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Volume 50,
Issue 1,
1980
Volume 50, Issue 1, 1980
- Review Article
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- Articles
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- Bacterial
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Host Range, Immunity and Antigenic Properties of Lambdoid Coliphage HK97
More LessSUMMARYTemperate coliphage HK97 was isolated from pig dung. Although HK97 is antigenically unrelated to coliphage λ, it has similar morphology, host range and immunity properties, and can recombine with it.
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- Animal
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Gradual Changes of Influenza Virions During Passage of Undiluted Material
More LessSUMMARYDefective influenza virions undergo gradual changes during passage of undiluted material: the amount of RNA in virions diminishes, the equimolarity of the RNA segments is lost and the interfering activity of defective virions decreases. Defective virions of early passages have a small deficiency in genetic material and at a high m.o.i. induce the synthesis of the complete set of virus-specific proteins. Defective virions from later passages are characterized by a considerable deficiency in genetic material and a decrease in the amount of high mol. wt. RNA segments. They do not demonstrate complementation and at a high m.o.i. do not induce the synthesis of virus-specific proteins, thus revealing analogous defects in the genome of the majority of defective virions. The characteristics of protein synthesis in cells infected with these virions are similar to those in uninfected cells.
During undiluted passage of influenza virus the cyclic production of defective and infectious virions takes place.
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Characterization of a New Adenovirus Type 5 Assembly Intermediate
More LessSUMMARYAdenovirus H5 (Ad5) DNA–protein complexes were extracted with ammonium sulphate (0.2 m) from virus-infected HeLa cell nuclei at 18 h after infection. Analysis of the material by centrifugation through discontinuous sucrose gradients in heavy water revealed the existence of several populations of molecules which were identified, in order of increasing buoyant density, as mature DNA–protein complexes, replication complexes, assembly intermediates and virions. When observed under the electron microscope, some of the assembly intermediates showed a capsid with a tail of entirely double-stranded (ds) DNA, or of dsDNA continued by a portion of single-stranded (ss) DNA thickened by a coat of E-72 K DNA binding protein. Singly or doubly-forked Ad5 replicating DNA molecules partially packaged in virus capsids were also observed. It is suggested that these molecules could be assembly intermediates, i.e. one of the first steps of assembly corresponding to virus DNA entering pre-formed capsids or their precursors. The fact that replication was still going on at one end of many of the DNA molecules in the intermediates, while encapsidation was taking place at the other, raises the possibility of a coupled DNA replication-packaging process in the formation of adenovirions.
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Hepatitis B e Antigen Polypeptides Isolated from Sera of Individuals Infected with Hepatitis B Virus: Comparison with HBeAg Polypeptide Derived from Dane Particles
More LessSUMMARYHepatitis B e antigen (HBeAg) occurs in the serum of individuals infected with hepatitis B virus both free and in association with IgG. Utilizing a succession of steps involving salt precipitation, affinity chromatography, ion-exchange chromatography and isoelectrofocusing, we isolated free and IgG-bound forms of HBeAg from the sera of infected individuals with an overall gain in specific activity of 3000-fold and 540-fold, respectively. Polypeptide profiles of purified HBeAg preparations were studied by SDS–polyacrylamide gel electrophoresis in the presence of 2-mercaptoethanol. Both free and IgG-bound preparations revealed polypeptides with mol. wt. of 15500 (P15.5) and 16500 (P16.5), and HBeAg activity was detected corresponding to their positions. The HBeAg polypeptides (P15.5/16.5) derived from sera were physicochemically different from the two polypeptides with HBeAg activity (P19 and P45) liberated from Dane particle cores by the conventional method involving incubation with Nonidet P40 and 2-mercaptoethanol. However, when core particles were prepared in the presence of a proteolytic enzyme, in addition to Nonidet P40 and 2-mercaptoethanol, they gave rise to HBeAg polypeptides with mol. wt. of 31000 (P31) and 15500. Furthermore, P31 split into P15.5 when heated at 100 °C for 2 min. On the basis of these results, P15.5 may be assumed to be the essential polypeptide bearing HBeAg activity in the serum and also in Dane particles.
