- Volume 65, Issue 4, 1984
Volume 65, Issue 4, 1984
- Articles
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- Bacterial
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A Cloning Strategy for the Bacteriophage T4 vs Gene
More LessSummaryThe product of the bacteriophage T4 vs gene, the τ peptide, has been shown to thermally stabilize temperature-sensitive valyl-tRNA synthetase (EC 6.1.1.9). To clone the bacteriophage T4 vs gene, recombinant pBR322-T4 DNA molecules were used to transform Escherichia coli CP 790302 (valS ts). Transformants that grew at 43.5 °C on selective medium exhibited some properties indicative of a phage-modified valyl-tRNA synthetase. The data are consistent with successful cloning of the vs gene and atypical modification of the valyl-tRNA synthetase in CP 790302.
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- Animal
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In vitro Mouse Cytomegalovirus Infection of Mouse Tracheal Epithelial Cells Requires the Presence of Other Cell Types
More LessSummaryRecently, methods have been developed to culture dissociated tracheal epithelial (TE) cells. Attempts were made to infect these epithelial cells with mouse cytomegalovirus (MCMV) to see if the dissociated epithelial cells share characteristics of infection with MCMV-infected tracheal organ cultures. When isolated TE cells were incubated with MCMV at multiplicities between 0.10 and 2.0, no infection or minimal infection resulted. Centrifugation of virus onto the TE cell sheets also resulted in only minimal infection. If MCMV-infected mouse embryo fibroblasts were added to cultures of TE cells, however, the TE cells subsequently became infected. TE cells could also be infected by incubating MCMV with mixed cultures of uninfected fibroblasts and TE cells. The epithelial nature of infected cells was confirmed by electron microscopy. Reconstruction experiments demonstrated that fibroblast-mediated infection of mouse TE cells with MCMV was not simply due to the large amounts of virus provided by the infected fibroblasts. It is suggested that cell-cell contact, or fusion with infected cells is required for productive infection of TE cells with MCMV.
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Rat Cytomegalovirus: Studies on the Viral Genome and the Proteins of Virions and Nucleocapsids
More LessSUMMARYThe nucleocapsids (N-capsids) isolated from the nuclei of rat cytomegalovirus (RCMV)-infected rat embryo fibroblasts (REF) are composed of three major proteins: 142 × 103 (142K), 40K and 32K mol. wt. Nucleocapsids isolated from the cytoplasmic fraction (C-capsids) are composed of proteins found in N-capsids and five major and seven minor new protein species. Most of the proteins present in C-capsids are found in the extracellular enveloped virions, although the ratios vary. Proteins that are abundantly present, particularly in virions (mol. wt. 125K, 116K, 87K, 79K, 71K, 68K, 62K, 50K, 43K and 28K), are probably the major constituents of the viral envelope. The DNA recovered from extracellular virions was purified to homogeneity and by equilibrium centrifugation in CsCl one density class of 1.716(±0.001) g/ml was found. Contour length measurements showed one size class of a linear double-stranded DNA corresponding to an average mol. wt. of 144(±9) × 106 which is in good agreement with data obtained by restriction endonuclease analysis (REA), which yielded mol. wt. values of 132(±9) × 106 (HindIII), 138(±2) × 106 (EcoRI) and 137 × 106 (BglII). The REA patterns also revealed the presence of 0.25 m and 0.5 m fragments, which might indicate, in analogy with other cytomegalo- and herpesviruses, the existence of four different configurations of the RCMV genome. The infectivity of RCMV DNA was determined in subconfluent REF monolayers. A cytopathic effect characteristic of RCMV was observed 6 days post-transfection and up to 60 plaques/µg DNA were obtained. Using DNA—DNA filter hybridization the degree of homology between the genomes of RCMV and murine or human CMV was examined. Under stringent conditions (50% formamide) values of 12(±2)% and 3(±1)% were found whereas under non-stringent conditions (20% formamide) values of 21(±2)% and 6(±1)% were obtained, respectively.
