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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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GM3 containing AVNs are sequestered in peripheral cellular regions in HeLa/CD169 cells. (a–d) Darkfield images of HeLa/CD169 cells 20h after an initial 10 min exposure to GM3 containing (a–b) AVN1 or (c–d) AVN2. (e–f) Corresponding fluorescence images of HeLa/CD169 with GM3 containing AVN2 after nucleus (blue) and Lysotracker (red) staining. The AVNs show an enrichment in regions that do not costain with the Lysotracker. (g) Darkfield, (h) CD169-mCherry fluorescent image and (i) overlay of GM3-AVN2 on HeLa/CD169-mCherry cells 20h after incubation, which confirms increased CD169 concentrations at locations of AVN2 enrichment. The images in (a–i) show representative cells from five independent experiments. (j–l) Overlaid darkfield (green) and fluorescence Lysotracker images (red) of HeLa/CD169 cells after (j) 4h of incubation with EGF functionalized 80 nm Au NPs, (k) 20h after incubation with pegylated 80 nm Au NPs, or (l) 20h after 10min incubation with fluorescently labeled GM3-liposomes. Scale bars are 10µm.
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Figure 6: GM3 containing AVNs are sequestered in peripheral cellular regions in HeLa/CD169 cells. (a–d) Darkfield images of HeLa/CD169 cells 20h after an initial 10 min exposure to GM3 containing (a–b) AVN1 or (c–d) AVN2. (e–f) Corresponding fluorescence images of HeLa/CD169 with GM3 containing AVN2 after nucleus (blue) and Lysotracker (red) staining. The AVNs show an enrichment in regions that do not costain with the Lysotracker. (g) Darkfield, (h) CD169-mCherry fluorescent image and (i) overlay of GM3-AVN2 on HeLa/CD169-mCherry cells 20h after incubation, which confirms increased CD169 concentrations at locations of AVN2 enrichment. The images in (a–i) show representative cells from five independent experiments. (j–l) Overlaid darkfield (green) and fluorescence Lysotracker images (red) of HeLa/CD169 cells after (j) 4h of incubation with EGF functionalized 80 nm Au NPs, (k) 20h after incubation with pegylated 80 nm Au NPs, or (l) 20h after 10min incubation with fluorescently labeled GM3-liposomes. Scale bars are 10µm.

Mentions: We included a comparison of the spectral content of AVNs and cellular background by analyzing the relative intensity distribution on the red (R), green (G), and blue (B) color channels of the digital camera used to acquire the cell images. While the intensity of the cellular background is equally distributed across the R, G, B channels, the relative contributions from the R and G channels are locally increased in areas containing AVNs. We conclude that, due to their spectral characteristics and their high intensity (see insets), AVNs built around an 80 nm Au core can be differentiated with high fidelity even from grainy background in the investigated single cell layer. We took advantage of the large optical cross-sections of GM3 functionalized AVNs to determine their spatial distribution in HeLa/CD169 cells 20 h after initial AVN binding. These experiments revealed a characteristic spatial redistribution of the AVNs from an initial random distribution to a preferential spatial enrichment in discrete spots at the cell periphery. The representative darkfield images of GM3 containing AVN1 (Fig. 6a – b) and AVN2 (Fig. 6c – d) treated HeLa/CD169 cells acquired 20 h after AVN exposure illustrate the formation of distinct NP clusters at the cell periphery. In the case of AVN2 we also performed fluorescence staining experiments to image the acidified membrane compartments and the cell nuclei (Fig. 6e – f). The correlated darkfield/fluorescence images reveal that the locations of high AVN concentration do not co-localize with Lysotracker. The preferential localization of AVN clusters at the HeLa/CD169 cell periphery in Lysotracker negative compartments excludes conventional lysosomal compartments as origin of the large-scale AVN clustering, similar to the localization pattern observed with HIV Gag-eGFP VLPs. As in the case of the VLPs, we observed this phenotype in approximately 1/3 of all investigated cells. We attribute the variability in the cellular response to AVNs and VLPs to the intrinsic heterogeneity of immortalized cancer cell lines,58,59 as well as to variations in the CD169 expression level on the single cell level. The observation of similar spatio-temporal distributions for both AVN1 and AVN2 implies that the intact lipid bilayer membrane of AVN1 is not required to trigger GM3-CD169-mediated cellular processes, but that the presentation of GM3 in a single leaflet tethered to a solid core, as is the case for AVN2, is sufficient to successfully reproduce HIV Gag VLP behavior in HeLa/CD169 cells.


