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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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SEM/FIB of HeLa/CD169 cells 20h after exposure to GM3 containing AVN2. (a) Darkfield image of a representative cell with AVN2 clusters. (b) SEM image of the same cell after drying and coating with a 5nm thin Au/Pd layer. (c) Magnified SEM image of the cell surface in the area of interest marked with a white square in (a–b). The magnified view shows AVN2 particles and clusters located on the cell surface. (d) After removal of ~10nm of cellular material through focused ion beam milling, more clustered (red arrow) and individual (green arrow) AVN2 particles emerge. (e) After an additional removal of ~60 nm, the AVN2 clusters (red arrow) and monomers (green arrow) are all completely excavated. (f) SEM image of the area of interest recorded with a tilt angle of 52° after removal of another ~20nm cellular material. No additional AVN2 are exposed. Scale bars are 5µm for (a–b), and 2µm for (c–f).
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Figure 8: SEM/FIB of HeLa/CD169 cells 20h after exposure to GM3 containing AVN2. (a) Darkfield image of a representative cell with AVN2 clusters. (b) SEM image of the same cell after drying and coating with a 5nm thin Au/Pd layer. (c) Magnified SEM image of the cell surface in the area of interest marked with a white square in (a–b). The magnified view shows AVN2 particles and clusters located on the cell surface. (d) After removal of ~10nm of cellular material through focused ion beam milling, more clustered (red arrow) and individual (green arrow) AVN2 particles emerge. (e) After an additional removal of ~60 nm, the AVN2 clusters (red arrow) and monomers (green arrow) are all completely excavated. (f) SEM image of the area of interest recorded with a tilt angle of 52° after removal of another ~20nm cellular material. No additional AVN2 are exposed. Scale bars are 5µm for (a–b), and 2µm for (c–f).

Mentions: The multimodal Au core of AVNs makes it possible to investigate the peripheral accumulation of AVNs observed in the optical microscope via scanning electron microscopy (SEM) and focused ion beam (FIB) milling.63 SEM detects the secondary electrons generated upon impact from a primary electron beam. Since the secondary electrons have a very short escape depth,64 SEM imaging is highly surface specific. Fig. 8 shows an optical darkfield (a) and correlated SEM (b) image of a representative HeLa/CD169 cell 20 h after addition of AVN2. The optical image indicates a large number of bound AVNs and their clustering in a peripheral cell area (white square in Fig. 8a). A SEM image of the entire cell is shown in Fig. 8b. Prior to any FIB milling the number of detected NPs in this area is low, and those NPs that are detected are located right at the cell edge where the cell is very thin (Fig. 8c). To obtain further information about the relative localization of the AVN clusters with regard to the cell membrane, we removed cellular material through FIB milling in the area under investigation. Fig. 8d and the magnified view show that already after the removal of only 10 nm of cellular material, additional AVN clusters (red arrows) as well as individual AVNs (green arrows) become detectable in areas that did not contain NPs in Fig. 8c. Some clusters are only faintly visible, indicating that they are still mostly contained in the cell matrix. An additional milling of 60 nm further excavates the large AVN clusters (Fig. 8e). A tilted view (52°) of the area after removal of approximately 90 nm of cellular material is included in Fig. 8f. When we milled even deeper, the AVNs were gradually removed and no additional AVNs were observed in the investigated cell section.


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)

SEM/FIB of HeLa/CD169 cells 20h after exposure to GM3 containing AVN2. (a) Darkfield image of a representative cell with AVN2 clusters. (b) SEM image of the same cell after drying and coating with a 5nm thin Au/Pd layer. (c) Magnified SEM image of the cell surface in the area of interest marked with a white square in (a–b). The magnified view shows AVN2 particles and clusters located on the cell surface. (d) After removal of ~10nm of cellular material through focused ion beam milling, more clustered (red arrow) and individual (green arrow) AVN2 particles emerge. (e) After an additional removal of ~60 nm, the AVN2 clusters (red arrow) and monomers (green arrow) are all completely excavated. (f) SEM image of the area of interest recorded with a tilt angle of 52° after removal of another ~20nm cellular material. No additional AVN2 are exposed. Scale bars are 5µm for (a–b), and 2µm for (c–f).
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Figure 8: SEM/FIB of HeLa/CD169 cells 20h after exposure to GM3 containing AVN2. (a) Darkfield image of a representative cell with AVN2 clusters. (b) SEM image of the same cell after drying and coating with a 5nm thin Au/Pd layer. (c) Magnified SEM image of the cell surface in the area of interest marked with a white square in (a–b). The magnified view shows AVN2 particles and clusters located on the cell surface. (d) After removal of ~10nm of cellular material through focused ion beam milling, more clustered (red arrow) and individual (green arrow) AVN2 particles emerge. (e) After an additional removal of ~60 nm, the AVN2 clusters (red arrow) and monomers (green arrow) are all completely excavated. (f) SEM image of the area of interest recorded with a tilt angle of 52° after removal of another ~20nm cellular material. No additional AVN2 are exposed. Scale bars are 5µm for (a–b), and 2µm for (c–f).
Mentions: The multimodal Au core of AVNs makes it possible to investigate the peripheral accumulation of AVNs observed in the optical microscope via scanning electron microscopy (SEM) and focused ion beam (FIB) milling.63 SEM detects the secondary electrons generated upon impact from a primary electron beam. Since the secondary electrons have a very short escape depth,64 SEM imaging is highly surface specific. Fig. 8 shows an optical darkfield (a) and correlated SEM (b) image of a representative HeLa/CD169 cell 20 h after addition of AVN2. The optical image indicates a large number of bound AVNs and their clustering in a peripheral cell area (white square in Fig. 8a). A SEM image of the entire cell is shown in Fig. 8b. Prior to any FIB milling the number of detected NPs in this area is low, and those NPs that are detected are located right at the cell edge where the cell is very thin (Fig. 8c). To obtain further information about the relative localization of the AVN clusters with regard to the cell membrane, we removed cellular material through FIB milling in the area under investigation. Fig. 8d and the magnified view show that already after the removal of only 10 nm of cellular material, additional AVN clusters (red arrows) as well as individual AVNs (green arrows) become detectable in areas that did not contain NPs in Fig. 8c. Some clusters are only faintly visible, indicating that they are still mostly contained in the cell matrix. An additional milling of 60 nm further excavates the large AVN clusters (Fig. 8e). A tilted view (52°) of the area after removal of approximately 90 nm of cellular material is included in Fig. 8f. When we milled even deeper, the AVNs were gradually removed and no additional AVNs were observed in the investigated cell section.

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