Microdomains of the C-type lectin DC-SIGN are portals for virus entry into dendritic cells

Cambi, Alessandra; Lange, Frank de; Maarseveen, Noortje M van; Nijhuis, Monique; Joosten, Ben; Dijk, E.M.H.P. van; Bakker, B.I. de; Fransen, J.A.M.; Bovée-Geurts, Petra H. M.; Leeuwen, Frank N. van; Hulst, Niek F. van; Figdor, Carl G.

doi:10.1083/jcb.200306112

Cited by 223 publications

(260 citation statements)

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“…39: 1923Immunol. -1928 Alessandra Cambi et al We and others previously demonstrated that on DC, DC-SIGN may reside in cholesterol-and glycosphingolipid-enriched lipid rafts [9,19]. In agreement with these observations, we show that lipid raft disruption upon extraction of plasma membrane cholesterol by methyl-b-cyclodextrin treatment affects DC-SIGNmediated binding to ligand-coated beads both in DC and in the CHO cells stably expressing DC-SIGN (CHO-DC-SIGN) (Fig.…”

Section: Short Communicationsupporting

confidence: 78%

“…Although DC-SIGN has been shown to bind to several viruses, its capacity to directly mediate viral entry has often been debated [7,8]. Recently, we demonstrated that on the plasma membrane of DC, DC-SIGN is organized in nanoclusters, some of which colocalize with lipid rafts, that specifically confer to the receptor its capacity of binding and internalizing viruses as well as Ag conjugated to fluorescent nanoparticles [9,10]. These findings were substantiated by a biophysical study by Neumann et al, who showed that these nanoclusters are enriched near the leading edge of living DC, but are preferentially endocytosed at lamellar sites posterior to the leading edge, suggesting a directed Binding of HIV-1 to DC-SIGN leads to non-fusogenic uptake of virions by DC, which is required for enhancement of T-cell infection [12] and is dependent on the cytoplasmic domain of the DC-SIGN molecule [13].…”

Section: Introductionmentioning

confidence: 99%

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The C‐type lectin DC‐SIGN internalizes soluble antigens and HIV‐1 virions via a clathrin‐dependent mechanism

et al. 2009

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Dendritic cells (DC), professional Ag-presenting cells located in mucosae and lymphoid organs, operate at the interface of innate and adaptive immunity and are likely the first cells to encounter invading HIV-1. Although the C-type lectin DC-Specific ICAM-3-grabbing nonintegrin (DC-SIGN) binds to several viruses, including HIV-1, its direct involvement in viral entry remains controversial. Despite its central role in DC function, little is known about the underlying molecular mechanism(s) of DC-SIGN-mediated Ag uptake. Here, we analyzed the early stages of DC-SIGN-mediated endocytosis and demonstrate that both membrane cholesterol and dynamin are required. Confocal microscopy and clathrin RNAi showed that DC-SIGN-mediated internalization occurs via clathrin-coated pits. Electron microscopy of ultrathin sections showed the involvement of DC-SIGN in clathrin-dependent HIV-1 internalization by DC. Currently, DC-specific C-type lectins are considered potential target in anti-tumor clinical trials. Detailed information about how different Ag are internalized via these receptors will facilitate the rational design of targeted therapeutic strategies.Key words: C-type lectin . DC . Endocytosis . Targeting . Virus Supporting Information available online IntroductionDendritic cells (DC) are professional Ag-presenting cells that operate at the interface of innate and adaptive immunity [1].Since DC are located in the mucosae and the lymphoid tissues, they are likely to be the first cells to encounter invading HIV-1 particles [2]. DC express several PRR that specifically recognize distinct pathogen-associated molecular patterns displayed on microbial surfaces including C-type lectin receptors (CLR) [3] and TLR [4].DC-specific ICAM-3-grabbing non-integrin (DC-SIGN; CD209) is a CLR initially described as an ICAM-3 binding protein [5] as well as a major HIV-1 receptor on DC [6]. In addition, DC-SIGN binds several other viruses as well as fungi, bacteria, and parasites [3]. Although DC-SIGN has been shown to bind to several viruses, its capacity to directly mediate viral entry has often been debated [7,8]. Recently, we demonstrated that on the plasma membrane of DC, DC-SIGN is organized in nanoclusters, some of which colocalize with lipid rafts, that specifically confer to the receptor its capacity of binding and internalizing viruses as well as Ag conjugated to fluorescent nanoparticles [9,10]. These findings were substantiated by a biophysical study by Neumann et al., who showed that these nanoclusters are enriched near the leading edge of living DC, but are preferentially endocytosed at lamellar sites posterior to the leading edge, suggesting a directed Binding of HIV-1 to DC-SIGN leads to non-fusogenic uptake of virions by DC, which is required for enhancement of T-cell infection [12] and is dependent on the cytoplasmic domain of the DC-SIGN molecule [13]. SHORT COMMUNICATIONAg internalized by DC-SIGN can be presented via both MHC class I and class II [14,15]. In addition, DC-SIGN has been shown to promote both MHC class I-a...

