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Glycosphingolipid-functionalized nanoparticles recapitulate CD169-dependent HIV-1 uptake and trafficking in dendritic cells.

Yu X, Feizpour A, Ramirez NG, Wu L, Akiyama H, Xu F, Gummuluru S, Reinhard BM - Nat Commun (2014)

Bottom Line: This distribution is reminiscent of CD169-dependent HIV-1 sequestration in mature DCs.Our results highlight GM3-CD169 binding as a gp120-independent signal for sequestration and preservation of HIV-1 infectivity.They also indicate that plasmonic AVNs offer improved features over liposome-based systems and represent a versatile tool for probing specific virus-cell interactions.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and The Photonics Center, Boston University, Boston, Massachusetts 02215, USA.

ABSTRACT
Ganglioside GM3, a host-derived glycosphingolipid incorporated in the membrane of human immunodeficiency virus-1 (HIV-1) viral particles, mediates interactions between HIV-1 and Siglec1/CD169, a protein expressed on dendritic cells (DCs). Such interactions, which seem to be independent of viral envelope glycoprotein gp120, are poorly understood. Here we develop a model system consisting of self-assembled artificial virus nanoparticles (AVNs) that are free of viral glycoproteins or other host-derived glycolipids and glycoproteins. These plasmonic AVNs contain a membrane of defined composition wrapped around a solid metal core. GM3-containing AVNs are captured by CD169-expressing HeLa cells or mature DCs, and are sequestered within non-lysosomal tetraspanin-positive compartments. This distribution is reminiscent of CD169-dependent HIV-1 sequestration in mature DCs. Our results highlight GM3-CD169 binding as a gp120-independent signal for sequestration and preservation of HIV-1 infectivity. They also indicate that plasmonic AVNs offer improved features over liposome-based systems and represent a versatile tool for probing specific virus-cell interactions.

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Sequestration of GM3 containing AVN2 particles in mature DCs. (a) The color darkfield image of a representative DC after 1h of incubation shows a distinct AVN cluster in a peripheral position. The overlay of (b) monochromatic darkfield and (c) Lysotracker images in (d) shows that AVNs are not sequestered into lysosomes. (e) Color darkfield, (f) monochromatic darkfield, and (g) fluorescence images of DCs coincubated with Gag-eGFP VLPs for 1h. (h) The overlay of darkfield and fluorescence images confirms that areas enriched in AVNs and VLPs colocalize. (i) Color darkfield, (j) monochromatic darkfield and (k) fluorescence images of DCs incubated with AVNs and then fluorescently labeled for CD81. (l) The overlay images reveal that AVN containing compartments are associated with CD81. Scale bars are 5µm. All DC experiments were independently repeated with primary cells from at least two donors.
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Figure 7: Sequestration of GM3 containing AVN2 particles in mature DCs. (a) The color darkfield image of a representative DC after 1h of incubation shows a distinct AVN cluster in a peripheral position. The overlay of (b) monochromatic darkfield and (c) Lysotracker images in (d) shows that AVNs are not sequestered into lysosomes. (e) Color darkfield, (f) monochromatic darkfield, and (g) fluorescence images of DCs coincubated with Gag-eGFP VLPs for 1h. (h) The overlay of darkfield and fluorescence images confirms that areas enriched in AVNs and VLPs colocalize. (i) Color darkfield, (j) monochromatic darkfield and (k) fluorescence images of DCs incubated with AVNs and then fluorescently labeled for CD81. (l) The overlay images reveal that AVN containing compartments are associated with CD81. Scale bars are 5µm. All DC experiments were independently repeated with primary cells from at least two donors.

Mentions: Although HeLa/CD169 cells are a useful model system to test and calibrate AVNs, it is unclear to what degree the observed spatial segregation of AVNs in HeLa/CD169 cells is relevant in DCs that mediate HIV-1 trans-infection. To clarify this question, primary monocyte derived DCs were matured with E.coli lipopolysaccharides (LPS) and then incubated with AVNs. While binding of Gal-Cer or blank AVNs to mature DCs was very low (see Supplementary Fig. 6), GM3 containing AVNs bound efficiently to mature DCs. Fig. 7 shows darkfield and fluorescence images of mature DCs that were continuously incubated with GM3 containing AVN1 for 1h under culture conditions similar to those for HeLa/CD169 cells. We observed that GM3-AVNs captured by mature DCs were redistributed and collected in tightly focused spatial locations. For mature DCs, 1 h of incubation with AVNs was sufficient to result in a strongly polarized AVN distribution with large NP clusters preferentially located at the cell periphery (Fig. 7a). Darkfield and fluorescent Lysotracker costaining experiments (Fig. 7b – d) confirm that the AVN enriched compartments in mature DCs are distinct from lysosomal compartments since no colocalization was observed between AVNs and lysosomal marker. While in HeLa/CD169 cells, multiple smaller AVN clusters are frequently found within a single cell, in mature DCs we preferentially observed the formation of one single compartment highly enriched in AVNs. Although the data obtained with both mature DCs and HeLa/CD169 cells confirm the existence of a cellular process that results in a spatial coalescence of bound AVNs, CD169-mediated trafficking and localization of AVNs in peripheral non-lysosomal compartments occurs with faster kinetics and is more pronounced in mature DCs.


