The cytoplasmic tail of the influenza C virus glycoprotein HEF negatively affects transport to the cell surface.
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The surface glycoprotein, HEF, of influenza C virus (C/Johannesburg/1/66) has been shown to undergo a post-translation conformational change that is evident in a dramatic change of electrophoretic mobility. If the corresponding gene is expressed in the absence of other viral proteins, this folding process does not occur at all or only very inefficiently. A chimaeric protein, HEF-HA(Tail), in which the short cytoplasmic tail (Arg-Thr-Lys) of HEF was replaced by the cytoplasmic tail of the haemagglutinin of an influenza A virus (fowl plague virus) was constructed. In contrast to the wild-type protein, the chimaeric protein was detected on the cell surface. No further improvement of the surface expression was observed when both the transmembrane domain and the cytoplasmic tail were replaced by the corresponding domains of either the influenza A haemagglutinin or gp40, an endogenous protein of MDCK cells. For the HEF-HA(Tail) construct this study shows that a substantial amount of the protein is converted to the 100 kDa mature form that is observed in virus-infected cells. The HEF-HA expressed on the cell surface reacted positively in esterase and haemadsorption assays, indicating that it was present in a biologically active form. The results show that the short cytoplasmic tail of HEF has a negative effect on the folding and surface transport of this protein. How this effect may be prevented during a virus infection is discussed. (+info)
The use of a quantitative fusion assay to evaluate HN-receptor interaction for human parainfluenza virus type 3.
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Sialic acid is the receptor determinant for the human parainfluenza virus type 3 (HPF3) hemagglutinin-neuraminidase (HN) glycoprotein, the molecule responsible for binding of the virus to cell surfaces. In order for the fusion protein (F) of HPF3 to promote membrane fusion, HN must interact with its receptor. In addition to its role in receptor binding and fusion promotion, the HPF3 HN molecule contains receptor-destroying (sialidase) activity. The putative active sites are in the extracellular domain of this type II integral membrane protein. However, HN is not available in crystalline form; the exact locations of these sites, and the structural requirements for binding to the cellular receptor, which has not yet been isolated, are unknown. Nor have small molecular synthetic inhibitors of attachment or fusion that would provide insight into these processes been identified. The strategy in the present study was to develop an assay system that would provide a measure of a specific step in the viral cycle-functional interaction between viral glycoproteins and the cell during attachment and fusion-and serve to screen a variety of substances for inhibitory potential. The assay is based on our previous finding that CV-1 cells persistently infected (p.i.) with HPF3 do not fuse with one another but that the addition of uninfected CV-1 cells, supplying the critical sialic acid containing receptor molecules that bind HN, results in rapid fusion. In the present assay two HeLa cell types were used: we persistently infected HeLa-LTR-betagal cells, assessed their fusion with uninfected HeLa-tat cells, and then quantitated the beta-galactosidase (betagal) produced as a result of this fusion. The analog alpha-2-S-methyl-5-N-thioacetylneuraminic acid (alpha-Neu5thioAc2SMe) interfered with fusion, decreasing betagal production by 84% at 50 mM and by 24% at 25 mM. In beginning to extend our studies to different types of molecules, we tested an unsaturated derivative of sialic acid, 2,3-dehydro-2-deoxy-n-acetyl neuraminic acid (DANA), which is known to inhibit influenza neuraminidase by virtue of being a transition-state analog. We found that 10 mM DANA inhibited neuraminidase activity in HPF3 viral preparations. More significantly, this compound was active in our assay of HN-receptor interaction; 10 mM DANA completely blocked fusion and betagal production, and hemadsorption inhibition by DANA suggested that DANA blocks attachment. In plaque reduction assays performed with the compounds, the active analog alpha-Neu5thioAc2SMe reduced plaque formation by 50% at a 50 mM concentration; DANA caused a 90% inhibition in the plaque reduction assay at a concentration of 25 mM. Our results indicate that specific sialic acid analogs that mimic the cellular receptor determinant of HPF3 can block virus cell interaction and that an unsaturated n-acetyl-neuraminic acid derivative with affinity to the HN site responsible for neuraminidase activity also interferes with HN-receptor binding. Strategies suggested by these findings are now being pursued to obtain information regarding the relative locations of the active sites of HN and to further elucidate the relationship between the receptor-binding and receptor-destroying activities of HN during the viral life cycle. The quantitative assay that we describe is of immediate applicability to large-scale screening for potential inhibitors of HPF3 infection in vivo. (+info)
Gliding mutants of Mycoplasma mobile: relationships between motility and cell morphology, cell adhesion and microcolony formation.
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The present study characterizes gliding motility mutants of Mycoplasma mobile which were obtained by UV irradiation. They were identified by their abnormal colony shapes in 0.1% agar medium, showing a reduced number of satellite colonies compared to the wild-type. A total of ten mutants were isolated based on their colony phenotype. Using dark-field and electron microscopy, two classes of mutants, group I and group II, were defined. Cells of group I mutants had irregular, flexible and sometimes elongated head-like structures and showed a tendency to aggregate. Neither binding to glass nor gliding motility was observed in these mutants. Cells of group II mutants were rather spherical in shape, with the long axis reduced to 80% and the short axis enlarged to 120% of that of wild-type cells, respectively. Their gliding speed was 20% faster than that of wild-type cells. Three of the ten mutants remained unclassified. Mutant m6 had a reduced binding activity to glass and a reduced gliding motility with 50% of the speed of the wild-type strain. The ability of wild-type and mutant colonies to adsorb erythrocytes was found to correlate with the binding activity required for gliding, indicating that mycoplasma gliding depends on cytadherence-associated components. Finally, the ability to form microcolonies on surfaces was shown to correlate with the gliding activity, suggesting a certain role of gliding motility in the parasitic life-cycle of mycoplasmas. (+info)
The production of a temperature-sensitive persistent measles virus infection.
