. Charley and J. GelfiSUMMARYSeveral strains of the enteropathogenic coronavirus transmissible gastroenteritis virus (TGEV) have been shown to replicate in alveolar macrophages maintained in vitro. A distinct cytopathic effect was observed at a multiplicity of infection ≥0.1. Infected cells released infectious virus. The extent of both virus production and cell destruction was highly dependent upon the virus input. At low input, cell viability was affected only slightly, and a delayed and persistent virus production could be observed. TGEV infection of macrophages also led to a marked synthesis of type I interferon. Thus, the possibility that alveolar macrophages act as an extra-intestinal target for TGEV must be considered.
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Properties of Defective Interfering Particles of Sindbis Virus Generated in Vertebrate and Mosquito Cells
More LessSUMMARYDefective interfering (DI) particles of Sindbis virus were generated during serial, undiluted passage of a cloned virus stock in both chick embryo fibroblast (CEF) cells and Aedes albopictus mosquito cells. DI particle-containing stocks were identified by their ability to interfere with standard virus replication and to synthesize small DI RNA species in the cell type in which they were generated. DI RNA species generated during serial passage in CEF cells were able to replicate in both vertebrate and A. albopictus cells. Some of the DI RNA species generated during serial passage in A. albopictus cells replicated efficiently in both cell types while others, sometimes present in the same DI particle stock, failed to replicate in cells of vertebrate origin. These results suggest that evolution of DI RNA species during serial passage of Sindbis virus in mosquito cells may result in the loss or alteration of nucleotide sequences required for RNA synthesis in vertebrate cells.
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Heterogeneity of Sindbis Virus Glycoprotein E1 and its Modification by Host Cell Transformation
More LessSUMMARYThe electrophoretic properties of glycoprotein E1 of Sindbis virus grown in Rous sarcoma virus-transformed cells were compared with those of Sindbis virus grown in untransformed cells. Isoelectric focusing in a gel containing 9.5 m-urea and 2% Nonidet P40 indicated that the glycoprotein E1 of Sindbis virus from the untransformed cells was electrochemically heterogeneous and consisted of four components, whose isoelectric points (pIs) ranged from 6.2 to 6.7; the E1 of Sindbis virus from the transformed cells contained an additional component with a pI of 6.1. After treatment with neuraminidase the observed charge heterogeneity disappeared regardless of whether the virus had come from the transformed or the untransformed cells. The five components were digested with Pronase and compared by gel filtration on Bio-Gel P-6; the four components with relatively higher pIs released high-mannose-type and complex-type oligosaccharides which differed in their content of sialic acid. In contrast, the pI 6.1 component, which existed only in the transformed cell-derived SV glycoprotein E1, released only complex-type oligosaccharide. These results indicate that the properties of glycoprotein E1 derived from both cell types are basically similar but that a small proportion of the E1 molecules from the transformed cells, with carbohydrate chains elongated by attachment of sialic acid residues, lacks simple-type oligosaccharides either because they are not attached or because they have been processed to the complex type. Glycoprotein E2 appeared as at least three components in nonequilibrium pH gradient electrophoresis. However, no significant difference was observed between the cell types.
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Purification, Morphology and Antigenic Characterization of Measles Virus Envelope Components
More LessSUMMARYMeasles virus haemagglutinin (H), fusion (F) and matrix (M) components were purified by affinity chromatography using monoclonal antibodies coupled to CNBr-activated Sepharose. H and M proteins were purified to homogeneity as determined by polyacrylamide gel electrophoresis by a single cycle of adsorption-desorption. The corresponding purification of the F protein required two cycles of adsorption-desorption. After the second adsorption an extra wash with 1 m-guanidine-HCl was employed to remove the contaminating cellular actin. Electron microscopic examination of the purified envelope proteins showed that, at pH 6.0, the H peplomers had a truncated conical shape (width 6.5 to 4 nm, length 16 nm), and the F peplomers had a club-like shape (dimensions of the oval head 6 × 9 nm, length 15 nm). Lengths of both peplomers were measured excluding their undiscernible hydrophobic part. The M component at pH 3.0 appeared as a rounded particle (diam. 8 nm, central accumulation of contrast 1.5 nm) suggested to include four to six M polypeptides. Rabbit hyperimmune sera were prepared against all three purified envelope components. These sera reacted only with the homologous antigen in radioimmunoprecipitation assays. Both antisera against the H and F components neutralized the virus and blocked virus-specific haemolysis, but only anti-H serum inhibited haemagglutination.