Glycosphingolipid-functionalized nanoparticles recapitulate CD169-dependent HIV-1 uptake and trafficking in dendritic cells.

Yu X, Feizpour A, Ramirez NG, Wu L, Akiyama H, Xu F, Gummuluru S, Reinhard BM - Nat Commun (2014)

Sequestration of GM3 containing AVN2 particles in mature DCs. (a) The color darkfield image of a representative DC after 1h of incubation shows a distinct AVN cluster in a peripheral position. The overlay of (b) monochromatic darkfield and (c) Lysotracker images in (d) shows that AVNs are not sequestered into lysosomes. (e) Color darkfield, (f) monochromatic darkfield, and (g) fluorescence images of DCs coincubated with Gag-eGFP VLPs for 1h. (h) The overlay of darkfield and fluorescence images confirms that areas enriched in AVNs and VLPs colocalize. (i) Color darkfield, (j) monochromatic darkfield and (k) fluorescence images of DCs incubated with AVNs and then fluorescently labeled for CD81. (l) The overlay images reveal that AVN containing compartments are associated with CD81. Scale bars are 5µm. All DC experiments were independently repeated with primary cells from at least two donors.
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Figure 7: Sequestration of GM3 containing AVN2 particles in mature DCs. (a) The color darkfield image of a representative DC after 1h of incubation shows a distinct AVN cluster in a peripheral position. The overlay of (b) monochromatic darkfield and (c) Lysotracker images in (d) shows that AVNs are not sequestered into lysosomes. (e) Color darkfield, (f) monochromatic darkfield, and (g) fluorescence images of DCs coincubated with Gag-eGFP VLPs for 1h. (h) The overlay of darkfield and fluorescence images confirms that areas enriched in AVNs and VLPs colocalize. (i) Color darkfield, (j) monochromatic darkfield and (k) fluorescence images of DCs incubated with AVNs and then fluorescently labeled for CD81. (l) The overlay images reveal that AVN containing compartments are associated with CD81. Scale bars are 5µm. All DC experiments were independently repeated with primary cells from at least two donors.
Mentions: Although HeLa/CD169 cells are a useful model system to test and calibrate AVNs, it is unclear to what degree the observed spatial segregation of AVNs in HeLa/CD169 cells is relevant in DCs that mediate HIV-1 trans-infection. To clarify this question, primary monocyte derived DCs were matured with E.coli lipopolysaccharides (LPS) and then incubated with AVNs. While binding of Gal-Cer or blank AVNs to mature DCs was very low (see Supplementary Fig. 6), GM3 containing AVNs bound efficiently to mature DCs. Fig. 7 shows darkfield and fluorescence images of mature DCs that were continuously incubated with GM3 containing AVN1 for 1h under culture conditions similar to those for HeLa/CD169 cells. We observed that GM3-AVNs captured by mature DCs were redistributed and collected in tightly focused spatial locations. For mature DCs, 1 h of incubation with AVNs was sufficient to result in a strongly polarized AVN distribution with large NP clusters preferentially located at the cell periphery (Fig. 7a). Darkfield and fluorescent Lysotracker costaining experiments (Fig. 7b – d) confirm that the AVN enriched compartments in mature DCs are distinct from lysosomal compartments since no colocalization was observed between AVNs and lysosomal marker. While in HeLa/CD169 cells, multiple smaller AVN clusters are frequently found within a single cell, in mature DCs we preferentially observed the formation of one single compartment highly enriched in AVNs. Although the data obtained with both mature DCs and HeLa/CD169 cells confirm the existence of a cellular process that results in a spatial coalescence of bound AVNs, CD169-mediated trafficking and localization of AVNs in peripheral non-lysosomal compartments occurs with faster kinetics and is more pronounced in mature DCs.

Bottom Line: This distribution is reminiscent of CD169-dependent HIV-1 sequestration in mature DCs.Our results highlight GM3-CD169 binding as a gp120-independent signal for sequestration and preservation of HIV-1 infectivity.They also indicate that plasmonic AVNs offer improved features over liposome-based systems and represent a versatile tool for probing specific virus-cell interactions.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and The Photonics Center, Boston University, Boston, Massachusetts 02215, USA.

ABSTRACT
Ganglioside GM3, a host-derived glycosphingolipid incorporated in the membrane of human immunodeficiency virus-1 (HIV-1) viral particles, mediates interactions between HIV-1 and Siglec1/CD169, a protein expressed on dendritic cells (DCs). Such interactions, which seem to be independent of viral envelope glycoprotein gp120, are poorly understood. Here we develop a model system consisting of self-assembled artificial virus nanoparticles (AVNs) that are free of viral glycoproteins or other host-derived glycolipids and glycoproteins. These plasmonic AVNs contain a membrane of defined composition wrapped around a solid metal core. GM3-containing AVNs are captured by CD169-expressing HeLa cells or mature DCs, and are sequestered within non-lysosomal tetraspanin-positive compartments. This distribution is reminiscent of CD169-dependent HIV-1 sequestration in mature DCs. Our results highlight GM3-CD169 binding as a gp120-independent signal for sequestration and preservation of HIV-1 infectivity. They also indicate that plasmonic AVNs offer improved features over liposome-based systems and represent a versatile tool for probing specific virus-cell interactions.

Show MeSH
Related in: MedlinePlus