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

Characterization of AVNs. Zeta potential (ZP) and average hydrodynamic diameter of (a) AVN1 and (b) AVN2 without glycosphingolipids (Blank) or containing 3% Gal-Cer, or 3% GM3. The presented data were obtained from three independent experiments. Darkfield image, fluorescence image, overlay, and colocalization statistics (clockwise top left to bottom right) for (c) AVN1 and (d) AVN2. 1000 particles were evaluated for each colocalization statistics. Representative TEM image of (e) AVN1 and (f) AVN2. The insets in (e) and (f) contain magnified views of selected AVNs, which show a distinct corona formed by the self-assembled lipids around the NPs. Scale bars are 10µm in (c) and (d) and 20 nm in (e) and (f).
© Copyright Policy
Related In: Results  -  Collection

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

Figure 3: Characterization of AVNs. Zeta potential (ZP) and average hydrodynamic diameter of (a) AVN1 and (b) AVN2 without glycosphingolipids (Blank) or containing 3% Gal-Cer, or 3% GM3. The presented data were obtained from three independent experiments. Darkfield image, fluorescence image, overlay, and colocalization statistics (clockwise top left to bottom right) for (c) AVN1 and (d) AVN2. 1000 particles were evaluated for each colocalization statistics. Representative TEM image of (e) AVN1 and (f) AVN2. The insets in (e) and (f) contain magnified views of selected AVNs, which show a distinct corona formed by the self-assembled lipids around the NPs. Scale bars are 10µm in (c) and (d) and 20 nm in (e) and (f).

Mentions: We characterized the generated AVNs by measuring their hydrodynamic diameters and zeta potentials (Fig. 3a – b). The hydrodynamic radius, reported as the peak in the size distribution obtained through dynamic light scattering, for GM3 containing AVN1 is rhyd(AVN1)= 77.8 ± 5.3(s.d.) nm compared to rhyd(AVN2)= 50.7 ± 3.0(s.d.) nm for GM3 containing AVN2. The observed difference in rhyd between AVN1 and AVN2 is too large to be accounted for only by differences in the membrane shell. Instead, the measured size difference indicates some self-association of the 80 nm Au NPs in the case of AVN1. Consistent with an increased level of NP clustering,41,54,55 the UV-Vis spectra (see Supplementary Fig. 1) confirm a red-shift of the ensemble-averaged plasmon resonance wavelength of approximately 8 nm for AVN1 when compared with AVN2. Both of the AVNs have zeta potentials between −20 ~ −30 mV, which is close to the published value for HIV-1 particles of −20 mV under identical experimental conditions.52 Due to their resonant interaction with the incident light, the Au NPs used in this work are extraordinarily bright and have a characteristic green-orange color. Liposomes and other organic contaminations with comparable sizes, in contrast, are dim (see Supplementary Fig. 2) or appear as broadband scatterers, which makes them easily discernable from metal NPs. Correlated darkfield/fluorescence microscopy (see Supplementary Fig. 3) is, therefore, an appropriate method for validating successful lipid wrapping around the NPs. Fig. 3c and d show darkfield and fluorescence images of surface-immobilized AVN1 and AVN2 for representative preparations. Consistent with a successful formation of AVNs that contain both lipid and noble metal NP components, the images show > 90% colocalization of fluorescence and darkfield signals for all AVN preparations. The exact colocalization statistics for approximately 1000 particles of each type are summarized in the insets. Control experiments performed with pegylated Au NPs incubated with TopFluor cholesterol in the absence of lipids did not yield any measurable fluorescence signal (see Supplementary Fig. 4). High-resolution TEM images of AVN1 and AVN2 (Fig. 3e – f) show a distinct corona around the NPs, which is additional proof of successful membrane assembly around the NPs. For AVN2 the corona is 4–5 nm thin. Together with the small difference between rhyd(AVN2) and the hydrodynamic radius of the citrate stabilized Au NPs (rhyd = 49.0 ± 1.2(s.d.) nm), the thin corona indicates the addition of a single lipid layer to the octadecane thiol functionalized NPs in the AVN2 assembly process.