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Establishment and Maintenance of Persistent Infection by the Phlebovirus Toscana in Vero Cells
More LessSUMMARYPersistent infections were established by serial undiluted passages of the Toscana virus of the genus Phlebovirus in Vero cells. Persistence was maintained through more than 70 passages over a period of 2 years. The persistently infected cells were morphologically similar to the parental Vero cells and released variable amounts of infectious virus. A small percentage of the persistently infected cells produced infectious virus while a larger percentage was shown to possess viral antigens by fluorescent antibody staining. Temperature sensitivity and host cell interferon production were not involved in establishment or maintenance of persistence. The persistently infected cultures were resistant to superinfection with homologous Toscana virus, but they were susceptible to superinfection with heterologous viruses of different genera or families. Toscana virus persistently infected cells showed a selective graded resistance to the replication of other Phleboviruses. Additionally, the virus from persistently infected cells interfered with the replication of standard Toscana virus when they co-infected normal cells. The characteristics of the persistently infected cultures are compatible with some of the characteristics described for the persistence mediated by defective-interfering particles of other viruses.
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Action of Human Lymphoblastoid Interferon on HeLa Cells Infected with RNA-containing Animal Viruses
More LessSUMMARYProtein synthesis in interferon-treated HeLa cells infected by different animal RNA viruses has been studied. Synthesis of vesicular stomatitis virus (VSV) proteins was not detected in cells treated with concentrations of HuIFN-α(Ly) above 10 IU/ml. No specific inhibition of glycosylation of the G protein was observed. In addition, inhibition of host protein synthesis in IFN-treated cells occurred when high multiplicities of VSV were used, even though no viral protein synthesis was detected. For other viruses, such as Newcastle disease virus, Semliki Forest virus, encephalomyocarditis virus and poliovirus, treatment of cells with interferon also led to inhibition of viral protein synthesis. However, influenza virus and reovirus protein synthesis in interferon-treated cells stayed at control levels. The finding of viral translation in influenza virus and reovirus-infected cells treated with interferon suggests that, at least for these two systems, the antiviral state is not mediated by the bulk inhibition of viral protein synthesis through the dsRNA-activated protein kinase, or the 2′–5′ oligo(A) system.
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Antiviral Activity of Interferon in Rats and the Effect of Immune Suppression
More LessSUMMARYThe antiviral effect of interferon was studied in a number of experimental virus infections in the rat. Interferon was shown to protect rats infected with pseudorabies virus, herpes simplex virus or vesicular stomatitis virus. The antiviral activity was not inhibited by immune suppression induced by azothioprine, prednisolone or cyclosporin A treatment. Cyclophosphamide completely blocked the in vivo activity of interferon in rats.
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A Structural Investigation of the Epstein—Barr (EB) Virus Membrane Antigen Glycoprotein, gp340
More LessSUMMARYEpstein—Barr (EB) virus membrane antigen (MA) glycoprotein (gp340) purified by a molecular weight-based technique has been subjected to biochemical analysis. Following treatment with glycosidases or tunicamycin during synthesis, the carbohydrate moiety was found to be made up of both O-linked and N-linked types and to constitute about 50% of the molecular mass. Digestion studies with neuraminidase and oligosaccharidase have indicated that the molecule is heavily sialated with most of the sialic acid located on the O-linked sugars. The high carbohydrate content of gp340 appears to confer resistance to proteolysis; thus, V8 protease was only effective at concentrations above 1 mg/ml when three large fragments of mol. wt. 330K, 190K and 160K were generated. Removal of sialic acid before V8 protease digestion did not alter this pattern nor affect the antigenicity of the digestion fragments. Antigenicity of the intact molecule was likewise unaffected by removal of sialic acid nor were the O-linked and N-linked carbohydrate moieties essential for this property. The binding of virus-neutralizing human sera and monoclonal antibody by gp340 from which either O-linked or N-linked sugars had been removed seems to indicate that the sites on the molecule that generate the neutralizing antibodies are present in the protein component. The significance of these results is discussed in relation to the development of a subunit vaccine against EB virus.
