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Biomedical subjects

D S Lyles

Publications and source records attributed to D S Lyles.

At least 55 records · Page 3Linked to original sources
PubMed1986

Dissociation of newly synthesized Sendai viral proteins from the cytoplasmic surface of isolated plasma membranes of infected cells.

The interaction of Sendai viral proteins with the membranes of infected cells during budding of progeny virions was studied. BHK cells infected with Sendai virus were labeled with [35S]methionine, and the plasma membranes were purified on polycationic polyacrylamide beads. The isolated membranes were incubated with various agents which perturb protein structure to dissociate viral proteins from the membranes. Incubation of membranes with thiocyanate and guanidine removed both the M and nucleocapsid proteins. Urea (6 M) removed the nucleocapsid proteins but removed M protein only in the presence of 0.1 or 1.0 M KCl. In contrast, high salt concentrations alone eluted only the M protein, leaving the nucleocapsid proteins completely membrane bound. About 65% of the M protein was eluted in the presence of 4 M KCl. The remaining membrane-associated M protein was resistant to further extraction by 4 M KCl. Thus, M protein forms two types of interaction with the membrane, one of them being a more extensive association with the membrane than the other.

Animals↗
PubMed1985

Labeling of the cytoplasmic domain of the influenza virus hemagglutinin with fluorescein reveals sites of interaction with membrane lipid bilayers.

The hemagglutinin (HA) glycoprotein of influenza virus was labeled in its cytoplasmic domain with fluorescein. Reactive amino groups in the external domain were blocked by modification of the intact virus with the membrane-impermeable reagent isethionyl acetimidate. The HA was then solubilized with the detergent octyl glucoside, and the single lysine in the cytoplasmic domain was reacted with fluorescein isothiocyanate. This protocol resulted in the incorporation of 1.3 mol of fluorescein/mol of HA. Using a virus strain lacking lysine in the cytoplasmic domain of HA, it was determined that 0.47 mol of fluorescein/mol of HA was located at an additional site(s). The fluorescein groups at both sites exist in an environment of reduced polarity as shown by a shift in excitation and emission maxima and a shift in the pKa of the fluorescein groups. The fluorescence polarization and the pKa of the fluorescein groups were greater when the HA was incorporated into liposomes than when in detergent solution. These data indicate that the fluorescein groups interact directly with the lipid bilayer, probably in the phospholipid head-group region. The fluorescence properties of the labeled HA were not responsive to the gel to liquid-crystal phase transition in the lipid bilayer. These results indicate that the boundary between the cytoplasmic domain and the hydrophobic sequence that anchors the protein to the lipid bilayer is located in the head-group region of the bilayer.

Electrophoresis, Polyacrylamide Gel↗
PubMed1985

Role of the nuclear envelope in synthesis, processing, and transport of membrane glycoproteins.

The outer nuclear membrane is morphologically similar to rough endoplasmic reticulum. The presence of ribosomes bound to its cytoplasmic surface suggests that it could be a site of synthesis of membrane glycoproteins. We have examined the biogenesis of the vesicular stomatitis virus G protein in the nuclear envelope as a model for the biogenesis of membrane glycoproteins. G protein was present in nuclear membranes of infected Friend erythroleukemia cells immediately following synthesis and was transported out of nuclear membranes to cytoplasmic membranes with a time course similar to transport from rough endoplasmic reticulum (t 1/2 = 5-7 min). Temperature-sensitive mutations in viral membrane proteins which block transport of G protein from endoplasmic reticulum also blocked transport of G protein from the nuclear envelope. Friend erythroleukemia cells and NIH 3T3 cells differed in the fraction of newly synthesized G protein found in nuclear membranes, apparently reflecting the relative amount of nuclear membrane compared to endoplasmic reticulum available for glycoprotein synthesis. Nuclear membranes from erythroleukemia cells appeared to have the enzymatic activities necessary for cleavage of the signal sequence and core glycosylation of newly synthesized G protein. Signal peptidase activity was detected by the ability of detergent-solubilized membranes of isolated nuclei to correctly remove the signal sequence of human preplacental lactogen. RNA isolated from the nuclear envelope was highly enriched for G protein mRNA, suggesting that G protein was synthesized on the outer nuclear membrane rather than redistributing to nuclear membranes from endoplasmic reticulum before or during cell fractionation. These results suggest a mechanism for incorporation of membrane glycoproteins into the nuclear envelope and suggest that in some cell types the nuclear envelope is a major source of newly synthesized membrane glycoproteins.

Animals↗
PubMed1985

Neuraminidase activity is not the cause of influenza virus-induced neutrophil dysfunction.

