scholarly journals A Defect in Influenza A Virus Particle Assembly Specific to Primary Human Macrophages

mBio ◽  
2018 ◽  
Vol 9 (5) ◽  
Author(s):  
Sukhmani Bedi ◽  
Takeshi Noda ◽  
Yoshihiro Kawaoka ◽  
Akira Ono
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Influenza A ◽  
Cytochalasin D ◽  
Virus Release ◽  
Cell Type ◽  
Monocytic Cell ◽  
Monocytic Cell Line ◽  
Particle Assembly ◽  
Human Macrophages

ABSTRACTInfluenza A virus (IAV) propagates efficiently in epithelial cells, its primary target in the respiratory tract. In contrast, productive infection of most IAV strains is either blocked or highly inefficient in macrophages. The exact nature of the defect in IAV replication in human macrophages remains unknown. In this study, we showed that even compared to a monocytic cell line differentiated to macrophage-like cells, primary human monocyte-derived macrophages (MDM) are inefficient in IAV production, despite comparable levels of expression of viral glycoproteins at the plasma membrane. Correlative fluorescence scanning electron microscopy revealed that formation of budding structures at the cell surface is inefficient in MDM even though clustering of a viral glycoprotein, hemagglutinin (HA), is observed, suggesting that a step in IAV particle assembly is blocked in MDM. Using anin situproximity ligation assay, we further determined that HA associates with neuraminidase (NA) but fails to associate with another viral transmembrane protein, M2, at the MDM plasma membrane. Notably, the defects in HA-M2 association and particle assembly in MDM were reversed upon cytochalasin D treatment that inhibits actin polymerization. These results suggest that HA-M2 association on the plasma membrane is a discrete step in IAV production, which is susceptible to suppression by actin cytoskeleton in MDM. Virus release remained inefficient in MDM upon cytochalasin D treatment, suggesting the presence of an additional defect(s) in virus release in this cell type. Overall, our study revealed the presence of multiple cell-type-specific mechanisms negatively regulating IAV production at the plasma membrane in MDM.IMPORTANCEIdentification of host cell determinants promoting or suppressing replication of viruses has been aided by analyses of host cells that impose inherent blocks on viral replication. In this study, we show that primary human MDM, which are not permissive to IAV replication, fail to support virus particle formation. This defect is specific to primary human macrophages, since a human monocytic cell line differentiated to macrophage-like cells supports IAV particle formation. We further identified association between two viral transmembrane proteins, HA and M2, on the cell surface as a discrete assembly step, which is defective in MDM. Defective HA-M2 association and particle budding, but not virus release, in MDM are rescued by disruption of actin cytoskeleton, revealing a previously unknown, negative role for actin, which specifically targets an early step in the multistep IAV production. Overall, our study uncovered a host-mediated restriction of association between viral transmembrane components during IAV assembly.

scholarly journals A Defect in Influenza A Virus Particle Assembly Specific to Primary Human Macrophages

2017 ◽  
Author(s):  
Sukhmani Bedi ◽  
Takeshi Noda ◽  
Yoshihiro Kawaoka ◽  
Akira Ono
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Cell Line ◽  
Influenza A Virus ◽  
Influenza A ◽  
Actin Polymerization ◽  
Monocytic Cell ◽  
Monocytic Cell Line ◽  
Particle Assembly ◽  
Human Macrophages

AbstractThe primary target of Influenza A virus (IAV) is epithelial cells in the respiratory tract. In contrast to epithelial cells, productive infection of most IAV strains is either blocked or highly inefficient in macrophages. The exact nature of the defect in IAV replication in human macrophages remains unknown. In this study, we showed that primary human monocyte-derived macrophages (MDM) are inefficient in IAV release even when compared to a monocytic cell line differentiated to macrophage-like cells, despite comparable levels of expression of viral glycoproteins at the plasma membrane. Correlative fluorescence scanning electron microscopy revealed that formation of budding structures at the cell surface is inefficient in MDM even though clustering of a viral glycoprotein, hemagglutinin (HA), is observed, suggesting that IAV particle assembly is blocked in human MDM. Using anin situproximity ligation assay, we further determined that association between HA and the viral ion channel protein M2 is defective at the plasma membrane of MDM. In contrast, HA and another glycoprotein neuraminidase (NA) associate with each other on the MDM surface efficiently. Notably, the defect in association between HA and M2 in MDM was reversed upon inhibition of actin polymerization by cytochalasin D. Altogether, these results suggest that HA-M2 association on the plasma membrane is a discrete step in the IAV assembly process, which is separable from the association between HA and NA and susceptible to suppression by actin cytoskeleton. Overall, our study revealed the presence of a cell-type-specific mechanism negatively regulating IAV assembly at the plasma membrane.ImportanceIdentification of host cell determinants promoting or suppressing replication of many viruses has been aided by analyses of host cells that impose inherent blocks on viral replication. In this study, we show that primary human MDM are not permissive to IAV replication due to a defect at the virus particle formation step. This defect is specific to primary human macrophages, since a human monocytic cell line differentiated to macrophage-like cells supports IAV particle formation. We further identified association between two viral transmembrane proteins, HA and M2, on the cell surface as a discrete assembly step, which is defective in MDM. Defective HA-M2 association in MDM is rescued by disruption of the actin cytoskeleton, revealing a previously unknown, negative role for actin polymerization, which is generally thought to play positive roles in IAV assembly. Overall, our study uncovered a host-mediated restriction of association between viral transmembrane components during IAV assembly.