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Immunological and Morphological Properties of HBeAg Subtypes (HBeAg/1 and HBeAg/2) in Hepatitis B Virus Core Particles
More LessSUMMARYA polypeptide of 21500 mol. wt., structurally associated with hepatitis B virus core particles, was shown to have two kinds of HBeAg antigenicity (HBeAg/1 and HBeAg/2). This was revealed by transferring a single core peptide from polyacrylamide gels to nitrocellulose sheets (Western blotting), which reacted with anti-HBeAg/1 and anti-HBeAg/2. Selective discrimination of the two HBe antigens was achieved by radioimmunoassay (RIA). When highly purified core particles were incubated at 37°C in a 0.1% SDS-0.1% 2-mercaptoethanol solution, only HBeAg/1 was released after 5 min incubation and the release of HBeAg/2 occurred only after prolonging incubation for 30 min. The course of degradation was also detected by CsCl density gradient centrifugation. These results indicate that HBeAg/1 is less closely associated with core particles than is HBeAg/2. Electron microscopy showed that the core particles from which HBeAg/1 was removed were more labile than the original preparation when incubated at 56 °C in aqueous solution, or at 37 °C in Sarkosyl solutions; when placed in 1 m-NaCl or -CsCl solution, the particles swelled to a larger diameter than untreated cores.
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Purification of the Scrapie Agent by Density Gradient Centrifugation
More LessSUMMARYPlasma membrane-enriched preparations from scrapie-infected and healthy hamster brains, as well as preparations of neural retina, were sonicated, then separated by rate-zonal sedimentation in 10 to 25% Nycodenz® gradients. Gradient fractions were extracted with 0.5% Triton X-100 and re-fractionated by equilibrium density centrifugation in linear 25 to 40% CsCl gradients. Infectivity was highest in a fraction having a density of 1.280 g/ml and which contained a visible band of material. Digestion of the Nycodenz fractions with proteinase K before detergent extraction and CsCl fractionation resulted in a shift in the visible band to a density of 1.235 g/ml with most of the scrapie infectivity remaining at 1.280 g/ml. When labelled with 125I after 40-fold concentration, this 1.280 g/ml CsCl fraction from the proteinase K-treated gradients contained only a single band of protein(s) having a mol. wt. near 30000. No differences were seen between proteins in healthy or scrapie-infected preparations.
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The Reticuloendothelial System in Scrapie Pathogenesis
More LessSUMMARYA single injection of 1 mg dextran sulphate 500 per mouse considerably prolonged incubation times and produced survivors, even when given 3 days after intravenous or intraperitoneal scrapie infection. Since this compound could be detected in mononuclear phagocytes of spleen and lymph nodes for up to 7 months, it is suggested that it impairs a particular step in the infectious process in these cells of the lymphoreticular system. Blockage of the reticuloendothelial system by trypan blue and silica did not alter the pathogenesis.
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Friend Erythroleukaemia Cell Mutants Defective in Viral Gene Expression
More LessSUMMARYCellular mutants defective in the expression of viral polypeptides were isolated from the Friend erythroleukaemia cell line 745a by immunoselection with anti-ecotropic murine leukaemia virus serum in the presence of complement. The mode of appearance of the antiserum-resistant cells showed an interesting pattern which is discussed in the text. One of the mutants obtained contained no detectable gp70 or p15(E) while retaining gPr90env; defects appeared to reside in the processing of the gPr90env to gp70 and p15(E). In another type of mutant, gPr90env was not detected. All these mutants retained spleen focus-forming virus (SFFV)-specific gp55 and gag precursor Pr68gag. The mutants superinfected with the helper virus produced the helper and SFFV also, indicating that these mutations did not affect the replication of the exogenously infecting virus.