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Bovine Cytomegaloviruses: Identification and Differential Properties
J. Storz, B. Ehlers, W. J. Todd and H. LudwigSUMMARYBiological properties and restriction enzyme patterns of the slowly replicating herpesviruses isolated from cattle affected with different diseases in North America and Europe were analysed. These virus isolates induced identical plaques that developed within 7 to 9 days in bovine foetal spleen cells and within 5 days in actively growing Georgia bovine kidney cells. These virus isolates were found to be antigenically related when tested in the indirect immunofluorescence test, and antigenic relationships with bovine herpesvirus 1 (BHV-1), BHV-2, BHV-3 or BHV-6 were not detected. The genomes of these strains were shown to have virtually identical cleavage sites when treated with restriction enzymes EcoRI, BamHI, SstII, SphI and HindIII. The resulting restriction enzyme patterns differed strikingly from those of BHV-1, BHV-2, BHV-3 and BHV-6. Because the herpesviruses tested become enveloped on the nuclear as well as on endoplasmic membranes, a process through which they induce cytoplasmic vesicles filled with enveloped viral particles, and because of the unique cytoplasmic inclusions that are induced, we classify them tentatively as bovine cytomegaloviruses.
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A Quantitative Study of the Effects of Several Nucleoside Analogues on Established Herpes Encephalitis in Mice
More LessSUMMARYMice with established herpes encephalitis were used to compare the effects of chemotherapy using three different nucleoside analogues. Encephalitis was produced by intranasal inoculation of a type 1 strain of herpes simplex virus. Without chemotherapy all mice died within 5 to 7 days of inoculation. Oral acyclovir (ACV) was a successful preventative measure if commenced within 2 days of inoculation but much less effective if the onset of treatment was further delayed. From the third day, when central nervous system infection had definitely become established, ACV only reduced mortality if given intraperitoneally (i.p.) at regular 6-hourly intervals. Comparison with bromovinyldeoxyuridine (BVdU) and the new nucleoside analogue dihydroxypropoxy-methylguanine (DHPG) using the same 6-hourly i.p. regimen revealed that BVdU was poorly effective, despite better activity in vitro, whereas DHPG was the most successful. Virus was rapidly eradicated from all parts of the brain by DHPG therapy, and by day 10, no infectious virus remained in the brains of treated mice, no virus antigens were observed and no trace of virus DNA could be detected in neural tissues by Southern blotting.
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Fate of Frog Virus 3 DNA Replicated in the Nucleus of Arginine-deprived CHO Cells
More LessSUMMARYIn a productive infection, frog virus 3 (FV 3) DNA was synthesized in both the cell nucleus and cytoplasm. The infection was aborted in arginine-starved Chinese hamster ovary cells and viral DNA replication was then restricted to the nuclear compartment. It has been demonstrated that the newly synthesized FV 3 DNA present in the nucleus is of genomic size. After the addition of arginine, this DNA is transferred into the cytoplasm and can be encapsidated. The formation of infectious particles occurred even if an inhibitor of DNA synthesis was added simultaneously with arginine. Although the synthesis of early FV 3 polypeptides and their intracellular distribution were comparable in the presence or in the absence of arginine, the production of late species was greatly reduced by arginine deprivation; one of these late proteins must be involved in the passage from the nuclear to the cytoplasmic phase of FV 3 DNA replication. This system made it possible to carry out an independent analysis of the nuclear events that comprise the first stage of FV 3 multiplication.