scholarly journals CD4-CCR5 interaction in intracellular compartments contributes to receptor expression at the cell surface

Blood ◽  
2009 ◽  
Vol 113 (9) ◽  
pp. 1938-1947 ◽  
Author(s):  
Lamia Achour ◽  
Mark G. H. Scott ◽  
Hamasseh Shirvani ◽  
Alain Thuret ◽  
Georges Bismuth ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Receptor Expression ◽  
Cell Surface Expression ◽  
Dependent Manner ◽  
Small Interfering Rnas ◽  
Concentration Dependent Manner ◽  
Monocytic Cell ◽  
Monocytic Cell Line ◽  
Intracellular Compartments

The association of CD4, a glycoprotein involved in T-cell development and antigen recognition, and CC chemokine receptor 5 (CCR5), a chemotactic G protein–coupled receptor, which regulates trafficking and effector functions of immune cells, forms the main receptor for HIV. We observed that the majority of CCR5 is maintained within the intracellular compartments of primary T lymphocytes and in a monocytic cell line, contrasting with its relatively low density at the cell surface. The CCR5-CD4 association, which occurs in the endoplasmic reticulum, enhanced CCR5 export to the plasma membrane in a concentration-dependent manner, whereas inhibition of endogenous CD4 with small interfering RNAs decreased cell-surface expression of endogenous CCR5. This effect was specific for CCR5, as CD4 did not affect cellular distribution of CXCR4, the other HIV coreceptor. These results reveal a previously unappreciated role of CD4, which contributes to regulating CCR5 export to the plasma membrane.

Multimerization of monocyte chemoattractant protein-1 is not required for glycosaminoglycan-dependent transendothelial chemotaxis

2001 ◽  
Vol 358 (3) ◽  
pp. 737-745 ◽  
Author(s):  
Simi ALI ◽  
Adrian C. V. PALMER ◽  
Sarah J. FRITCHLEY ◽  
Yvonne MALEY ◽  
John A. KIRBY
Keyword(s):  
Cell Surface ◽  
Fusion Protein ◽  
Radioligand Binding ◽  
Heparan Sulphate ◽  
Placental Alkaline Phosphatase ◽  
Monocytic Cell ◽  
Monocytic Cell Line ◽  
Monocyte Chemoattractant Protein 1 ◽  
Monocyte Chemoattractant ◽  
Monocyte Chemoattractant Protein

Chemokines interact with specific G-protein-coupled cell-surface receptors and with glycosaminoglycans (GAGs), such as heparan sulphate. Although chemokines often form multimers in solution, this process may be enhanced following interaction with GAGs on the cell surface, or within the extracellular matrix. However, the significance of multimerization for chemokine function remains controversial. In the present study, a fusion protein was prepared between the prototypical human CC chemokine, monocyte chemoattractant protein-1 (MCP-1; also known as CCL-2) and a large secreted placental alkaline phosphatase (SEAP) moiety. This fusion protein (MCP-1–SEAP) remained monomeric under conditions that promote oligomerization of the native chemokine. Radioligand binding showed that both native MCP-1 and MCP-1–SEAP competed for the same site on the surface of HEK-293 cells expressing the CCR2b chemokine receptor. The interaction between either chemokine species and endothelial cell surface GAGs was antagonized by the addition of the heparan sulphate-like molecule, heparin. Both MCP-1 and MCP-1–SEAP induced a Ca2+-flux in the THP-1 monocytic cell line, and were equally effective at promoting transendothelial chemotaxis of mononuclear immune cells, with maximal migration being produced by treatment with 12nM of either species. In each case this chemotactic response was almost completely antagonized by the addition of heparin. The importance of interaction between either native MCP-1 or MCP-1–SEAP and cell-surface GAGs for transcellular migration was demonstrated by the almost complete absence of leucocyte chemotaxis across monolayers of GAG-deficient mutant cells. In summary, this study shows that multimerization is neither necessary for, nor potentiates, the biological activity of MCP-1. However, the results do clearly demonstrate the importance of the interaction between MCP-1 and cell-surface heparan sulphate for transmonolayer leucocyte chemotaxis.

scholarly journals Antibodies to tubulin and actin bind to the surface of a human monocytic cell line, U937.