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A persistent infection of measles virus was established in HEp2 cells. All cells contained virus antigen when tested by specific immunofluorescence and approx. 50% were positive by haemadsorption. Infectious virus released into the supernatant medium was usually equivalent to no more than 0-001 p.f.u./cell, but between 10 and 40% of the infected cells produced plaques when plated on Vero cells. Passage of persistently infected cultures in the presence of measles antibody had no effect on the proportion of antigen-positive cells. Virus obtained from the supernatant medium of persistently infected cultures was temperature sensitive at 39-5 degrees C when tested on Vero cells whereas the original non-persistent virus produced infections on Vero cells at 39-8 degrees C. On passage of the persistently infected culture at 39-5 degrees C most of the surface antigens disappeared within 24 h whereas the intracellular virus antigens had not totally disappeared until the 5th passage. (+info)
Detection of antibody and complement complexed in vivo on membranes of human cancer cells by mixed hemadsorption techniques.
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The mixed hemadsorption (MHA) techniques demonstrated antibody and complement fixed in vivo to the surface of human cancer cells. Tumors from 12 cancer patients and normal tissues from 5 cancer patients and 8 patients with cerebrovascular or cardiac diseases were collected from biopsy and autopsy for in vitro testing. Antiserum to human whole immunoglobulins and antiserum to human C3 were used in the MHA techniques. Positive MHA patterns were demonstrated on the surface of cancer cells by both methods. Positive reactions ranged from 12 to 32% in mixed hemadsorption for anitbody detection and from 10 to 34% in mixed hemadsorption for complement component 3 detection. Normal tissues obtained from cancer patients or from patients who died of causes other than cancer rarely exhibited distinct MHA reactivity. Collectively, the data suggest that most human cancers are antigenic in the autologous host and that tumor-associated antigens of cancer cells react in vivo with their humoral antibody to fix complement. (+info)
Hemagglutinin residues of recent human A(H3N2) influenza viruses that contribute to the inability to agglutinate chicken erythrocytes.
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To identify the molecular determinants contributing to the inability of recent human influenza A(H3N2) viruses to agglutinate chicken erythrocytes, phenotypic revertants were selected upon passage in eggs or MDCK cells. The Leu194Ile or Val226Ile substitutions were detected in their hemagglutinin (HA) sequence concomitantly with the phenotypic reversion. Remarkably, as little as 3.5% of variants bearing a Val226Ile substitution was found to confer the ability to agglutinate chicken erythrocytes to the virus population. Hemadsorption assays following transient expression of mutated HA proteins showed that the successive Gln226 --> Leu --> Ile --> Val changes observed on natural isolates resulted in a progressive loss of the ability of the HA to bind chicken erythrocytes. The Val226Ile change maintained the preference of the HA for SAalpha2,6Gal over SAalpha2,3Gal and enhanced binding of the HA to alpha2,6Gal receptors present on chicken erythrocytes. In contrast, simultaneous Ser193Arg and Leu194Ile substitutions that were found to confer the ability to agglutinate sheep erythrocytes increased the affinity of the HA for SAalpha2,3Gal. (+info)
High resolution investigations with the scanning and transmission electron microscope of haemadsorption binding sites of mumps virus-infected HeLa cells.
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Specific changes at the surface of HeLa cells infected with mumps virus were investigated in parallel with the scanning and transmission electron microscope. The distribution of haemadsorption binding sites and virus-induced antigens at cell surfaces was simultaneously studied by labelling virus-specific antigens with peroxidase-conjugated antibodies after haemadsorption. New information was obtained upon the three-dimensional aspect of the red blood cells, the topographical distribution of their binding sites on the infected cells, and the specific structures at the cell surface which are involved in the process of haemadsorption. (+info)
Changes in the virus-host cell relationship in a stable non-virogenic cell line persistently infected with measles virus (BGM/MV).
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A non-virogenic African green monkey kidney cell line BGM/MV persistently infected with a neurotropic mouse brain-adapted strain of measles virus, was found to have undergone significant changes in the virus-host cell relationship between passages 35 and 119. Rather than the stable non-cytopathic relationship previously reported in which approximately 100% of the cells contained measles antigens and less than 1% of the cells expressed cell surface measles antigen, we observed cyclic manifestations of c.p.e. together with changes in the percentage of cells expressing intracellular and cell surface measles antigens. Treatment of BGM/MV cells with actinomycin D effected an increase in the percentage of cells expressing cell surface virus haemagglutinin (HA) at times when the percentage of cells with surface HA was less than the percentage of cells with intracellular measles antigens. Superinfection studies employing measles virus and vesicular stomatitis virus revealed a consonant cyclic refractivity and essentially no refractivity, respectively. Endogenous, infectious measles virus was not detected nor was interferon. It was concluded that a host cell factor other than interferon was modulating the cyclic expression of the measles virus infection. (+info)