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Vesicular stomatitis virus enters cells through vesicles incompletely coated with clathrin that depend upon actin for internalization.

Cureton DK, Massol RH, Saffarian S, Kirchhausen TL, Whelan SP - PLoS Pathog. (2009)

Bottom Line: The mechanisms by which viruses co-opt the clathrin machinery for efficient internalization remain uncertain.By analysis of multiple independent virus internalization events, we show that VSV induces the nucleation of clathrin for its uptake, rather than depending upon random capture by formation of a clathrin-coated pit.This work provides new mechanistic insights into the process of virus internalization as well as uptake of unconventional cargo by the clathrin-dependent endocytic machinery.

View Article: PubMed Central - PubMed

Affiliation: Departments of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts, United States of America.

ABSTRACT
Many viruses that enter cells by clathrin-dependent endocytosis are significantly larger than the dimensions of a typical clathrin-coated vesicle. The mechanisms by which viruses co-opt the clathrin machinery for efficient internalization remain uncertain. Here we examined how clathrin-coated vesicles accommodate vesicular stomatitis virus (VSV) during its entry into cells. Using high-resolution imaging of the internalization of single viral particles into cells expressing fluorescent clathrin and adaptor molecules, we show that VSV enters cells through partially clathrin-coated vesicles. We found that on average, virus-containing vesicles contain more clathrin and clathrin adaptor molecules than conventional vesicles, but this increase is insufficient to permit full coating of the vesicle. We further show that virus-containing vesicles depend upon the actin machinery for their internalization. Specifically, we found that components of the actin machinery are recruited to virus-containing vesicles, and chemical inhibition of actin polymerization trapped viral particles in vesicles at the plasma membrane. By analysis of multiple independent virus internalization events, we show that VSV induces the nucleation of clathrin for its uptake, rather than depending upon random capture by formation of a clathrin-coated pit. This work provides new mechanistic insights into the process of virus internalization as well as uptake of unconventional cargo by the clathrin-dependent endocytic machinery.

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AP-2 is a functional adapter for VSV internalization.(A) An image of BSC-1 cells (left) stably expressing σ2-eGFP (green) treated with non-targeting (NT) or μ2-adaptin siRNAs and exposed to Alexa 568 labeled tf (red). Images were acquired from the bottom and middle of the NT and μ2 siRNA-treated cells, respectively. Dashed white lines demark the cell boundaries. A series of kymograph views showing VSV (blue) internalization in siRNA treated cells (right). Note the lack of virus internalization in cells lacking σ2-eGFP. (B) A graph of the % of bound VSV particles that were internalized by 7 cells treated with the NT siRNA and 5 cells treated with the μ2 siRNA and defective for transferrin uptake. (C) A graph depicting the effect of μ2 depletion on VSV gene expression. Cells were doubly transfected with the indicated siRNAs and inoculated with rVSV-LUC at an MOI of 0.5. Virus particles were removed after 1 h, and luminescence was quantified at 4 h p.i. Values represent the mean+/−the standard deviation of 2 independent experiments.
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ppat-1000394-g002: AP-2 is a functional adapter for VSV internalization.(A) An image of BSC-1 cells (left) stably expressing σ2-eGFP (green) treated with non-targeting (NT) or μ2-adaptin siRNAs and exposed to Alexa 568 labeled tf (red). Images were acquired from the bottom and middle of the NT and μ2 siRNA-treated cells, respectively. Dashed white lines demark the cell boundaries. A series of kymograph views showing VSV (blue) internalization in siRNA treated cells (right). Note the lack of virus internalization in cells lacking σ2-eGFP. (B) A graph of the % of bound VSV particles that were internalized by 7 cells treated with the NT siRNA and 5 cells treated with the μ2 siRNA and defective for transferrin uptake. (C) A graph depicting the effect of μ2 depletion on VSV gene expression. Cells were doubly transfected with the indicated siRNAs and inoculated with rVSV-LUC at an MOI of 0.5. Virus particles were removed after 1 h, and luminescence was quantified at 4 h p.i. Values represent the mean+/−the standard deviation of 2 independent experiments.

Mentions: Using siRNA to target the μ2 subunit of AP-2 in cells constitutively expressing σ2-eGFP, we showed that AP-2 depletion completely inhibited clathrin-dependent VSV internalization (Figure 2). Loss of μ2 can be followed by disappearance of the σ2-eGFP signal from coated pits, as assembly of one AP-2 heterotetramer depends on all four of its subunits. As expected, cells lacking detectable σ2-eGFP (>95% of the population) failed to take up an AP-2 dependent cargo, transferrin, which remained trapped at the cell surface (Figure 2A, red signal). Moreover, inspection of 37 virus particles attached to 5 different cells depleted for AP-2 showed that VSV remained attached at the cell surface (Figure 2A, blue signal). By contrast, cells treated with a control siRNA displayed normal levels of σ2-eGFP, robust transferrin uptake, and maintained the capacity to internalize VSV (42/63 particles; 8 cells) (Figure 2A, B). In other cells, traces of AP-2 expression remained following siRNA treatment. Under such conditions, we found that low levels of AP-2 accumulated beneath particles, and that virus was internalized by clathrin (not shown). Thus, low levels of AP-2 are sufficient for clathrin-dependent virus uptake. Using a recombinant VSV that expresses firefly luciferase as a marker of infection (rVSV-LUC), we further show that μ2 depletion resulted in diminished infection of cells as indicted by a 65% reduction in luciferase activity (Figure 2C). These data demonstrate that AP-2 is essential for the clathrin-dependent uptake of VSV.


