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Sorting of small infectious virus particles by flow virometry reveals distinct infectivity profiles.

Gaudin R, Barteneva NS - Nat Commun (2015)

Bottom Line: Historically, averaged bulk analysis of viral particles has been the primary method to quantitatively investigate these parameters, though this neglects heterogeneity within populations.Our study highlights significant differences in particle infectivity according to its nature, the type of producer cells and the lipid membrane composition at the budding site.Together, our results present the flow virometry assay as a powerful and versatile tool to define virus particle profiles.

View Article: PubMed Central - PubMed

Affiliation: 1] Program in Cellular and Molecular Medicine, Boston Children's Hospital, 200 Longwood Avenue, Boston, Massachusetts 02115, USA [2] Department of Cell Biology, Harvard Medical School, 200 Longwood Avenue, Boston, Massachusetts 02115, USA.

ABSTRACT
The nature and concentration of lipids and proteins at the surface of viruses are essential parameters for determining particle infectiveness. Historically, averaged bulk analysis of viral particles has been the primary method to quantitatively investigate these parameters, though this neglects heterogeneity within populations. Here we analyse the properties of Junin virus particles using a sensitive flow virometry assay and further sort virions while conserving their infectiveness. This method allows us to characterize the relationship between infectivity, virus size and RNA content and to compare particles secreted by Vero cells with those from physiologically relevant human primary macrophages. Our study highlights significant differences in particle infectivity according to its nature, the type of producer cells and the lipid membrane composition at the budding site. Together, our results present the flow virometry assay as a powerful and versatile tool to define virus particle profiles.

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Junin virus buds at CD9-enriched sites. JUNV-A647 viral particles stained with either a CD9 antibody conjugated to PE (a) or Cholera Toxin B (CTB) conjugated to FITC (b) were analyzed by flow virometry and plotted as a function of Alexa 647 GPC antibody staining. (c) JUNV particles stained with a GPC antibody coupled to an Alexa Fluor 647 as in Figure 1g, were adsorbed on glow discharged glass coverslip. Samples were blocked in 0.5% BSA in PBS for 30 min, incubated with mouse anti-CD9 antibody coupled to PE for 30 min and then washed extensively in 0.5% BSA in PBS. Coverslips were mounted on slides and image by spinning disk confocal microscopy. The micrograph represents CD9-PE (red) and JUNV-A647 (cyan) and the red channel was shifted by 6 pixels up in order to better appreciate colocalization of the two fluorophores. Over 500 JUNV-A647 particles were counted and Pearson correlation with CD9-PE was evaluated at 0.77 +/− 0.1. Bar = 5 μm. (d) Vero cells infected with JUNV for 24 hrs were fixed, permeabilized and stained with a CD9 antibody conjugated to PE and the GPC specific GD01 antibody conjugated to Alexa Fluor 488. Images were acquired by spinning disk confocal microscopy and represent the plasma membrane of an infected cell. The images show CD9 staining (upper panel), GPC staining (middle panel) and the overlay of the two channels (bottom panel). The pink arrows highlight GPC positive spots, likely corresponding to budding events that are enriched in CD9. Bar = 5 μm.
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Figure 5: Junin virus buds at CD9-enriched sites. JUNV-A647 viral particles stained with either a CD9 antibody conjugated to PE (a) or Cholera Toxin B (CTB) conjugated to FITC (b) were analyzed by flow virometry and plotted as a function of Alexa 647 GPC antibody staining. (c) JUNV particles stained with a GPC antibody coupled to an Alexa Fluor 647 as in Figure 1g, were adsorbed on glow discharged glass coverslip. Samples were blocked in 0.5% BSA in PBS for 30 min, incubated with mouse anti-CD9 antibody coupled to PE for 30 min and then washed extensively in 0.5% BSA in PBS. Coverslips were mounted on slides and image by spinning disk confocal microscopy. The micrograph represents CD9-PE (red) and JUNV-A647 (cyan) and the red channel was shifted by 6 pixels up in order to better appreciate colocalization of the two fluorophores. Over 500 JUNV-A647 particles were counted and Pearson correlation with CD9-PE was evaluated at 0.77 +/− 0.1. Bar = 5 μm. (d) Vero cells infected with JUNV for 24 hrs were fixed, permeabilized and stained with a CD9 antibody conjugated to PE and the GPC specific GD01 antibody conjugated to Alexa Fluor 488. Images were acquired by spinning disk confocal microscopy and represent the plasma membrane of an infected cell. The images show CD9 staining (upper panel), GPC staining (middle panel) and the overlay of the two channels (bottom panel). The pink arrows highlight GPC positive spots, likely corresponding to budding events that are enriched in CD9. Bar = 5 μm.

