Limits...
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.

Show MeSH

Related in: MedlinePlus

Spatial redistribution of HIV Gag-eGFP VLPs after binding to HeLa/CD169 cells. The VLPs selectively bind to (a) HeLa/CD169 cells but not to (b) parental HeLa cells after 10min of incubation. (c–d) 20h after binding to HeLa/CD169, the initially randomly distributed VLPs are enriched in peripheral compartments that do not co-stain with Lysosome Tracker. Fluorescence images of (e) CD169-mCherry, (f) VLP, and (g) overlay 20h after initial incubation with HIV Gag-eGFP VLPs. The fluorescence data show a strong optical colocalization of CD169 and VLPs. The VLP segregation was confirmed in two independent imaging experiments. Scale bars are 5µm.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4109413&req=5

Figure 4: Spatial redistribution of HIV Gag-eGFP VLPs after binding to HeLa/CD169 cells. The VLPs selectively bind to (a) HeLa/CD169 cells but not to (b) parental HeLa cells after 10min of incubation. (c–d) 20h after binding to HeLa/CD169, the initially randomly distributed VLPs are enriched in peripheral compartments that do not co-stain with Lysosome Tracker. Fluorescence images of (e) CD169-mCherry, (f) VLP, and (g) overlay 20h after initial incubation with HIV Gag-eGFP VLPs. The fluorescence data show a strong optical colocalization of CD169 and VLPs. The VLP segregation was confirmed in two independent imaging experiments. Scale bars are 5µm.

Mentions: VLPs are currently the model systems of choice for investigating the viral glycoprotein-independent capture of HIV-1 particles,7–9 whose behavior defines logical benchmarks for the evaluation of our reverse engineering strategy. In a first round of calibration experiments, we evaluated the binding specificity of VLPs in HeLa cells transduced to constitutively express CD169 (HeLa/CD169). Cells were incubated for 10 min with 1×1010/mL VLPs in Dulbecco’s Modified Eagle Medium (DMEM), washed and transferred to the fluorescence microscope for an optical inspection of the binding. We found that VLPs bound efficiently to HeLa/CD169 (Fig. 4a) but not to CD169-negative parental HeLa cells (Fig. 4b). This selective binding confirms that VLP binding is CD169 specific. Previous studies by Puryear et al13 and Izquierdo-Useros et al7,9 have demonstrated that GM3-mediated interactions of VLPs and infectious HIV-1 particles with CD169 on mature DCs induced a polarized virus distribution that resulted in an accumulation of the particles in non-lysosomal focal spots with preferential localization at the cell periphery. This characteristic spatial distribution was typically observed within 1–2 hours after virus binding with the exact time depending on the particle concentration. Interestingly, we observed a similar clustering of VLPs in HeLa/CD169 only when the cells were cultured for additional time after exposure to the VLPs. While the VLPs are initially randomly distributed across the surface of HeLa/CD169 cells (Fig. 4a), they experience a spatial redistribution as a function of time. In approximately 1/3 of the cells, this redistribution culminates after 20 h in a strong local enrichment of VLPs in spatially confined spots. In good agreement with previous observations in DCs,7,9,13 the locations of VLP enrichment do not co-stain with Lysotracker, which is a marker for acidified intracellular compartments, and the VLP enriched spots are often located at the cell periphery (Fig. 4c – d). We augmented the VLP tracking with simultaneous spatial mapping of the CD169-mCherry fusion protein and observed strong enrichment of CD169-mCherry at the site of VLP clustering (Fig. 4e – g), consistent with a role of CD169 as the receptor for HIV Gag VLPs.


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)

Spatial redistribution of HIV Gag-eGFP VLPs after binding to HeLa/CD169 cells. The VLPs selectively bind to (a) HeLa/CD169 cells but not to (b) parental HeLa cells after 10min of incubation. (c–d) 20h after binding to HeLa/CD169, the initially randomly distributed VLPs are enriched in peripheral compartments that do not co-stain with Lysosome Tracker. Fluorescence images of (e) CD169-mCherry, (f) VLP, and (g) overlay 20h after initial incubation with HIV Gag-eGFP VLPs. The fluorescence data show a strong optical colocalization of CD169 and VLPs. The VLP segregation was confirmed in two independent imaging experiments. Scale bars are 5µm.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4109413&req=5

Figure 4: Spatial redistribution of HIV Gag-eGFP VLPs after binding to HeLa/CD169 cells. The VLPs selectively bind to (a) HeLa/CD169 cells but not to (b) parental HeLa cells after 10min of incubation. (c–d) 20h after binding to HeLa/CD169, the initially randomly distributed VLPs are enriched in peripheral compartments that do not co-stain with Lysosome Tracker. Fluorescence images of (e) CD169-mCherry, (f) VLP, and (g) overlay 20h after initial incubation with HIV Gag-eGFP VLPs. The fluorescence data show a strong optical colocalization of CD169 and VLPs. The VLP segregation was confirmed in two independent imaging experiments. Scale bars are 5µm.
Mentions: VLPs are currently the model systems of choice for investigating the viral glycoprotein-independent capture of HIV-1 particles,7–9 whose behavior defines logical benchmarks for the evaluation of our reverse engineering strategy. In a first round of calibration experiments, we evaluated the binding specificity of VLPs in HeLa cells transduced to constitutively express CD169 (HeLa/CD169). Cells were incubated for 10 min with 1×1010/mL VLPs in Dulbecco’s Modified Eagle Medium (DMEM), washed and transferred to the fluorescence microscope for an optical inspection of the binding. We found that VLPs bound efficiently to HeLa/CD169 (Fig. 4a) but not to CD169-negative parental HeLa cells (Fig. 4b). This selective binding confirms that VLP binding is CD169 specific. Previous studies by Puryear et al13 and Izquierdo-Useros et al7,9 have demonstrated that GM3-mediated interactions of VLPs and infectious HIV-1 particles with CD169 on mature DCs induced a polarized virus distribution that resulted in an accumulation of the particles in non-lysosomal focal spots with preferential localization at the cell periphery. This characteristic spatial distribution was typically observed within 1–2 hours after virus binding with the exact time depending on the particle concentration. Interestingly, we observed a similar clustering of VLPs in HeLa/CD169 only when the cells were cultured for additional time after exposure to the VLPs. While the VLPs are initially randomly distributed across the surface of HeLa/CD169 cells (Fig. 4a), they experience a spatial redistribution as a function of time. In approximately 1/3 of the cells, this redistribution culminates after 20 h in a strong local enrichment of VLPs in spatially confined spots. In good agreement with previous observations in DCs,7,9,13 the locations of VLP enrichment do not co-stain with Lysotracker, which is a marker for acidified intracellular compartments, and the VLP enriched spots are often located at the cell periphery (Fig. 4c – d). We augmented the VLP tracking with simultaneous spatial mapping of the CD169-mCherry fusion protein and observed strong enrichment of CD169-mCherry at the site of VLP clustering (Fig. 4e – g), consistent with a role of CD169 as the receptor for HIV Gag VLPs.

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