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Murine Leukaemia Virus p30 Heterogeneity as Revealed by Two-dimensional Gel Electrophoresis Is Not an Artefact of the Technique
More LessSUMMARYWe have utilized two-dimensional (2D) gel electrophoresis [the first dimension being a linear pH gradient (5 to 8) and the second an 8 to 15% acrylamide gradient] to characterize the virion protein, p30, from several strains of purified murine leukaemia virus (MuLV). In all cases, we found that there was a predominant (70 to 90%) Coomassie Brilliant Blue-staining p30 spot, as well as several other species which differed in pI. The major p30 spot differed in pI among different MuLV strains and the minor spots varied depending on the host cell used to grow the virus. Specifically, (i) Moloney (M)-MuLV/NIH-3T3 showed two spots, a major one at pI 6.3 and a more acidic one, (ii) AKR/NIH-3T3, AKR/mouse embryo, and Gross/NIH-3T3 showed four spots, with the two basic, minor spots of AKR/NIH-3T3 appearing relatively decreased in intensity, and (iii) Rauscher (R)-MuLV/JLS-V9 (BALB/c) showed two spots, a major one with greater than 90% of the estimated Coomassie Brilliant Blue stain at a pI of 6.5 and a minor, acidic one. The major spots of AKR and m-MuLV viruses also differed in pI. The major spot of the AKR and Gross N-tropic viruses had a pI of 6.7 while that of NB-tropic virus m-MuLV had a pI of 6.3. The possibility that the heterogeneity observed in p30 was an artefact of the 2D gel technique had to be considered since urea was used to denature proteins in the first dimension of the gel. This possibility was made unlikely by our finding that another technique, chromato-focusing, gave the same results. Specifically, m-MuLV/JLS-V9 p30, when separated on chromatofocusing columns under non-denaturing conditions yielded three peaks, each of which directly corresponded to the three spots (pI:6.1, 6.3, 6.6) observed on 2D gels. Furthermore, tryptic peptide maps of the major (pI 6.3) and one of the minor (pI 6.6) m-MuLV spots, although very similar in peptide composition, showed about five clearly defined differences. These results indicate (i) that the p30s of several N- and NB-tropic viruses are heterogeneous in pI, and (ii) for one particular MuLV, the p30 heterogeneity can be explained by a difference in amino acid composition. These findings of p30 charge heterogeneity may reflect either the presence of several different p30s in each virus particle and/or a heterogeneity in the virus population.
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Structural Differences in Envelope Glycoproteins Associated with Rat Leukaemia Virus Produced by Novikoff Hepatocellular Carcinoma and Spontaneously Transformed Wistar Rat Embryo Cells
SUMMARYImmunochemical and immunocytochemical techniques have been used to characterize viral glycoproteins and endogenous rat leukaemia viruses (RaLV) produced both by Novikoff hepatocellular carcinoma cells and spontaneously transformed Wistar rat embryo cells (WRC). Results from immunocytochemical analysis demonstrated that RaLV produced by Novikoff and WRC cells could be distinguished by their unique patterns of reactivity with xenoantisera raised against virus particles or viral glycoproteins. This differential labelling was unexpected since all the antisera tested had been shown by immunoprecipitation and immunodepletion analysis to be reactive with viral glycoproteins expressed on the cell surface. Since no significant differences in cell surface-associated viral glycoproteins and those shed from the cell surface were detected by pulse iodination analysis, it was concluded that the apparent discrepancy between immunoferritin labelling and immunoprecipitation analysis resulted from differences in antigen accessibility on intact virions caused by structural differences in the viral glycoproteins expressed on Novikoff and WRC cells. This conclusion was supported by results from ferritin-lectin labelling, affinity chromatography and neuraminidase digestion studies which demonstrated differences in the saccharide moieties on both virion and cell surface-associated viral glycoproteins. Further evidence of structural differences was provided by limited digestion with trypsin and V8 protease of the M r 64000 (Nov gp64) and M r 68000 (WRC gp68) viral glycoproteins immunoprecipitated from Novikoff and WRC cells, respectively, with either monospecific anti-Rauscher murine leukaemia virus anti-gp70 serum or monospecific antiserum against Nov gp64 (anti-gp64). Results from digestion studies showed that all the major cleavage fragments from WRC gp68 were of higher molecular weight than their Nov gp64-derived counterparts. Evidence that Nov gp64 and WRC gp68 both share structural homology with other murine viral gp70s was suggested by results from immunoprecipitation analysis with anti-gp70 and anti-gp64 sera under reducing and non-reducing conditions which demonstrated the presence of an interchain disulphide bond in both glycoproteins and showed that at least some of these molecules exist on the cell surface as disulphide-linked heterodimers of M r 78000 and 82000.