Influenza viruses have been shown to decrease the ability of polymorphonuclear leukocytes (PMN) to respond to a variety of stimuli. This study was done to determine if viral neuraminidase was responsible for decreased PMN function. Treatment of human PMN with purified neuraminidases from influenza virus, Vibrio cholerae, or Clostridium perfringens did not significantly affect the ability of human PMN to respond to stimulation. Occasional virus preparations that lacked the ability to depress PMN function did not differ in neuraminidase activity from viruses capable of causing depression. These results demonstrate that neuraminidase activity is not the cause of influenza virus-induced PMN dysfunction.

Centrifugation, Density Gradient↗
PubMed1985

Expression of influenza virus NS2 nonstructural protein in bacteria and localization of NS2 in infected eucaryotic cells.

The nonstructural NS2 protein of influenza A/PR/8/34 virus was efficiently expressed in bacteria, and monospecific antisera were prepared against the bacterially synthesized polypeptide. These antisera were cross-reactive among the NS2 proteins of various influenza A viruses. However, they did not react with the NS2 of influenza B/Lee/40 virus nor with other proteins of influenza A viruses such as NS1. Antisera against NS2 were used to determine that the NS2 protein is localized in the cell nucleus during influenza virus infection, as shown by immunofluorescence microscopy. Cells infected with simian virus 40 recombinants containing the influenza virus NS gene revealed that both the NS1 and NS2 proteins appeared in the nucleus, even in the absence of expression of other influenza virus-specific components.

Animals↗
PubMed1984

Characterization of the effect of influenza virus on polymorphonuclear leukocyte membrane responses.

Depressed chemotactic activity of polymorphonuclear leukocytes (PMNL) infected with influenza virus could be due to changes occurring at the plasma membrane. The present study examined the effect of unopsonized influenza virus on chemotaxis, adherence, receptor binding, shape change, membrane fluidity, and release of specific granules from PMNL. Chemotactic activity of PMNL under-agarose to the chemoattractants, zymosan-activated serum ( ZAS ) and N-formyl-methionyl-leucyl-phenylalanine (fMLP), and adherence of PMNL to a plastic surface were markedly decreased in virus-treated cells as compared to control cells. The binding of fMLP to the PMNL was increased in virus-treated cells compared with control cells. Exposure of cells to virus, ZAS , or fMLP caused 35%-50% of the cells to become bipolar in shape, whereas less than 5% of the cells exposed to buffer became bipolar. Influenza virus did not alter membrane fluidity as measured by electron spin resonance spectroscopy with the probe 5-doxyl stearate. Virus-treated PMNL stimulated with FMLP or Staphylococcus aureus exhibited a marked decrease in the amount of lactoferrin released into phagosomes, onto the cells' outer membrane, and into the extracellular medium as compared to control cells. The possible relationship between inhibition of lysosomal enzyme degranulation and decreased chemotactic activity and adherence of PMNL is discussed.

Cell Adhesion↗
PubMed1983

Antigenic determinants of vesicular stomatitis virus: analysis with antigenic variants.

Antigenic variants of vesicular stomatitis virus (VSV) serotypes New Jersey and Indiana (VSV-NJ, VSV-Ind) were selected by using a panel of monoclonal antibodies (MAb) specific for the major surface glycoprotein (G-protein). The reactivity of antigenic variants with the panel of MAb confirmed observations made by competitive binding assays that four distinct antigenic sites (A-D)NJ on the VSV-NJ G-protein and four partially overlapping sites (A, B1, B2, C)Ind on the VSV-Ind G-protein are involved in virus neutralization. Furthermore, subregions within the A epitopes of both serotypes were detected by variant analysis. The frequency of variation at most epitopes was 1 in 10(5) for VSV-NJ and 1 in 10(6) for VSV-Ind. The A3 and C determinants of VSV-Ind, however, defined by MAb that exhibited overlap in binding to other epitopes, appeared to be relatively invariant. Multiple mutations may be necessary to abolish antibody binding at these sites. Overlap of the C group of anti-VSV-Ind MAb with the A epitopes was assigned to the A2 subregion, because variants selected with A2 MAb show reduced binding of C MAb. Heterogeneous antisera from a primary immune response could detect differences in reactivity between variants at the A epitopes and wild-type VSV-NJ or VSV-Ind, suggesting the A epitope is immunodominant. Hyperimmune sera could detect a small difference between ANJ and BNJ variants compared to wild-type VSV-NJ, but could not distinguish between VSV-Ind variants and wild-type VSV-Ind.

Antibodies, Monoclonal↗
PubMed1983

Cytotoxic T lymphocytes reactive with vesicular stomatitis virus: analysis of specificity with monoclonal antibodies directed to the viral glycoprotein.