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)

Characterization of AVNs. Zeta potential (ZP) and average hydrodynamic diameter of (a) AVN1 and (b) AVN2 without glycosphingolipids (Blank) or containing 3% Gal-Cer, or 3% GM3. The presented data were obtained from three independent experiments. Darkfield image, fluorescence image, overlay, and colocalization statistics (clockwise top left to bottom right) for (c) AVN1 and (d) AVN2. 1000 particles were evaluated for each colocalization statistics. Representative TEM image of (e) AVN1 and (f) AVN2. The insets in (e) and (f) contain magnified views of selected AVNs, which show a distinct corona formed by the self-assembled lipids around the NPs. Scale bars are 10µm in (c) and (d) and 20 nm in (e) and (f).
© Copyright Policy
Related In: Results  -  Collection

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

Figure 3: Characterization of AVNs. Zeta potential (ZP) and average hydrodynamic diameter of (a) AVN1 and (b) AVN2 without glycosphingolipids (Blank) or containing 3% Gal-Cer, or 3% GM3. The presented data were obtained from three independent experiments. Darkfield image, fluorescence image, overlay, and colocalization statistics (clockwise top left to bottom right) for (c) AVN1 and (d) AVN2. 1000 particles were evaluated for each colocalization statistics. Representative TEM image of (e) AVN1 and (f) AVN2. The insets in (e) and (f) contain magnified views of selected AVNs, which show a distinct corona formed by the self-assembled lipids around the NPs. Scale bars are 10µm in (c) and (d) and 20 nm in (e) and (f).
Mentions: We characterized the generated AVNs by measuring their hydrodynamic diameters and zeta potentials (Fig. 3a – b). The hydrodynamic radius, reported as the peak in the size distribution obtained through dynamic light scattering, for GM3 containing AVN1 is rhyd(AVN1)= 77.8 ± 5.3(s.d.) nm compared to rhyd(AVN2)= 50.7 ± 3.0(s.d.) nm for GM3 containing AVN2. The observed difference in rhyd between AVN1 and AVN2 is too large to be accounted for only by differences in the membrane shell. Instead, the measured size difference indicates some self-association of the 80 nm Au NPs in the case of AVN1. Consistent with an increased level of NP clustering,41,54,55 the UV-Vis spectra (see Supplementary Fig. 1) confirm a red-shift of the ensemble-averaged plasmon resonance wavelength of approximately 8 nm for AVN1 when compared with AVN2. Both of the AVNs have zeta potentials between −20 ~ −30 mV, which is close to the published value for HIV-1 particles of −20 mV under identical experimental conditions.52 Due to their resonant interaction with the incident light, the Au NPs used in this work are extraordinarily bright and have a characteristic green-orange color. Liposomes and other organic contaminations with comparable sizes, in contrast, are dim (see Supplementary Fig. 2) or appear as broadband scatterers, which makes them easily discernable from metal NPs. Correlated darkfield/fluorescence microscopy (see Supplementary Fig. 3) is, therefore, an appropriate method for validating successful lipid wrapping around the NPs. Fig. 3c and d show darkfield and fluorescence images of surface-immobilized AVN1 and AVN2 for representative preparations. Consistent with a successful formation of AVNs that contain both lipid and noble metal NP components, the images show > 90% colocalization of fluorescence and darkfield signals for all AVN preparations. The exact colocalization statistics for approximately 1000 particles of each type are summarized in the insets. Control experiments performed with pegylated Au NPs incubated with TopFluor cholesterol in the absence of lipids did not yield any measurable fluorescence signal (see Supplementary Fig. 4). High-resolution TEM images of AVN1 and AVN2 (Fig. 3e – f) show a distinct corona around the NPs, which is additional proof of successful membrane assembly around the NPs. For AVN2 the corona is 4–5 nm thin. Together with the small difference between rhyd(AVN2) and the hydrodynamic radius of the citrate stabilized Au NPs (rhyd = 49.0 ± 1.2(s.d.) nm), the thin corona indicates the addition of a single lipid layer to the octadecane thiol functionalized NPs in the AVN2 assembly process.

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