Vesicular stomatitis virus enters cells through vesicles incompletely coated with clathrin that depend upon actin for internalization.

Cureton DK, Massol RH, Saffarian S, Kirchhausen TL, Whelan SP - PLoS Pathog. (2009)

AP-2 is a functional adapter for VSV internalization.(A) An image of BSC-1 cells (left) stably expressing σ2-eGFP (green) treated with non-targeting (NT) or μ2-adaptin siRNAs and exposed to Alexa 568 labeled tf (red). Images were acquired from the bottom and middle of the NT and μ2 siRNA-treated cells, respectively. Dashed white lines demark the cell boundaries. A series of kymograph views showing VSV (blue) internalization in siRNA treated cells (right). Note the lack of virus internalization in cells lacking σ2-eGFP. (B) A graph of the % of bound VSV particles that were internalized by 7 cells treated with the NT siRNA and 5 cells treated with the μ2 siRNA and defective for transferrin uptake. (C) A graph depicting the effect of μ2 depletion on VSV gene expression. Cells were doubly transfected with the indicated siRNAs and inoculated with rVSV-LUC at an MOI of 0.5. Virus particles were removed after 1 h, and luminescence was quantified at 4 h p.i. Values represent the mean+/−the standard deviation of 2 independent experiments.
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Related In: Results  -  Collection

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ppat-1000394-g002: AP-2 is a functional adapter for VSV internalization.(A) An image of BSC-1 cells (left) stably expressing σ2-eGFP (green) treated with non-targeting (NT) or μ2-adaptin siRNAs and exposed to Alexa 568 labeled tf (red). Images were acquired from the bottom and middle of the NT and μ2 siRNA-treated cells, respectively. Dashed white lines demark the cell boundaries. A series of kymograph views showing VSV (blue) internalization in siRNA treated cells (right). Note the lack of virus internalization in cells lacking σ2-eGFP. (B) A graph of the % of bound VSV particles that were internalized by 7 cells treated with the NT siRNA and 5 cells treated with the μ2 siRNA and defective for transferrin uptake. (C) A graph depicting the effect of μ2 depletion on VSV gene expression. Cells were doubly transfected with the indicated siRNAs and inoculated with rVSV-LUC at an MOI of 0.5. Virus particles were removed after 1 h, and luminescence was quantified at 4 h p.i. Values represent the mean+/−the standard deviation of 2 independent experiments.
Mentions: Using siRNA to target the μ2 subunit of AP-2 in cells constitutively expressing σ2-eGFP, we showed that AP-2 depletion completely inhibited clathrin-dependent VSV internalization (Figure 2). Loss of μ2 can be followed by disappearance of the σ2-eGFP signal from coated pits, as assembly of one AP-2 heterotetramer depends on all four of its subunits. As expected, cells lacking detectable σ2-eGFP (>95% of the population) failed to take up an AP-2 dependent cargo, transferrin, which remained trapped at the cell surface (Figure 2A, red signal). Moreover, inspection of 37 virus particles attached to 5 different cells depleted for AP-2 showed that VSV remained attached at the cell surface (Figure 2A, blue signal). By contrast, cells treated with a control siRNA displayed normal levels of σ2-eGFP, robust transferrin uptake, and maintained the capacity to internalize VSV (42/63 particles; 8 cells) (Figure 2A, B). In other cells, traces of AP-2 expression remained following siRNA treatment. Under such conditions, we found that low levels of AP-2 accumulated beneath particles, and that virus was internalized by clathrin (not shown). Thus, low levels of AP-2 are sufficient for clathrin-dependent virus uptake. Using a recombinant VSV that expresses firefly luciferase as a marker of infection (rVSV-LUC), we further show that μ2 depletion resulted in diminished infection of cells as indicted by a 65% reduction in luciferase activity (Figure 2C). These data demonstrate that AP-2 is essential for the clathrin-dependent uptake of VSV.

Bottom Line: The mechanisms by which viruses co-opt the clathrin machinery for efficient internalization remain uncertain.By analysis of multiple independent virus internalization events, we show that VSV induces the nucleation of clathrin for its uptake, rather than depending upon random capture by formation of a clathrin-coated pit.This work provides new mechanistic insights into the process of virus internalization as well as uptake of unconventional cargo by the clathrin-dependent endocytic machinery.

View Article: PubMed Central - PubMed

Affiliation: Departments of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts, United States of America.

ABSTRACT
Many viruses that enter cells by clathrin-dependent endocytosis are significantly larger than the dimensions of a typical clathrin-coated vesicle. The mechanisms by which viruses co-opt the clathrin machinery for efficient internalization remain uncertain. Here we examined how clathrin-coated vesicles accommodate vesicular stomatitis virus (VSV) during its entry into cells. Using high-resolution imaging of the internalization of single viral particles into cells expressing fluorescent clathrin and adaptor molecules, we show that VSV enters cells through partially clathrin-coated vesicles. We found that on average, virus-containing vesicles contain more clathrin and clathrin adaptor molecules than conventional vesicles, but this increase is insufficient to permit full coating of the vesicle. We further show that virus-containing vesicles depend upon the actin machinery for their internalization. Specifically, we found that components of the actin machinery are recruited to virus-containing vesicles, and chemical inhibition of actin polymerization trapped viral particles in vesicles at the plasma membrane. By analysis of multiple independent virus internalization events, we show that VSV induces the nucleation of clathrin for its uptake, rather than depending upon random capture by formation of a clathrin-coated pit. This work provides new mechanistic insights into the process of virus internalization as well as uptake of unconventional cargo by the clathrin-dependent endocytic machinery.

Show MeSH
Related in: MedlinePlus