A panel of monoclonal antibodies (MAb) reactive with the vesicular stomatitis virus (VSV) glycoprotein (G-protein) was used to inhibit lysis of infected target cells by cytotoxic T lymphocytes (CTL) reactive with VSV serotypes Indiana and New Jersey (VSV-Ind, VSV-NJ). MAb to distinct epitopes on the VSV-NJ G-protein were able to block lysis of VSV-NJ-infected targets using anti-VSV-NJ CTL. Furthermore, when a combination of MAb to three distinct epitopes was used, increased inhibition was observed when compared to single epitope blocking. Cross-reactive CTL generated using either VSV-NJ or VSV-Ind could be inhibited by MAb specific for the heterologous virus in blocking assays. Cross-reactive MAb also were able to block both serotype-specific and cross-reactive lysis. Antigenic variants selected with some of the MAb used in blocking studies were used to infect target cells. Reduction in lysis was not observed using variants with mutations at several epitopes. This result suggests that blocking by MAb involves steric hindrance of CTL-recognized determinants proximal to the MAb binding site.

Animals↗
PubMed1982

Influenza A virus-induced polymorphonuclear leukocyte dysfunction.

Previous studies have shown that influenza A virus can activate the polymorphonuclear leukocyte (PMN) respiratory burst and that upon subsequent stimulation of the cell there is depressed metabolic function. We examined the mechanism by which influenza virus causes PMN dysfunction by measuring the effect upon the chemiluminescent activity of cells of varying the type of influenza virus used, the period of time that cells were exposed to virus, and the secondary stimulus that was used. The various types of intact influenza virus elicited different amounts of chemiluminescent activity, but when cells were subsequently stimulated with phorbol myristate acetate, each virus caused equivalent depression of the PMN response. Purified glycoproteins incorporated into a liposome structure similarly stimulated the PMN chemiluminescence, yet did not induce PMN dysfunction. Depressed PMN function was noted after as little as 5 min of incubation of cells with virus and occurred to both receptor-dependent (zymosan, N-formylmethionyl-leucyl-phenylalanine, and phorbol myristate acetate) and -independent (calcium ionophore A23187) stimuli.

Humans↗
PubMed1982

Inhibition of neutrophil lysosome-phagosome fusion associated with influenza virus infection in vitro. Role in depressed bactericidal activity.

The present study examined the effect of various unopsonized strains of influenza A virus on release of myeloperoxidase (MPO) and acid phosphatase in polymorphonuclear leukocytes (PMNL). These results were correlated with the effect that these same viruses had on bactericidal activity in PMNL. Several strains of virus inhibited the fusion of azurophil granules with phagosomes containing Staphylococcus aureus. These same strains inhibited the extracellular release of MPO from PMNL (39-59%) and caused depressed killing (42-77%). In contrast, one of the influenza viruses (X-47a) did not inhibit PMNL MPO release or killing. The data indicate a close relationship between the ability of influenza virus to ablate normal intracellular lysosome-phagosome fusion with subsequent depression of bactericidal functions of PMNL.

Blood Bactericidal Activity↗
PubMed1981

Interaction of Sendai virus proteins with the cytoplasmic surface of erythrocyte membranes following viral envelope fusion.

Radiolabeled Sendai virus was allowed to fuse with human erythrocytes and inside out vesicles were prepared from the erythrocyte membranes. Viral proteins incorporated into inside out vesicles were released from the membrane following treatment with various agents which perturb protein structure. Disruption products were analyzed by sucrose gradient centrifugation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis to identify the viral proteins. The results indicated that 6 M guanidine, 40 mM lithium diiodosalicylate (pH 11-13), and 4 M potassium thiocyanate removed viral nucleocapsid and M proteins from the vesicles. Urea (6.0 M) and 5 mM p-chloromercuribenzenesulfonate removed only viral nucleocapsid proteins. KCl (1.0 or 0.1 M), 5 or 0.5 mM sodium phosphate, 10 mM EDTA (pH 10), 10 mM lithium diiodosalicylate, 1 M urea, 0.5 mM ATP, or freeze-thaw cycles did not release any viral proteins. By contrast, treatment of inside out vesicles with Triton X-100 solubilizes the lipid bilayer, releasing viral integral membrane glycoproteins and leaving viral nucleocapsids intact. Nucleocapsids isolated from virions do not adsorb nonspecifically to control inside out vesicles. These data imply that Sendai viral nucleocapsid and M proteins interact extensively with the cytoplasmic surface of infected cell membranes, in the manner of peripheral membrane proteins, whereas the glycoproteins, HN and F1, behave as integral membrane proteins.