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Different Sizes of Restriction Endonuclease Fragments from the Terminal Repetitions of the Herpes Simplex Virus Type 1 Genome Latent in Trigeminal Ganglia of Mice
More LessSUMMARYTrigeminal ganglion DNA from mice latently infected with herpes simplex virus type 1 was analysed by restriction enzyme digestion, agarose gel electrophoresis and blot hybridization to 32P-labelled viral DNA. Viral DNA from parental virions and from virions obtained as a consequence of reactivation by ganglion neurectomy were similarly analysed. The BamHI restriction fragments of parental and reactivated virions were almost indistinguishable from each other, and several of the larger BamHI fragments of viral DNA were also found in latently infected ganglia at unaltered sizes. In contrast, fractionation of EcoRI fragments of latently infected ganglion DNA by reverse phase column (RPC-5) chromatography, followed by gel electrophoresis and blot hybridization to a viral DNA probe from the S component terminal repetition, revealed the presence of several terminal fragments at discrete sizes ranging from 1 kb to 15 kb, quite unlike the 5.7 kb terminal EcoRI K fragment of virion-derived DNA. These results indicate that structural changes occur in the viral genome concomitantly with the establishment of latency, such as may result from extensive gene rearrangement or integration into cellular DNA.
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Cassia Yellow Blotch Virus: a New Bromovirus from an Australian Native Legume, Cassia pleurocarpa
More LessSUMMARYCassia yellow blotch virus (CYBV) is a previously undescribed bromovirus infecting the Australian indigenous legume, Cassia pleurocarpa, in western Queensland. Although CYBV infected test plants in several families, symptoms were usually restricted to local lesions on the inoculated leaves. Systemic infection occurred in only eight leguminous species and in the non-legume, Nicotiana clevelandii. Chenopodium amaranticolor was a suitable host for local lesion assays and CYBV was readily purified from N. clevelandii. Purified CYBV preparations, when negatively stained, contained isometric particles 25 to 27 nm in diameter with a penetrated central core of approx. 10 nm. The virions sedimented as a single component with a sedimentation coefficient (s 20,w) of 85S and had an isoelectric point of 3.6. At pH 7.0, the virions sedimented more slowly than at pH 5.0 and 6.5 probably because they swelled. Purified virions contained a single coat protein species with a molecular weight of 20800 comprising 196 amino acid residues as estimated by FITMOL analysis of the amino acid composition. The virion genome consisted of four single-stranded RNA molecules of which only the three largest were required for infectivity. CYBV appeared to be serologically unrelated to three other bromoviruses and this lack of relationship correlated with differences in their coat protein amino acid compositions.
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Comparisons among Satellite RNA Species from Five Isolates of Tomato Black Ring Virus and One Isolate of Myrobalan Latent Ringspot Virus
More LessSUMMARYThe satellite RNA species from five isolates of tomato black ring virus (TBRV), belonging to two serotypes, and one isolate of myrobalan latent ringspot virus (MLRV) were compared. Each was translated in wheat germ extracts to give a single prominent polypeptide of mol. wt. 48000 (TBRV satellites) or 45000 (MLRV satellite). Analysis of the translation products by peptide mapping, and of the satellite RNA molecules by oligonucleotide mapping, showed that the MLRV satellite RNA was distinct from any of the others. The TBRV satellites had some similarities to each other but were of two types that differed in nucleotide sequence and that were associated with TBRV helper isolates of the different serotypes.
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A Physical Map for the Heliothis zea SNPV Genome
More LessSUMMARYRestriction sites on the Heliothis zea nuclear polyhedrosis virus (HzSNPV) genome were mapped for the endonucleases BamHI, EcoRI, HindIII, SstI, XhoI and KpnI. XhoI-F was used as the zero-point of the physical map because it hybridized to the HindIII-V fragment which codes for the 3′ end of the polyhedrin gene of the Autographa californica nuclear polyhedrosis virus (AcMNPV). The DNA homology between eight AcMNPV cloned fragments and HzSNPV DNA was mapped using Southern hybridization and the locations of HzSNPV DNA sequences sharing homology with AcMNPV gene sequences are discussed.
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