Mentions: Since FVA enables us to define specific virions properties, we next investigated how the cholesterol content at the plasma membrane influences JUNV budding and infectivity. Cholesterol is an important component of cellular membranes that favors protein cluster microdomains by rigidifying lipid bilayers and could therefore influence GPC and cellular components incorporation into viral particles. We first examined the viral particle surface distributions of the Tetraspanin CD9, marker of the Tetraspanin-enriched microdomains (TEM) and the Cholera toxin subunit B (CTB), which binds to GM1 gangliosides and localizes primarily to lipid rafts. Both of these plasma membrane subdomains are enriched in cholesterol19,20. FVA analysis of JUNV-A647 particles co-stained with either an anti-CD9 antibody conjugated to Phycoerythrin (CD9-PE) or a CTB recombinant protein coupled to Fluorescein isothiocyanate (CTB-FITC) revealed that JUNV particles contain high levels of CD9 and CTB (Fig. 5a,b). Single staining controls showed no crosstalk between fluorophores (Supplementary Fig. 4a,b). Immunofluorescence and confocal spinning disk microscopy of JUNV-A647 particles spread on glass and stained with CD9-PE antibody confirm that CD9 is indeed enriched at the surface of JUNV particles (Fig. 5c; Pearson correlation coefficient: 0.77 − 0.1). Moreover, staining of infected cells with GPC and CD9 antibodies also illustrate that clusters of GPC at the plasma membrane are found in CD9-enriched regions (pink arrows in Fig. 5d). These results further suggest that virions bud at specialized regions of the plasma membrane.


Sorting of small infectious virus particles by flow virometry reveals distinct infectivity profiles.

Gaudin R, Barteneva NS - Nat Commun (2015)

