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 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 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.

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.

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.

Differential Uptake of Functionalized Polystyrene Nanoparticles by Human Macrophages and a Monocytic Cell Line

ACS Nano ◽  
2011 ◽  
Vol 5 (3) ◽  
pp. 1657-1669 ◽  
Author(s):  
Oleg Lunov ◽  
Tatiana Syrovets ◽  
Cornelia Loos ◽  
Johanna Beil ◽  
Michael Delacher ◽  
...  
Keyword(s):  
Cell Line ◽  
Polystyrene Nanoparticles ◽  
Monocytic Cell ◽  
Monocytic Cell Line ◽  
Human Macrophages

scholarly journals Changes of cell-surface thiols and intracellular signaling in human monocytic cell line THP-1 treated with diphenylcyclopropenone

2010 ◽  
Vol 35 (6) ◽  
pp. 871-879 ◽  
Author(s):  
Morihiko Hirota ◽  
Akira Motoyama ◽  
Mie Suzuki ◽  
Masashi Yanagi ◽  
Masato Kitagaki ◽  
...  
Keyword(s):  
Cell Surface ◽  
Cell Line ◽  
Intracellular Signaling ◽  
Monocytic Cell ◽  
Monocytic Cell Line ◽  
Human Monocytic Cell Line ◽  
Human Monocytic Cell

Effect of SP-A and surfactant lipids on expression of cell surface markers in the THP-1 monocytic cell line

1997 ◽  
Vol 272 (6) ◽  
pp. L1070-L1077 ◽  
Author(s):  
S. G. Kremlev ◽  
D. S. Phelps
Keyword(s):  
Cell Surface ◽  
Cell Line ◽  
Fluorescent Antibody ◽  
Intercellular Adhesion Molecule ◽  
Surface Markers ◽  
Cell Surface Markers ◽  
Simultaneous Treatment ◽  
Monocytic Cell ◽  
Monocytic Cell Line ◽  
The Impact

Pulmonary surfactant and its lipid components inhibit cell proliferation and cytokine expression. Surfactant protein A (SP-A) can stimulate these same functions. We assessed the impact of SP-A and surfactant lipids on the expression of the cell surface markers, CD14, CD54 (intercellular adhesion molecule-1), and CD11b, by the human monocytic cell line THP-1 using fluorescent antibody staining and fluorescence-activated cell sorting. Under basal conditions CD14 and CD54 were undetectable, and CD11b was expressed at low levels. Incubation of the cells in 1,25(OH)2D3 alone, or with low doses of surfactant lipids, increased CD14, CD54, and CD11b. Expression was increased further by SP-A. However, the SP-A-induced increases in cell markers were blocked by simultaneous treatment with lipid. The results suggest that the ability of the macrophage to participate in an inflammatory response is enhanced by SP-A alone or by surfactant containing a higher than normal proportion of SP-A. They further suggest that the addition of lipids results in a phenotype less prone to initiate an inflammatory reaction.

Identification of Novel Receptors on Myeloma Cells and Monocytes That Contribute to Myeloma Tumor Proliferation and Angiogenesis.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2507-2507 ◽  
Author(s):  
Melinda S. Gordon ◽  
Hee Jin Lee ◽  
Richard A. Campbell ◽  
Haiming Chen ◽  
Benjamin Bonavida ◽  
...  
Keyword(s):  
Cell Surface ◽  
Cell Line ◽  
Solid Tumors ◽  
Peripheral Blood ◽  
Tumor Vascularization ◽  
Monocytic Cell ◽  
Monocytic Cell Line ◽  
Surface Receptors ◽  
Anaplastic Lymphoma ◽  
Cell Signals

Abstract Hyper-activation of multiple cell signaling pathways, including Akt, IRS-1, JAK/ STAT3, NF-kB, Wnt and MAPK, contribute to myeloma cell survival and/or proliferation, and myeloma tumor vascularization. We discovered the novel expression of two cell surface receptors that are able to activate these cell signals but were not previously known to be expressed by hematopoietic cells or to be involved in hematological malignancies. We analyzed bone marrow (BM) and peripheral blood (PB) mononuclear cells (MCs) from multiple myeloma (MM) patients and normal donors for the expression of new surface receptors by flow cytometry. We found that the malignant cells from myeloma patients (N = 10) and the MM cell lines RPMI 8226 and U266 express the receptor protein tyrosine phosphatase b/z (RPTPb/z). RPTPb/z contributes to the growth of solid tumors, including glioblastoma, neuroblastoma and melanoma by aberrant accumulation of b-catenin, leading to activation of Wnt, NF-kB and Akt-mediated cell signals. We interfered with RPTPb/z stimulation by its ligand, pleiotrophin (PTN), using specific anti-PTN antibody and showed significant inhibition of MM proliferation in vitro by MTT assay. Importantly, when this antibody was administered to MM tumor-bearing mice in vivo a significant decrease in tumor volume was detected. We have also discovered that peripheral blood monocytes from MM patients (N = 9) and normal donors (N = 7), and a monocytic cell line (THP-1), display untranslocated anaplastic lymphoma kinase (ALK) on the cell surface. Dysregulated ALK expression on a variety of solid tumors is associated with tumor growth, vascularization and metastasis. ALK+ monocytes and THP-1 cells, but not ALK− peripheral B or T lymphocytes or the ALK− monocytic cell line U937, respond to ALK stimulation by modulating hematopoietic genes and inducing the expression of endothelial genes as well as exhibiting changes in morphology typical of early vascular endothelial cells (Chen et al, 2004, Blood104: 1472). ALK cross-linking leads to activation of the signaling intermediates IRS-1, PI3′K/Akt, STAT3 and MAPK, that are known to play key roles in angiogenesis. Thus RPTPb/z and ALK, or their downstream signal mediators, could be important new molecular targets for anti-myeloma (RPTPb/z) or anti-angiogenic (ALK) therapy. We are defining the signaling pathway(s) leading from RPTPb/z stimulation to MM cellular proliferation and survival, and from ALK to tumor vascularization via monocytic transdifferentiation. These studies provide potential new targets that should lead to new therapeutic strategies for treating MM.

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