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)

GM3 containing AVNs are sequestered in peripheral cellular regions in HeLa/CD169 cells. (a–d) Darkfield images of HeLa/CD169 cells 20h after an initial 10 min exposure to GM3 containing (a–b) AVN1 or (c–d) AVN2. (e–f) Corresponding fluorescence images of HeLa/CD169 with GM3 containing AVN2 after nucleus (blue) and Lysotracker (red) staining. The AVNs show an enrichment in regions that do not costain with the Lysotracker. (g) Darkfield, (h) CD169-mCherry fluorescent image and (i) overlay of GM3-AVN2 on HeLa/CD169-mCherry cells 20h after incubation, which confirms increased CD169 concentrations at locations of AVN2 enrichment. The images in (a–i) show representative cells from five independent experiments. (j–l) Overlaid darkfield (green) and fluorescence Lysotracker images (red) of HeLa/CD169 cells after (j) 4h of incubation with EGF functionalized 80 nm Au NPs, (k) 20h after incubation with pegylated 80 nm Au NPs, or (l) 20h after 10min incubation with fluorescently labeled GM3-liposomes. Scale bars are 10µm.
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Figure 6: GM3 containing AVNs are sequestered in peripheral cellular regions in HeLa/CD169 cells. (a–d) Darkfield images of HeLa/CD169 cells 20h after an initial 10 min exposure to GM3 containing (a–b) AVN1 or (c–d) AVN2. (e–f) Corresponding fluorescence images of HeLa/CD169 with GM3 containing AVN2 after nucleus (blue) and Lysotracker (red) staining. The AVNs show an enrichment in regions that do not costain with the Lysotracker. (g) Darkfield, (h) CD169-mCherry fluorescent image and (i) overlay of GM3-AVN2 on HeLa/CD169-mCherry cells 20h after incubation, which confirms increased CD169 concentrations at locations of AVN2 enrichment. The images in (a–i) show representative cells from five independent experiments. (j–l) Overlaid darkfield (green) and fluorescence Lysotracker images (red) of HeLa/CD169 cells after (j) 4h of incubation with EGF functionalized 80 nm Au NPs, (k) 20h after incubation with pegylated 80 nm Au NPs, or (l) 20h after 10min incubation with fluorescently labeled GM3-liposomes. Scale bars are 10µm.
Mentions: We included a comparison of the spectral content of AVNs and cellular background by analyzing the relative intensity distribution on the red (R), green (G), and blue (B) color channels of the digital camera used to acquire the cell images. While the intensity of the cellular background is equally distributed across the R, G, B channels, the relative contributions from the R and G channels are locally increased in areas containing AVNs. We conclude that, due to their spectral characteristics and their high intensity (see insets), AVNs built around an 80 nm Au core can be differentiated with high fidelity even from grainy background in the investigated single cell layer. We took advantage of the large optical cross-sections of GM3 functionalized AVNs to determine their spatial distribution in HeLa/CD169 cells 20 h after initial AVN binding. These experiments revealed a characteristic spatial redistribution of the AVNs from an initial random distribution to a preferential spatial enrichment in discrete spots at the cell periphery. The representative darkfield images of GM3 containing AVN1 (Fig. 6a – b) and AVN2 (Fig. 6c – d) treated HeLa/CD169 cells acquired 20 h after AVN exposure illustrate the formation of distinct NP clusters at the cell periphery. In the case of AVN2 we also performed fluorescence staining experiments to image the acidified membrane compartments and the cell nuclei (Fig. 6e – f). The correlated darkfield/fluorescence images reveal that the locations of high AVN concentration do not co-localize with Lysotracker. The preferential localization of AVN clusters at the HeLa/CD169 cell periphery in Lysotracker negative compartments excludes conventional lysosomal compartments as origin of the large-scale AVN clustering, similar to the localization pattern observed with HIV Gag-eGFP VLPs. As in the case of the VLPs, we observed this phenotype in approximately 1/3 of all investigated cells. We attribute the variability in the cellular response to AVNs and VLPs to the intrinsic heterogeneity of immortalized cancer cell lines,58,59 as well as to variations in the CD169 expression level on the single cell level. The observation of similar spatio-temporal distributions for both AVN1 and AVN2 implies that the intact lipid bilayer membrane of AVN1 is not required to trigger GM3-CD169-mediated cellular processes, but that the presentation of GM3 in a single leaflet tethered to a solid core, as is the case for AVN2, is sufficient to successfully reproduce HIV Gag VLP behavior in HeLa/CD169 cells.

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