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Purification and Characterization of GP90, One of the Envelope Glycoproteins of Respiratory Syncytial Virus
More LessSUMMARYThe large glycoprotein, GP90, of respiratory syncytial virus (RSV) was purified by affinity chromatography using a monoclonal antibody. Hyperimmune rabbit antiserum directed specifically to the purified GP90 neutralized RSV to high titre but did not inhibit fusion of previously infected cells. 125I-labelled GP90 bound rapidly to HEp-2 cells in an unsaturable manner; binding was inhibited by the hyperimmune rabbit antiserum.
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Preparation and Analysis of the Nucleocapsid Proteins of Vesicular Stomatitis Virus and Sendai Virus, and Analysis of the Sendai Virus Leader-NP Gene Region
More LessSUMMARYA procedure is presented for isolating the nucleocapsid proteins, N and NP from vesicular stomatitis virus and Sendai virus respectively, in soluble form. These proteins were suitable for the determination of their blocked amino-terminal peptide sequences by gas-liquid chromatography/mass spectrometry at the low nanomole level. The N protein prepared by this procedure was previously shown to retain some of its expected biological activity. The sequence of 626 nucleotides from the 3′ end of the Sendai virus genome, which includes the first one-third of the NP gene, was determined. Using this information, primer extension studies on intracellular Sendai virus mRNAs allowed the determination of the structure of the leader-NP intervening sequence and the 5′ end of the NP mRNA. Comparison of the amino termini of the nucleocapsid proteins with their respective mRNA sequences revealed that these proteins are similarly processed in vivo.
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Mechanism of Rabies Virus Entry into CER Cells
F. Superti, M. Derer and H. TsiangSUMMARYThe early steps of rabies virus (CVS) infection in vitro were studied in chicken embryo-related (CER) cells. The infection was monitored by looking for specific intracytoplasmic viral inclusions using anti-rabies fluorescein isothiocyanate at 24 h after the addition of virus. The attachment of rabies virus to CER cells was shown to be inhibited by pretreatment of the cells with neuraminidase. These cells recovered their susceptibility to rabies virus infection 6 h after removal of the enzyme. Treatment of CER cells with neuraminidase after the viral attachment step did not inhibit infection. The subsequent delivery of infectious virions into acid prelysosomal vacuoles or lysosomes was studied using lysosomotropic agents. Ammonium chloride and chloroquine were used to prevent the virus fusion step thus preventing infection. Both drugs were shown to inhibit the early steps of infection, NH4 Cl having a much earlier effect than chloroquine. The two drugs had no effect on the attachment step nor did NH4Cl inhibit virus multiplication. The use of metabolic inhibitors (2-deoxy-d-glucose and sodium azide) shows that the entry of rabies virus into CER cells does not require the involvement of cellular energy processes. In electron microscopy studies, the presence of rabies virus particles was detected in coated pits and coated vesicles as well as in uncoated vesicles, and later in lysosomes. These data indicate that the mechanism by which rabies virus enters CER cells is probably through adsorptive endocytosis and does not require the participation of cellular metabolic active processes.
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Antibody-mediated Infection of P388D1 Cells with 17D Yellow Fever Virus: Effects of Chloroquine and Cytochalasin B
More LessSUMMARYAntibody-mediated 17D yellow fever virus infection of a murine macrophage-like cell line, P388D1, was not inhibited by concentrations of cytochalasin B which did inhibit phagocytosis of antibody-coated sheep red blood cells. Chloroquine did not affect antibody-mediated enhancement of viral growth but inhibited viral replication equally whether infection was carried out in the presence or absence of antibody. The results suggest that the route of internalization of virus and subsequent intracellular pathways of replication are similar for both conditions of infection.
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Newcastle Disease Virus: the Effect of Monoclonal Antibody in the Overlay on Virus Penetration and the Immunoselection of Variants
More LessSUMMARYMonoclonal antibody to the haemagglutinin—neuraminidase or the fusion glycoprotein of Newcastle disease virus blocked virus penetration which otherwise occurred within 90 s of the temperature being increased from 4 °C to 37 °C. These antibodies regularly immunoselected variant plaques only when incorporated into the agarose overlay. Variant viruses then formed plaques whereas the growth of non-neutralized normal virus was suppressed.