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Detection of IgM Antibodies to Cytomegalovirus (CMV) Using an Enzyme-labelled Antigen (ELA)
More LessSUMMARYWe have applied a peroxidase enzyme-labelled antigen (ELA) for the detection of IgM antibodies to cytomegalovirus: microtitre plates were coated with anti-IgM immunoglobulin. The IgM fraction of human serum was selectively bound to the precoated plates and the virus-specific IgM antibody was then detected by the enzyme-labelled antigen. A very efficient technique for the labelling of virus antigen is described. The IgM antibody was detected simply and specifically, Rheumatoid factor IgM did not interfere with this test.
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Biological Characterization of the Virus Causing Leukoencephalitis and Arthritis in Goats
More LessSUMMARYThis study describes the biological properties of a strain of virus isolated from tissues of a goat with leukoencephalomyelitis–arthritis. The agent is a retrovirus, having a virion-associated reverse transcriptase enzyme and an antigenic determinant(s) which cross-reacts with the p30 of visna–maedi viruses. Morphogenesis of the virus is also similar to visna virus in terms of virus assembly and the multinucleated giant cell formation which accompanies replication of the latter virus. Despite its cytopathogenic property the goat agent was not lytic in goat cell culture, causing instead a productive infection which persisted through multiple subcultures of the cells. The virus replicated incompletely in sheep cell cultures but could be rescued from the latter, weeks after inoculation, by co-cultivation with goat cells. Our data suggest that this strain of goat leukoencephalitis virus is a variant of the ovine retroviruses with a host range limited to the goat.
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Metabolic Requirements for the Maturation of Respiratory Syncytial Virus
M. Peeples and S. LevineSUMMARYThe metabolic processes required for maturation of respiratory syncytial (RS) virus were determined by testing with metabolic inhibitors in HeLa cells that had been trypsinized 18 h.i. Although >90% of the virus synthesized by that time remained cell-associated, treatment with trypsin inactivated at least 90% of the cell-associated virus. The trypsinized cells, when re-plated in virus growth medium, immediately resumed virus synthesis and this continued exponentially for at least 10 h, during which as little as 1 to 2 p.f.u./cell of new infectious virus could be detected. Treatment of these cells with 6-azauridine revealed that no further synthesis of virus RNA is required for a full yield of infectious virus. Treatment with cycloheximide, 2-deoxy-d-glucose and cytochalasin B suggested that neither protein synthesis nor glycosylation is required for the first 1 to 2 h following the resumption of virus production, but both processes are required for a full yield. These results suggest that with RS virus, the maturation process itself does not require RNA or protein synthesis or glycosylation.
Trypsin, in addition to inactivating accumulated virus, released non-infectious particulate material. The polypeptides of this material were similar to those of the RS virion except for a deficiency in glycoproteins. The trypsin apparently cleaved the virus glycoproteins on cell surfaces, which resulted in the inactivation of cell-associated virus and the release of a glycoprotein-deficient particle from cell surfaces.
The pool of virus glycoproteins is evidently the limiting factor in infectious virus production by cells trypsinized 18 h p.i. The non-glycosylated polypeptides of cell-associated virus are the same as those of free virus.
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Significance of Extracellular Enveloped Virus in the in vitro and in vivo Dissemination of Vaccinia
More LessSUMMARYThe significance of extracellular enveloped vaccinia (EEV) for the in vitro and in vivo dissemination of vaccinia virus was investigated. The quantity of in vitro released extracellular virus correlated very closely with the ability of 13 vaccinia strains to cause long-range spread of infection (comet formation) in cell cultures infected at low m.o.i. but was not correlated with plaque size. The kinetics of virus spread after low m.o.i. was related to the amount of virus released from primary infected cells but not to their content of intracellular naked vaccinia (INV). Most extracellular vaccinia virus from IHD-J-infected RK-13 cells banded in CsCl density gradients as EEV (88%) while very little banded as INV (2%). Antisera to the envelope prevented comet formation while antisera to INV did not.
CsCl centrifugation of blood-borne extracellular virus from rabbits infected intravenously with vaccinia virus after cyclophosphamide treatment revealed that 64% of the virus banded as EEV but only 11% as INV. High in vitro EEV-yielding vaccinia strains were able to spread from the respiratory tract to the brains of mice and cause death. Low in vitro EEV-yielding vaccinia strains were generally not able to disseminate in vivo or cause mouse mortality. The notable exception to this trend was strain WR, which, although releasing small amounts of virus in vitro, could nevertheless very effectively disseminate in vivo, causing a high rate of mouse mortality. Treatment with anti-envelope serum protected mice from a lethal vaccinia infection whereas antiserum to inactivated INV did not. These results indicate that the in vitro dissemination of vaccinia infection is mediated by EEV and implicate EEV as having a role in the in vivo dissemination.