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Section: Short Communicationsupporting

confidence: 78%

Section: Introductionmentioning

confidence: 99%

The C‐type lectin DC‐SIGN internalizes soluble antigens and HIV‐1 virions via a clathrin‐dependent mechanism

et al. 2009

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“…However, despite its ubiquitous nature, GLUT-1 has not been shown to facilitate viral binding and entry of HTLV-1 into DCs. Surface molecules on immature DCs have been reported to organize into microdomains of about 200 nm in the lipid raft regions of the plasma membrane (Cambi et al, 2004). The aggregation of surface molecules and putative receptors lends credence to the possibility that HTLV-1 binding may involve the formation of multiple bonds between the virus and the host cell target.…”

Section: Discussionmentioning

confidence: 97%

A novel high throughput quantum dot-based fluorescence assay for quantitation of virus binding and attachment

Kampani

Quann

Ahuja

et al. 2007

Journal of Virological Methods

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Quantum dots (QDots) are fluorescent semiconductor nanocrystals with a narrow emission spectrum, high quantum yield, and excellent photostability. These unique properties of QDots have been utilized to develop a fluorescent binding assay using biotinylated human T cell leukemia virus type 1 (biot-HTLV-1) conjugated with streptavidin-coated Qdots that enabled both qualitative and quantitative analyses of viral binding. The specificity and linearity of the assay was demonstrated utilizing T cells, the primary HTLV-1-susceptible cell population. Furthermore, differential binding of HTLV-1 was analyzed in additional cell types of clinical relevance including primary CD4 + and CD8 + T cells, dendritic cells (DCs), monocytes, bone marrow progenitor cells, and epithelial cells. DCs exhibited maximum binding affinity when compared to other examined cell types except the Jurkat and SUP-T1 T cell lines. Finally, blocking antibodies directed against a putative HTLV-1 receptor on DCs; DC-SIGN (dendritic cell-specific ICAM-3-grabbing non-integrin), were utilized to study the inhibition of HTLV-1 binding to target cells. Overall, these results demonstrated that this novel high throughput assay can be utilized to study the binding of a biotinylated virus and has implications for screening of viral binding inhibitors as well as host membrane proteins that may serve as receptors for viral entry. KeywordsHTLV-1; quantum dot; viral binding assay 1.IntroductionViral binding and attachment to a host cell membrane, while seemingly simplistic, is a complex area of research for a wide range of viruses. It is often viewed as the first step in infection, whereby a virion is able to attach to a target cell, fuse to the cell membrane, and deliver the contents of the capsid to the cytoplasm of the newly infected cell. The exact mechanism of binding to a host cell varies between viruses and is usually determined by the composition of attachment proteins located within the viral and cellular membranes. The population of cells † A part of this work was presented during 7th International Symposium on NeuroVirology (ISNV), Philadelphia, PA, USA, May 30 - Street, Philadelphia, PA 19102, USA, Tel.: 215-762-8586; Fax: 215-762-1955, E-mail: pjain@drexelmed.edu, Web site: http://www.drexelmed.edu/ Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. (Dhawan et al., 1991;Inghirami et al., 1988). Occasionally, radioactive labels are also utilized for the quantitative estimation of binding (Hubbard, 2003). However, organic fluorophores conventionally used for labeling nucleotides and proteins have poor pho...

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“…Although NSOM offers advantages such as the ability to correlate spectroscopic information with topographic information, it perturbs the sample. Nevertheless, in biological applications NSOM has enabled the correlation of topographic information with single-molecule fluorescence data from live cell membranes (32,33).…”

Section: Biomolecular Interactions Beyond the Classical Diffraction Lmentioning

confidence: 99%

Exploring Life by Single-Molecule Fluorescence Spectroscopy

Neuweiler¹,

Sauer²

2005

Anal. Chem.

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onsidering all the effort scientists put into visualizing individual atoms and molecules, it is amazing that it only takes a standard fluorescence microscope with high collection efficiency and suitable filters to detect the fluorescence emission of a single fluorophore. In <20 years, several innovations and technical breakthroughs have brought us to the point where we can study matter on a molecular scale-a skill unimaginable 50 years ago. Parallel to these developments, biochemistry and molecular and cellular biology have grown from infancy into prominent disciplines. New, exciting insights into biomolecular structure, self-assembly, dynamics, and regulation within living systems are results of refined methodological and experimental developments in biochemistry and molecular biology.We can now directly visualize and track individual molecules in their native environment; this allows us to study biological systems on a molecular scale. For example, single-molecule techniques are used to visualize the enzymatic mechanisms of DNA polymerase, which is responsible for the replication of genetic information. DNA polymerase synthesizes a DNA strand by incorporating nucleotides while moving like a locomotive on the complementary DNA template. We no longer calculate incorporation rates by looking at average DNA lengths; rather, we directly track the individual enzyme that is incorporating the nucleotides. In addition, we gain new insights into the working mechanism and performance of the enzyme.To see the difference between single-molecule and ensemble experiments, imagine that you are at a large station observing hundreds of passengers arriving on an unknown number of trains. From such an observation, you cannot answer questions about the individual routes the trains took, how many passengers boarded the train at which stations and at what times, or how many stops each train made. You observe only an average and can conclude only that trains typically transport hundreds of passengers at a time. If single-molecule spectroscopy is used to monitor biological reactions, individual properties can

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Microdomains of the C-type lectin DC-SIGN are portals for virus entry into dendritic cells

Cited by 223 publications

References 46 publications

The C‐type lectin DC‐SIGN internalizes soluble antigens and HIV‐1 virions via a clathrin‐dependent mechanism

The C‐type lectin DC‐SIGN internalizes soluble antigens and HIV‐1 virions via a clathrin‐dependent mechanism

A novel high throughput quantum dot-based fluorescence assay for quantitation of virus binding and attachment

Exploring Life by Single-Molecule Fluorescence Spectroscopy

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