1991 ◽  
Vol 39 (7) ◽  
pp. 981-985 ◽  
Author(s):  
S B Por ◽  
M A Cooley ◽  
S N Breit ◽  
R Penny ◽  
P W French
Keyword(s):  
Flow Cytometry ◽  
Cell Surface ◽  
Cell Line ◽  
Laser Scanning ◽  
Confocal Laser ◽  
Staining Procedure ◽  
Intermediate Filament Protein ◽  
U937 Cells ◽  
Monocytic Cell ◽  
Monocytic Cell Line

Intermittent reports of cytoskeleton proteins (actin and tubulin) on the cell surface have appeared over the last 13 years. Whereas most have concentrated on lymphocytes, this study provides evidence for the presence of these proteins on the surface of a human cultured monocyte-like cell line, U937. Both actin and tubulin were detected on the surface of U937 cells by flow cytometry, using an indirect staining procedure based on biotin-streptavidin-phycoerythrin, chosen for greater sensitivity. By use of this procedure, the majority of viable unstimulated U937 cells stained positively for actin and tubulin, although the level of fluorescence intensity was low. With an antibody specific for tyrosine-tubulin, most of the surface tubulin was also found to be tyrosinylated. For vimentin, an intermediate filament protein abundantly present in the cytoplasm of U937 cells, no staining could be detected. Confirmation of the flow cytometry data for surface actin and tubulin on unstimulated U937 cells was achieved by direct vesualization using a confocal laser scanning microscope. When U937 cells were activated with PMA and LPS, a marked reduction in the level of cell surface actin and tubulin occurred. The role of cell surface actin and tubulin on unstimulated U937 cells, in terms of monocyte function, remains to be elucidated.

Role of microfilaments in asialoglycoprotein processing in adult and developing liver

1990 ◽  
Vol 259 (4) ◽  
pp. G639-G645
Author(s):  
S. S. Kaufman ◽  
P. L. Blain ◽  
J. H. Park ◽  
D. J. Tuma
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Rate Constant ◽  
Cytochalasin D ◽  
Ligand Complex ◽  
Receptor Ligand ◽  
Surface Receptor ◽  
Old Rats ◽  
Initial Movement

To assess the role of microfilaments in receptor-mediated endocytosis of asialoglycoproteins, hepatocytes isolated from adult and 6-day-old rats were treated with the antimicrofilamentous agent cytochalasin D and then incubated with 125I-asialoorosomucoid (ASOR). Cytochalasin D (50 microM) reduced degradation of continuously endocytosed ASOR (7.5 micrograms/ml) equally in adult and neonate to approximately 20% of control. Internalization of surface-bound ASOR suggested at least two discrete sites at which ligand translocation was inhibited by drug at both ages: 1) initial movement of receptor-ligand complex from cell surface to interior and 2) postinternalization ligand transit to lysosomes. Inhibition of plasma membrane translocation was confirmed by calculation of endocytotic rate constant (Ke) values, which were decreased to approximately 20-30% of control after cytochalasin D treatment. In contrast, the antimicrotubular drug colchicine did not reduce Ke values significantly nor did colchicine in combination with cytochalasin D impede lysosome-directed transport more than cytochalasin D alone. These results indicate that internalization of occupied asialoglycoprotein surface receptor is microfilament dependent irrespective of postnatal age and that subsequent participation of microfilaments in asialoglycoprotein trafficking is closely related to that of microtubules.

scholarly journals NF-κB cis-Acting Motifs of the Human Immunodeficiency Virus (HIV) Long Terminal Repeat Regulate HIV Transcription in Human Macrophages

2001 ◽  
Vol 75 (23) ◽  
pp. 11408-11416 ◽  
Author(s):  
Susana Asin ◽  
Gary D. Bren ◽  
Eva M. Carmona ◽  
Nancie J. Solan ◽  
Carlos V. Paya
Keyword(s):  
Human Immunodeficiency Virus ◽  
Long Terminal Repeat ◽  
Cell Line ◽  
Dominant Negative ◽  
Terminal Repeat ◽  
Monocytic Cell ◽  
Monocytic Cell Line ◽  
Human Macrophages ◽  
Hiv Transcription ◽  
Immunodeficiency Virus

