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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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Related in: MedlinePlus

Live cell imaging of clathrin-dependent endocytosis of single VSV particles.(A) Split channel images of a single BSC-1 cell (left and also in Video S1) transiently expressing tom-LCa (grey) highlight 3 virus particles (blue, circled) during internalization by clathrin-coated pits (CCPs). Kymographs (right) of sections of the cell surface showing tom-LCa fluorescence over time for clathrin-coated vesicles (CCV) containing and lacking virus. (B) A graph of the % of virus particles captured by a CCP or internalized by a CCV was plotted for 146 particles that attached to 28 different cells during image acquisition. (C) A tile view of images of a BSC-1 cell co-expressing σ2-eGFP (green) and tom-LCa (red) cropped from a time-lapse movie (Video S2), showing VSV appearing at the cell surface (time, t = −18 s) relative to the point (t = 0) of clathrin detection above background. (D) A graph of the kinetics of AP-2 and clathrin recruitment to the CCPs of panel C. Fluorescence intensities were plotted relative to the time of clathrin detection, and are expressed as a % of the average maximum clathrin observed in all pits lacking virus. The points are a weighted average of fluorescence intensities calculated as described in Materials and Methods. (E) A graph of the average kinetics of LCa and σ2 recruitment to CCPs containing (right, from 4 cells) or lacking (left, from 2 of the 4 cells) virus. Average fluorescence intensity and time are expressed as a % relative to CCV lacking virus observed in the same cells. Fluorescence intensity was calculated at 8 equally-spaced intervals and is plotted+/−standard error. (F) A graph of the clathrin fluorescence relative to vesicle lifetime for CCPs lacking (open circles) or containing (blue) virus. Fluorescence was expressed as a % relative to the average maximum for tom-LCa in pits lacking VSV. The average lifetime for pits containing virus was 110+/−44 s (15 cells), which was statistically distinct (Student's t-test: p = 2e-12) to that for pits lacking virus (51+/−16 s from 8 of the same cells). The peak clathrin fluorescence intensities in pits containing and lacking virus was 155+/−69 and 100+/−34, respectively. The difference between these values is statistically significant (Student's t-test: p = 1e-6).
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ppat-1000394-g001: Live cell imaging of clathrin-dependent endocytosis of single VSV particles.(A) Split channel images of a single BSC-1 cell (left and also in Video S1) transiently expressing tom-LCa (grey) highlight 3 virus particles (blue, circled) during internalization by clathrin-coated pits (CCPs). Kymographs (right) of sections of the cell surface showing tom-LCa fluorescence over time for clathrin-coated vesicles (CCV) containing and lacking virus. (B) A graph of the % of virus particles captured by a CCP or internalized by a CCV was plotted for 146 particles that attached to 28 different cells during image acquisition. (C) A tile view of images of a BSC-1 cell co-expressing σ2-eGFP (green) and tom-LCa (red) cropped from a time-lapse movie (Video S2), showing VSV appearing at the cell surface (time, t = −18 s) relative to the point (t = 0) of clathrin detection above background. (D) A graph of the kinetics of AP-2 and clathrin recruitment to the CCPs of panel C. Fluorescence intensities were plotted relative to the time of clathrin detection, and are expressed as a % of the average maximum clathrin observed in all pits lacking virus. The points are a weighted average of fluorescence intensities calculated as described in Materials and Methods. (E) A graph of the average kinetics of LCa and σ2 recruitment to CCPs containing (right, from 4 cells) or lacking (left, from 2 of the 4 cells) virus. Average fluorescence intensity and time are expressed as a % relative to CCV lacking virus observed in the same cells. Fluorescence intensity was calculated at 8 equally-spaced intervals and is plotted+/−standard error. (F) A graph of the clathrin fluorescence relative to vesicle lifetime for CCPs lacking (open circles) or containing (blue) virus. Fluorescence was expressed as a % relative to the average maximum for tom-LCa in pits lacking VSV. The average lifetime for pits containing virus was 110+/−44 s (15 cells), which was statistically distinct (Student's t-test: p = 2e-12) to that for pits lacking virus (51+/−16 s from 8 of the same cells). The peak clathrin fluorescence intensities in pits containing and lacking virus was 155+/−69 and 100+/−34, respectively. The difference between these values is statistically significant (Student's t-test: p = 1e-6).

Mentions: To image the internalization of single VSV particles in live cells, we conjugated the fluorescent dye, Alexa Fluor 647 to purified virions. This labeling process did not significantly reduce viral titer as measured by plaque assay (Figure S1A). Examination of labeled VSV particles by spinning disc confocal fluorescent microscopy showed distinct, diffraction-limited puncta with a single Gaussian distribution of fluorescence intensities (Figure S1B). This result is consistent with a lack of aggregates and the presence of single virus particles. To examine how clathrin-coated pits internalize VSV, we infected BSC-1 cells stably expressing tomato-clathrin light chain A1 (tom-LCa) and acquired images from the bottom surface of cells using a spinning disc confocal microscope. Single VSV particles attached to cells, and over the 6–10 min. time course of 28 time-lapse videos, more than 90% (133/146) of the attached particles associated with tom-LCa (Figure 1A, B, S3). Moreover, the tom-LCa fluorescence signal increased over time and then abruptly disappeared as the vesicle uncoated (Figure 1A, C, Videos S1, S2). More than 70% (98/133) of the captured particles underwent rapid, directed movement shortly after disappearance of the clathrin signal, indicating intracellular transport of virus-containing vesicles (Videos S1, S2). The few particles that failed to associate with clathrin remained at the cell surface and did not exhibit this rapid, directed motion. Thus, clathrin-mediated endocytosis accounts for the uptake of most attached VSV particles.