Junin virus buds at CD9-enriched sites. JUNV-A647 viral particles stained with either a CD9 antibody conjugated to PE (a) or Cholera Toxin B (CTB) conjugated to FITC (b) were analyzed by flow virometry and plotted as a function of Alexa 647 GPC antibody staining. (c) JUNV particles stained with a GPC antibody coupled to an Alexa Fluor 647 as in Figure 1g, were adsorbed on glow discharged glass coverslip. Samples were blocked in 0.5% BSA in PBS for 30 min, incubated with mouse anti-CD9 antibody coupled to PE for 30 min and then washed extensively in 0.5% BSA in PBS. Coverslips were mounted on slides and image by spinning disk confocal microscopy. The micrograph represents CD9-PE (red) and JUNV-A647 (cyan) and the red channel was shifted by 6 pixels up in order to better appreciate colocalization of the two fluorophores. Over 500 JUNV-A647 particles were counted and Pearson correlation with CD9-PE was evaluated at 0.77 +/− 0.1. Bar = 5 μm. (d) Vero cells infected with JUNV for 24 hrs were fixed, permeabilized and stained with a CD9 antibody conjugated to PE and the GPC specific GD01 antibody conjugated to Alexa Fluor 488. Images were acquired by spinning disk confocal microscopy and represent the plasma membrane of an infected cell. The images show CD9 staining (upper panel), GPC staining (middle panel) and the overlay of the two channels (bottom panel). The pink arrows highlight GPC positive spots, likely corresponding to budding events that are enriched in CD9. Bar = 5 μm.
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Figure 5: Junin virus buds at CD9-enriched sites. JUNV-A647 viral particles stained with either a CD9 antibody conjugated to PE (a) or Cholera Toxin B (CTB) conjugated to FITC (b) were analyzed by flow virometry and plotted as a function of Alexa 647 GPC antibody staining. (c) JUNV particles stained with a GPC antibody coupled to an Alexa Fluor 647 as in Figure 1g, were adsorbed on glow discharged glass coverslip. Samples were blocked in 0.5% BSA in PBS for 30 min, incubated with mouse anti-CD9 antibody coupled to PE for 30 min and then washed extensively in 0.5% BSA in PBS. Coverslips were mounted on slides and image by spinning disk confocal microscopy. The micrograph represents CD9-PE (red) and JUNV-A647 (cyan) and the red channel was shifted by 6 pixels up in order to better appreciate colocalization of the two fluorophores. Over 500 JUNV-A647 particles were counted and Pearson correlation with CD9-PE was evaluated at 0.77 +/− 0.1. Bar = 5 μm. (d) Vero cells infected with JUNV for 24 hrs were fixed, permeabilized and stained with a CD9 antibody conjugated to PE and the GPC specific GD01 antibody conjugated to Alexa Fluor 488. Images were acquired by spinning disk confocal microscopy and represent the plasma membrane of an infected cell. The images show CD9 staining (upper panel), GPC staining (middle panel) and the overlay of the two channels (bottom panel). The pink arrows highlight GPC positive spots, likely corresponding to budding events that are enriched in CD9. Bar = 5 μm.
Mentions: Since FVA enables us to define specific virions properties, we next investigated how the cholesterol content at the plasma membrane influences JUNV budding and infectivity. Cholesterol is an important component of cellular membranes that favors protein cluster microdomains by rigidifying lipid bilayers and could therefore influence GPC and cellular components incorporation into viral particles. We first examined the viral particle surface distributions of the Tetraspanin CD9, marker of the Tetraspanin-enriched microdomains (TEM) and the Cholera toxin subunit B (CTB), which binds to GM1 gangliosides and localizes primarily to lipid rafts. Both of these plasma membrane subdomains are enriched in cholesterol19,20. FVA analysis of JUNV-A647 particles co-stained with either an anti-CD9 antibody conjugated to Phycoerythrin (CD9-PE) or a CTB recombinant protein coupled to Fluorescein isothiocyanate (CTB-FITC) revealed that JUNV particles contain high levels of CD9 and CTB (Fig. 5a,b). Single staining controls showed no crosstalk between fluorophores (Supplementary Fig. 4a,b). Immunofluorescence and confocal spinning disk microscopy of JUNV-A647 particles spread on glass and stained with CD9-PE antibody confirm that CD9 is indeed enriched at the surface of JUNV particles (Fig. 5c; Pearson correlation coefficient: 0.77 − 0.1). Moreover, staining of infected cells with GPC and CD9 antibodies also illustrate that clusters of GPC at the plasma membrane are found in CD9-enriched regions (pink arrows in Fig. 5d). These results further suggest that virions bud at specialized regions of the plasma membrane.

Bottom Line: Historically, averaged bulk analysis of viral particles has been the primary method to quantitatively investigate these parameters, though this neglects heterogeneity within populations.Our study highlights significant differences in particle infectivity according to its nature, the type of producer cells and the lipid membrane composition at the budding site.Together, our results present the flow virometry assay as a powerful and versatile tool to define virus particle profiles.

View Article: PubMed Central - PubMed

Affiliation: 1] Program in Cellular and Molecular Medicine, Boston Children's Hospital, 200 Longwood Avenue, Boston, Massachusetts 02115, USA [2] Department of Cell Biology, Harvard Medical School, 200 Longwood Avenue, Boston, Massachusetts 02115, USA.

ABSTRACT
The nature and concentration of lipids and proteins at the surface of viruses are essential parameters for determining particle infectiveness. Historically, averaged bulk analysis of viral particles has been the primary method to quantitatively investigate these parameters, though this neglects heterogeneity within populations. Here we analyse the properties of Junin virus particles using a sensitive flow virometry assay and further sort virions while conserving their infectiveness. This method allows us to characterize the relationship between infectivity, virus size and RNA content and to compare particles secreted by Vero cells with those from physiologically relevant human primary macrophages. Our study highlights significant differences in particle infectivity according to its nature, the type of producer cells and the lipid membrane composition at the budding site. Together, our results present the flow virometry assay as a powerful and versatile tool to define virus particle profiles.

Show MeSH
Related in: MedlinePlus