Detergents↗
PubMed1981

Structure of Sendai viral proteins in plasma membranes of virus-infected cells.

Purified plasma membranes attached to polycationic polyacrylamide beads by their external surface were isolated from BHK cells infected with Sendai virus. Each of the viral proteins could be identified in the membranes of infected cells. Proteolysis with trypsin, which digests only the cytoplasmic surface of these membranes (because the external surface is protected by its attachment to beads), revealed that the internal proteins, L, P, NP, and M, were present on the cytoplasmic surface of the membrane and that small segments of the viral envelope glycoproteins, HN and F0, were partially exposed on the cytoplasmic surface. Since the major portions of HN and F0 are known to be present on the external membrane surface, these glycoproteins are transmembrane proteins before Sendai virus budding in infected cells.

Animals↗
PubMed1981

Subcellular localization of the env-related glycoproteins in Friend erythroleukemia cells.

A scheme was developed for the subcellular fractionation of murine erythroleukemia cells transformed by Friend leukemia virus. The subcellular localization of the env-related glycoproteins was determined by immune precipitation with antiserum against gp70, the envelope glycoprotein of the helper virus, followed by gel electrophoresis. In cells labeled for 2 h with [35S]methionine, the glycoprotein encoded by the defective spleen focus-forming virus, gp55SFFV, was found primarily in the nuclear fraction and in fractions containing dense cytoplasmic membranes such as endoplasmic reticulum. A similar distribution was noted for gp85env, the precursor to gp70. The concentration of viral glycoproteins in the nuclear fraction could not be accounted for by contamination with endoplasmic reticulum. In pulse-chase experiments, neither glycoprotein underwent major redistribution. However, labeled gp85env disappeared from intracellular membranes with a half-time of 30 min to 1 h, whereas labeled gp55SFFV was stable during a 2-h chase. In plasma membrane preparations with very low levels of contamination with endoplasmic reticulum, gp70 was the major viral env-related glycoprotein detected; a minor amount of gp55SFFV and no gp85env could be detected. The unexpected result of these experiments is the amount of viral glycoproteins found in the nuclear fraction. Presence of viral proteins in the nucleus could be relevant to the mechanism of viral leukemogenesis.

Animals↗
PubMed1980

Antigen-liposome modification of target cells as a method to alter their susceptibility to lysis by cytotoxic T lymphocytes.

A method of liposome modification of cell surfaces to render unsuitable target cells susceptible to lysis by anti-viral cytotoxic T lymphocytes (CTLs) is described. Liposomes containing the hemagglutinin-neuraminidase (HN) and fusion (F) glycoproteins of Sendai virus as well as purified H-2Kk cells and rendering those cells susceptible to lysis by B10. A anti-Sendai virus or anti-H-2Kk CTLs. The absence from the modifying liposomes of the HN or F proteins or H-2Kk antigens eliminated the ability of the target cells to be recognized and lysed by either effector cell population. Vesicles containing HN, H-2Kk molecules, and inactive fusion protein (Fo) were not capable of increasing the susceptibility of h-2-negative target cells to lysis. Liposomes containing inactive fusion protein were similarly unable to render H-2-positive target cells susceptible to lysis by anti-Sendai virus CTLs, suggesting that fusion of the liposomes to the cell surface is a prerequisite to lysis. It did not appear that attachement of liposomes to the cell surface was sufficient for generation of susceptible targets, however, because attachment to the cell surface was observed, as long as the HN glycoprotein was present in the liposomes. These results indicate that purified H-2Kk glycoproteins are target antigens for anti-H-2k CTLs and that B10 . A anti-Sendai virus CTLs recognize in an H-2-restricted manner the HN, F, or both glycoproteins of Sendai virus in the context of the purified H-2Kk glycoproteins. This technique of liposome modification of cell surfaces has potential applications in the examination of CTL antigen recognition and immunotherapy of many viral and neoplastic diseases.

Animals↗
PubMed1980

Elicitation of anti-Sendai virus cytotoxic T lymphocytes by viral and H-2 antigens incorporated into the same lipid bilayer by membrane fusion and by reconstitution into liposomes.

We have investigated the minimal molecular requirements for elicitation of anti-Sendai virus cytotoxic T lymphocytes (CTL), and the minimal molecular requirements for the recognition and lysis processes associated with anti-Sendai virus CTL-target cell interactions. This report demonstrates a) that the hemagglutinin-neuraminidase and/or fusion glycoproteins of Sendai virus can elicit anti-Sendai virus CTL and b) that these glycoproteins and H-2 antigens must be within the same membrane lipid bilayer for effective elicitation of anti-Sendai-virus CTL and for effective recognition and lysis of target cells by anti-Sendai virus CTL.

Animals↗