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)

Live cell imaging of clathrin-dependent endocytosis of single VSV particles.(A) Split channel images of a single BSC-1 cell (left and also in Video S1) transiently expressing tom-LCa (grey) highlight 3 virus particles (blue, circled) during internalization by clathrin-coated pits (CCPs). Kymographs (right) of sections of the cell surface showing tom-LCa fluorescence over time for clathrin-coated vesicles (CCV) containing and lacking virus. (B) A graph of the % of virus particles captured by a CCP or internalized by a CCV was plotted for 146 particles that attached to 28 different cells during image acquisition. (C) A tile view of images of a BSC-1 cell co-expressing σ2-eGFP (green) and tom-LCa (red) cropped from a time-lapse movie (Video S2), showing VSV appearing at the cell surface (time, t = −18 s) relative to the point (t = 0) of clathrin detection above background. (D) A graph of the kinetics of AP-2 and clathrin recruitment to the CCPs of panel C. Fluorescence intensities were plotted relative to the time of clathrin detection, and are expressed as a % of the average maximum clathrin observed in all pits lacking virus. The points are a weighted average of fluorescence intensities calculated as described in Materials and Methods. (E) A graph of the average kinetics of LCa and σ2 recruitment to CCPs containing (right, from 4 cells) or lacking (left, from 2 of the 4 cells) virus. Average fluorescence intensity and time are expressed as a % relative to CCV lacking virus observed in the same cells. Fluorescence intensity was calculated at 8 equally-spaced intervals and is plotted+/−standard error. (F) A graph of the clathrin fluorescence relative to vesicle lifetime for CCPs lacking (open circles) or containing (blue) virus. Fluorescence was expressed as a % relative to the average maximum for tom-LCa in pits lacking VSV. The average lifetime for pits containing virus was 110+/−44 s (15 cells), which was statistically distinct (Student's t-test: p = 2e-12) to that for pits lacking virus (51+/−16 s from 8 of the same cells). The peak clathrin fluorescence intensities in pits containing and lacking virus was 155+/−69 and 100+/−34, respectively. The difference between these values is statistically significant (Student's t-test: p = 1e-6).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2667253&req=5

ppat-1000394-g001: Live cell imaging of clathrin-dependent endocytosis of single VSV particles.(A) Split channel images of a single BSC-1 cell (left and also in Video S1) transiently expressing tom-LCa (grey) highlight 3 virus particles (blue, circled) during internalization by clathrin-coated pits (CCPs). Kymographs (right) of sections of the cell surface showing tom-LCa fluorescence over time for clathrin-coated vesicles (CCV) containing and lacking virus. (B) A graph of the % of virus particles captured by a CCP or internalized by a CCV was plotted for 146 particles that attached to 28 different cells during image acquisition. (C) A tile view of images of a BSC-1 cell co-expressing σ2-eGFP (green) and tom-LCa (red) cropped from a time-lapse movie (Video S2), showing VSV appearing at the cell surface (time, t = −18 s) relative to the point (t = 0) of clathrin detection above background. (D) A graph of the kinetics of AP-2 and clathrin recruitment to the CCPs of panel C. Fluorescence intensities were plotted relative to the time of clathrin detection, and are expressed as a % of the average maximum clathrin observed in all pits lacking virus. The points are a weighted average of fluorescence intensities calculated as described in Materials and Methods. (E) A graph of the average kinetics of LCa and σ2 recruitment to CCPs containing (right, from 4 cells) or lacking (left, from 2 of the 4 cells) virus. Average fluorescence intensity and time are expressed as a % relative to CCV lacking virus observed in the same cells. Fluorescence intensity was calculated at 8 equally-spaced intervals and is plotted+/−standard error. (F) A graph of the clathrin fluorescence relative to vesicle lifetime for CCPs lacking (open circles) or containing (blue) virus. Fluorescence was expressed as a % relative to the average maximum for tom-LCa in pits lacking VSV. The average lifetime for pits containing virus was 110+/−44 s (15 cells), which was statistically distinct (Student's t-test: p = 2e-12) to that for pits lacking virus (51+/−16 s from 8 of the same cells). The peak clathrin fluorescence intensities in pits containing and lacking virus was 155+/−69 and 100+/−34, respectively. The difference between these values is statistically significant (Student's t-test: p = 1e-6).
Mentions: To image the internalization of single VSV particles in live cells, we conjugated the fluorescent dye, Alexa Fluor 647 to purified virions. This labeling process did not significantly reduce viral titer as measured by plaque assay (Figure S1A). Examination of labeled VSV particles by spinning disc confocal fluorescent microscopy showed distinct, diffraction-limited puncta with a single Gaussian distribution of fluorescence intensities (Figure S1B). This result is consistent with a lack of aggregates and the presence of single virus particles. To examine how clathrin-coated pits internalize VSV, we infected BSC-1 cells stably expressing tomato-clathrin light chain A1 (tom-LCa) and acquired images from the bottom surface of cells using a spinning disc confocal microscope. Single VSV particles attached to cells, and over the 6–10 min. time course of 28 time-lapse videos, more than 90% (133/146) of the attached particles associated with tom-LCa (Figure 1A, B, S3). Moreover, the tom-LCa fluorescence signal increased over time and then abruptly disappeared as the vesicle uncoated (Figure 1A, C, Videos S1, S2). More than 70% (98/133) of the captured particles underwent rapid, directed movement shortly after disappearance of the clathrin signal, indicating intracellular transport of virus-containing vesicles (Videos S1, S2). The few particles that failed to associate with clathrin remained at the cell surface and did not exhibit this rapid, directed motion. Thus, clathrin-mediated endocytosis accounts for the uptake of most attached VSV particles.

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