ABSTRACT The role of NF-κB in the reactivation of human immunodeficiency virus (HIV) from latency in CD4 T lymphocytes is well documented. However, its role in driving HIV transcription in human macrophages, which contain a constitutive nuclear pool of NF-κB, is less well understood. In this study we have investigated the role that the constitutive pool of NF-κB and the NF-κB cis-acting motifs of the HIV long terminal repeat (LTR) play in regulating HIV transcription in human monocytic cells and primary macrophages. Inhibition of the constitutive nuclear pool of NF-κB (RelA and RelB) in the promonocytic U937 cell line using dominant-negative IκBα significantly decreases HIV replication. Moreover, it is demonstrated that in the differentiated monocytic cell line THP1, which contains a constitutive nuclear pool of NF-κB (RelB),an HIV provirus containing mutations of the κB cis-acting sites in the LTR is transcriptionally impaired. Reduction of the constitutive pool of NF-κB in human macrophages by an adenovirus vector expressing a dominant-negative IκBα also reduces HIV transcription. Lastly, mutation of the NF-κB cis-acting sites in the LTR of an R5 HIV provirus completely abrogates the first cycle of HIV transcription. These studies indicate that thecis-acting NF-κB motifs of the HIV LTR are critical in initiating HIV transcription in human macrophages and suggest that the constitutive nuclear pool of NF-κB is important in regulating HIV transcription in these cells.

scholarly journals Regulated internalization of caveolae.

1994 ◽  
Vol 127 (5) ◽  
pp. 1199-1215 ◽  
Author(s):  
R G Parton ◽  
B Joggerst ◽  
K Simons
Keyword(s):  
Alkaline Phosphatase ◽  
Plasma Membrane ◽  
Cell Surface ◽  
Phosphatase Activity ◽  
Okadaic Acid ◽  
Alkaline Phosphatase Activity ◽  
Kinase Inhibitor ◽  
Cytochalasin D ◽  
Microtubule Organizing Center ◽  
Hypertonic Medium

Caveolae are specialized invaginations of the plasma membrane which have been proposed to play a role in diverse cellular processes such as endocytosis and signal transduction. We have developed an assay to determine the fraction of internal versus plasma membrane caveolae. The GPI-anchored protein, alkaline phosphatase, was clustered in caveolae after antibody-induced crosslinking at low temperature and then, after various treatments, the relative amount of alkaline phosphatase on the cell surface was determined. Using this assay we were able to show a time- and temperature-dependent decrease in cell-surface alkaline phosphatase activity which was dependent on antibody-induced clustering. The decrease in cell surface alkaline phosphatase activity was greatly accelerated by the phosphatase inhibitor, okadaic acid, but not by a protein kinase C activator. Internalization of clustered alkaline phosphatase in the presence or absence of okadaic acid was blocked by cytochalasin D and by the kinase inhibitor staurosporine. Electron microscopy confirmed that okadaic acid induced removal of caveolae from the cell surface. In the presence of hypertonic medium this was followed by the redistribution of groups of caveolae to the center of the cell close to the microtubule-organizing center. This process was reversible, blocked by cytochalasin D, and the centralization of the caveolar clusters was shown to be dependent on an intact microtubule network. Although the exact mechanism of internalization remains unknown, the results show that caveolae are dynamic structures which can be internalized into the cell. This process may be regulated by kinase activity and require an intact actin network.

scholarly journals Plasma membrane folds on the mast cell surface and their relationship to secretory activity.

1977 ◽  
Vol 74 (3) ◽  
pp. 690-697 ◽  
Author(s):  
S J Burwen ◽  
B H Satir
Keyword(s):  
Mast Cell ◽  
Plasma Membrane ◽  
Mast Cells ◽  
Cell Surface ◽  
Normal Population ◽  
Secretory Activity ◽  
Cell Type ◽  
Distinct Cell Type ◽  
Granule Membrane ◽  
Fold Formation

Changes in the surface morphology of secreting mast cells have been followed by scanning electron microscopy. Mast cells isolated from the rat peritoneal cavity have folds of plasma membrane that form snake-like ridges on their surfaces. Fold length varies considerably from cell to cell, whereas fold width and depth appear to remain relatively constant. To assess the possible relationship between secretory activity and surface folding, a seimquantitative method was used for measuring fold length in control and secreting populations. A positive correlation is found between secretion of histamine and the extent of membrane folds on the mast cell surface. The source of the membrane required for fold formation is probably secretory granule membrane incorporated into the plasma membranene as a result of exocytosis. Furthermore, a distinct cell type devoid of surface folds, designated as a raspberry-type cell, is found to occur as an integral part of a normal population of mast cells. This cell type is resistant to stimulation by polymyxin.

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