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Quantification of Plasminogen Activator Activity Associated with Herpesvirus-transformed Cells
More LessSUMMARYHerpesvirus-transformed cell lines were examined for plasminogen activator (PA) activity using a quantitative assay. Previous results of cold fibrin overlay assays indicated that herpesvirus-transformed hamster cell lines produce fibrinolytic activity. Quantification of this activity involved the use of an 125I-fibrin lysis assay in which medium previously incubated with transformed or normal cells was tested for its ability to lyse 125I-fibrin polymers. This assay indicated an enhanced production of plasminogen-dependent fibrinolytic activity by transformed hamster cells compared to hamster embryo fibroblasts. The kinetics of secretion failed to reveal a significant difference in plasminogen activator activity in cells transformed by either herpes simplex virus types 1 or 2 (HSV-1 or HSV-2); however, transformed cells exhibited a significant increase in activity over non-transformed cells. Further characterization of PA activity associated with cells transformed by HSV-1 or HSV-2 has revealed that the protease is secreted and can function extracellularly. Extracellular PA activity produced by HSV-transformed cells is detected more efficiently than the cell-associated enzyme. Extracellular PA can be induced in two different species of cells by infection with partially inactivated HSV-2. Lytic infection of human embryo lung cells by HSV-2 strain 333 did not enhance activity, but infection of these cells with virus inactivated by u.v. irradiation resulted in increased PA levels from the cells. Increased enzyme levels were also detected in hamster embryo fibroblasts infected with partially inactivated virus. Further investigation of this enzyme function may determine whether increased levels of protease will indicate oncogenic transformation in vitro by herpesviruses.
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Formation of the Semliki Forest Virus Membrane Glycoprotein Complexes in the Infected Cell
More LessSUMMARYIn Semliki Forest virus (SFV)-infected cells, all structural proteins are translated from a 26S mRNA using a single initiation site. The capsid protein which is made first is released into the cytoplasm whereas the two membrane proteins, p62 (the precursor for E2 and E3) and E1, are inserted into the rough endoplasmic reticulum membrane. Based on gradient centrifugation and cross-linking studies, it can be seen that the p62 and E1 polypeptides form a complex immediately after synthesis and migrate to the plasma membrane in the form of a p62–E1 complex. The processing of p62 to E2 and E3 is first seen 25 to 30 min after a 10 min pulse of radioactive amino acids. This cleavage can be inhibited by addition of antisera specific for E1 and E3, thus supporting the view that, as in the case of the related Sindbis virus, this cleavage occurs on the external face of the plasma membrane. Proteolytic digestion of crude vesicle preparations derived from plasma membranes, combined with peptide mapping, indicate that the carboxy-terminal end of E2 spans the cell plasma membrane, there being a portion of mol. wt about 3000 located towards the cytosol.
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Mutational Changes of the Protease Susceptibility of Glycoprotein F of Newcastle Disease Virus: Effects on Pathogenicity
More LessSUMMARYTwo chemically induced mutants with an alteration in the susceptibility of glycoprotein F to proteolytic cleavage have been isolated from the apathogenic strains of La Sota and Ulster of Newcastle disease virus. In contrast to the La Sota wild type, cleavage of the precursor F0 and activation of cell fusing activity and infectivity take place if the mutant is grown in MDBK and BHK21-F cells. The mutant is, therefore, able to undergo multiple replication cycles in cells nonpermissive for the wild type. This increase in host range is paralleled by an increase in pathogenicity for chick embryos. The increase in host range of the Ulster mutant is less distinct. This mutant, which does not differ in pathogenicity from its wild type, produces in MDBK cells incompletely activated virus containing predominantly glycoprotein HN in the uncleaved and glycoprotein F in the cleaved form. The data support the concept that the susceptibility of the virus glycoproteins to proteolytic activation is an important factor in determining the pathogenicity of this virus.