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Characterization of Three Highly Purified Influenza Virus Strains by Electron Microscopy
More LessSUMMARYMass determinations on highly purified influenza virus preparations were performed using the technique of scanning transmission electron microscopy. The masses of the three strains, X49, B/Singapore/222/79 and B/Hong Kong/8/73 were determined. The average value was 174 × 106 daltons with only small differences between the three strains. The mass of virus particles after removal of the protruding spike proteins, haemagglutinin and neuraminidase by bromelain treatment was determined to be 86 × 106 daltons. From the mass difference and the known molecular weight of the spike proteins the number of spikes was estimated to lie in the range 400 to 500.
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Electron Microscopic Observation of a Newly Isolated Flavivirus-like Virus from Field-caught Mosquitoes
More LessSUMMARYOf many unidentified virus strains which were isolated from field-caught mosquitoes by using C6/36 cells (a virus-sensitive clone of Aedes albopictus cells), three strains which formed small size plaques (SP virus) in C6/36 cells were investigated by electron microscopy. Although the SP virus strains did not react with antisera against known arboviruses in serological tests, they closely resembled flaviviruses in morphology. However, when they were compared to Japanese encephalitis (JE) virus, several differences in morphogenesis were observed. Proliferating membraneous structures and electron-dense amorphous areas involving precursors of the virus were observed only in cells infected with the SP virus strains. Enlarged areas of endoplasmic reticulum containing mature virions were often observed adjacent to these structures. Since the SP virus strains were isolated from wild mosquitoes and multiplied only in mosquito cells, it seems appropriate to classify them as insect viruses which resemble togaviruses morphologically.
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Mapping of Late Transcripts of Autographa californica Nuclear Polyhedrosis Virus by ‘Criss-Cross’ DNA-RNA Hybridization
More LessSUMMARYA ‘criss-cross’ DNA-RNA hybridization technique and Northern blot analysis were used to analyse late RNA transcripts in Autographa californica nuclear polyhedrosis virus (AcNPV)-infected Spodoptera frugiperda cells. Infected cells were pulse-labelled with [32P]orthophosphate at 24 to 30 h post-infection and cytoplasmic poly(A)+ RNA was isolated at 30 h post-infection. Labelled RNA was fractionated on preparative methylmercury gels and blotted under annealing conditions onto Southern blots of preparative DNA gels containing the AcNPV DNA digested with EcoRI, BamHI or HindIII restriction endonucleases. During hybridization the axis of electrophoresis of the DNA fragments was at right angles to the axis of electrophoresis of the RNA. The ‘criss-cross’ hybridization pattern was used to identify viral transcripts, to estimate their relative sizes and to map them on the genome. A transcription map for at least 12 late RNA species was drawn based on the consensus data obtained from several ‘criss-cross’ blot hybridizations and concurrent Northern blot analysis.
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Genomic Heterogeneity of Simian Rotavirus SA11
More LessSUMMARYA preparation of simian rotavirus SA11 was shown to contain, in addition to the normal 11 genome segments, an RNA species with electrophoretic mobility slightly higher than that of segment 4. Limiting dilution passages allowed the separation of two virus clones distinguishable from each other by the electrophoretic mobility of that genome segment. Possible implications of this finding in virus behaviour and in the comparison of rotaviruses by RNA electrophoresis are discussed.
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Reconstruction Experiments Demonstrating Selective Effects of Defective Interfering Particles on Mixed Populations of Vesicular Stomatitis Virus
More LessSummaryMutants of vesicular stomatitis virus have previously been isolated whose replication is not affected by the presence of defective interfering (DI) particles. We describe here reconstruction experiments in BHK-21 cells which show that DI particle-resistant viruses are strongly selected over wild-type virus in the presence of DI particles. This occurs both during undiluted lytic passage and persistent infection. This selection is evident even when the mutant virus is added initially at very low levels relative to wild-type virus. This indicates that DI particles can be important selective factors affecting viral evolution.
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