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Biochemical Characterization of a Yucaipa-like Virus
SUMMARYA Yucaipa-like virus (PLOC/Senegal/273/77) was grown in embryonated chicken eggs and used for biochemical investigations after purification. The genome of the virus is composed of one fragment of single-stranded (ss)RNA with an estimated mol. wt. of 5.6 × 106. There are six virus structural polypeptides with mol. wt. of 126000, 68000 (major), 60000, 52000 (major), 44000 and 39000 (major). The fatty acid composition of the virus envelope seems to be very selective since we found only fatty acids containing 14 and 16 carbon atoms.
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A New Structural Protein for Newcastle Disease Virus
More LessSUMMARYProteins induced in Newcastle disease virus (NDV)-infected chick embryo fibroblasts (CEF) and proteins incorporated into virions grown in ovo were analysed by modified versions of a two-dimensional polyacrylamide gel electrophoresis system. The following previously described NDV-induced proteins were detected and resolved from host proteins: L (mol. wt. approx. 200K), HN (75K), F0 (66K), F1 (56K), NP (55K) and M (39K). Two additional polypeptides, NAP (nucleocapsid-associated protein, mol. wt. 56K) and a 36 K mol. wt. protein were induced in NDV-infected cells. NAP but not 36K was found in purified virions. Radioactive labelling studies with 3H-glucosamine and 32P-orthophosphate demonstrated that neither NAP nor 36K is glycosylated, but that both are phosphorylated.
Variation in the isoelectric point and apparent mol. wt. of NAP among different strains was seen and exactly reproduced in both CEF and baby hamster kidney (BHK) cells. The synthesis of NDV-induced proteins including NAP and 36K was unaffected by actinomycin D, whereas the synthesis of host cell proteins was drastically reduced. These data are evidence that NAP and 36K are virus coded. Peptide analysis indicated that NAP, NP and F1 are unrelated polypeptides. The demonstration that NAP is virus coded, together with its phosphorylation and association with the nucleocapsid, suggest that NAP may be the NDV analogue of the P protein of Sendai virus.
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Cricket Paralysis Virus RNA has a 3′ Terminal Poly(A)
More LessSUMMARYThe single-stranded (ss) virion RNA of cricket paralysis virus, an invertebrate picornavirus, has a mol. wt. of approx. 2.8 × 106 and contains a 3′ terminal poly(A) 20 to 70 nucleotides in length.
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Studies on Recombination in Heterologous Crosses of Rous Sarcoma Virus
More LessSUMMARYTs mutants in src from the Prague and Schmidt-Ruppin strains of Rous sarcoma virus (RSV) were used to superinfect chicken and quail cells chronically infected with the Bryan strain of RSV. No wild-type recombinants and few double-defective virions were produced. These results indicate that genetic recombination does not occur with high frequency in these heterologous crosses even if all infectious virus is derived from doubly infected cells.
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Biological Properties of Polyoma DNA Fragments Cloned in Plasmid pBR322
More LessSUMMARYThe two HindIII fragments of polyoma virus DNA were cloned in the HindIII site of plasmid pBR322, and the biological activity of the recombinant plasmids was tested in tissue culture cells. A mixture of recombinant plasmids containing the HindIII-A and HindIII-B fragments was infectious, but only after cleavage with HindIII. Recombinant plasmids containing the HindIII-A fragment, but not those containing the HindIII-B fragment, induced the transformation of Fischer rat 3T3 cells. These findings indicate that about half of the early region of polyoma virus DNA is not essential for the initiation or the maintenance of transformation.
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Plaque Formation and Purification of BK Virus in Cultured Human Urinary Cells
More LessSUMMARYPrimary human urinary cells support growth and plaque formation by papovavirus BK, permitting plaque purification of the virus. After plaquing, the virus forms uniform plaques and its DNA has a homogeneous restriction enzyme fragment profile. Support of BK replication as well as morphological and cultural characteristics suggest an epithelial origin for the cells.
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Enhanced Production of Interferon from Human Lymphoblastoid (Namalwa) Cells Pre-treated with Sodium Butyrate
More LessSUMMARYPre-treatment of Namalwa cells with 1 mm-sodium butyrate enhanced interferon production induced by Sendai virus. This enhancement was reversed if the cells were incubated in butyrate-free medium. Butyrate treatment increased the rate of interferon production but not its duration. Yields of interferon from Namalwa cells were also enhanced in response to other inducers including the synthetic dsRNA, poly(rI).poly(rC). Butyrate pre-treatment also increased interferon yields from cells of the vervet monkey kidney cell line, V3, but cells of the human diploid fibroblast line, MRC-5, did not respond.
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