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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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Visual comparison of CCV formation and VSV entry.(A) Electron micrographs depicting successive stages of CCV formation (top) and VSV entry (bottom). Clathrin appears as an electron dense coat on the cytosolic side of the plasma membrane, and early stages of clathrin assembly are indistinguishable. Conventional pits then adopt a constricted U shape, while virus-containing structures form elongated clathrin-coated tubes. Pits lacking virus become fully coated and pinch off from the membrane as spherical vesicles, but viral pits mature into much larger, partially uncoated structures. In the indicated samples, cells were treated with 20 µM cytoD for 10 min., and incubated with rVSV at an M.O.I. of 5000 for 15 min. Cells were fixed and stained as described in Materials and Methods. (B) Model of VSV internalization by the clathrin and actin machinery. Conventional clathrin-coated vesicles (top) constitutively nucleate on the cell surface and grow by addition of clathrin and adaptor proteins until a fully-coated, constricted pit is formed. Pits are severed from the plasma membrane in a dynamin-dependent, but actin-independent manner, and the clathrin coat is rapidly disassembled by Hsc70 and auxilin. During clathrin-dependent internalization of VSV, pits preferentially form in close proximity to virus particles. The growing clathrin lattice imparts a curvature to the tip of the pit, but coat assembly ceases when the constricted edge of the pit meets the enclosed particle. The actin cytoskeletal machinery is then recruited by dynamin to drive further invagination of the particle. Dynamin, possibly in conjunction with actin, mediates fission of the virus-containing pit, and clathrin is uncoated.
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ppat-1000394-g008: Visual comparison of CCV formation and VSV entry.(A) Electron micrographs depicting successive stages of CCV formation (top) and VSV entry (bottom). Clathrin appears as an electron dense coat on the cytosolic side of the plasma membrane, and early stages of clathrin assembly are indistinguishable. Conventional pits then adopt a constricted U shape, while virus-containing structures form elongated clathrin-coated tubes. Pits lacking virus become fully coated and pinch off from the membrane as spherical vesicles, but viral pits mature into much larger, partially uncoated structures. In the indicated samples, cells were treated with 20 µM cytoD for 10 min., and incubated with rVSV at an M.O.I. of 5000 for 15 min. Cells were fixed and stained as described in Materials and Methods. (B) Model of VSV internalization by the clathrin and actin machinery. Conventional clathrin-coated vesicles (top) constitutively nucleate on the cell surface and grow by addition of clathrin and adaptor proteins until a fully-coated, constricted pit is formed. Pits are severed from the plasma membrane in a dynamin-dependent, but actin-independent manner, and the clathrin coat is rapidly disassembled by Hsc70 and auxilin. During clathrin-dependent internalization of VSV, pits preferentially form in close proximity to virus particles. The growing clathrin lattice imparts a curvature to the tip of the pit, but coat assembly ceases when the constricted edge of the pit meets the enclosed particle. The actin cytoskeletal machinery is then recruited by dynamin to drive further invagination of the particle. Dynamin, possibly in conjunction with actin, mediates fission of the virus-containing pit, and clathrin is uncoated.

Mentions: We previously established the direct relationship between coat size and clathrin or AP-2 fluorescent intensity associated with endocytic coated pits and vesicles [1]. We compared the amount of clathrin associated with vesicles containing and lacking VSV. On average, virus-containing vesicles contained 1.5-fold more clathrin, with a maximum of 2.5-fold more, and in some instances, less clathrin than vesicles lacking virus (Figure 1F). Although pits internalizing VSV tend to have more clathrin, the increase is insufficient to coat fully a vesicle containing a viral particle. The size of VSV particles is 180×70 nm, which could fit within a spherical vesicle with an internal radius of 90 nm, or within a prolate spheroid with a polar radius of 90 nm and an equatorial radius of 35 nm. The surface area of such vesicles is respectively 9 and 3 times greater than that of a conventional clathrin-coated vesicle with an internal radius of 30 nm. Thus to be completely coated with clathrin, virus-containing vesicles would require at least 3 times the amount of clathrin observed on a conventional vesicle. Our data are not consistent with this level of clathrin on vesicles internalizing VSV, which leads us to conclude that clathrin does not fully coat such vesicles. Further support for this conclusion is provided by electron micrographs that show VSV present within clathrin-coated structures that contain an elongated tubular neck that lacks the characteristic clathrin density (Figure 8A). These images are also consistent with earlier electron micrographs that show VSV entering cells where clathrin is apparent on only a portion of the vesicle [15],[16],[17]. However, it was uncertain from this earlier work as to whether the static EM images represent an intermediate stage of the clathrin assembly process during virus internalization. By examining this process in real time in live cells, our data provides compelling evidence that virus enters cells in vesicles that lack a full clathrin coat.


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)

Visual comparison of CCV formation and VSV entry.(A) Electron micrographs depicting successive stages of CCV formation (top) and VSV entry (bottom). Clathrin appears as an electron dense coat on the cytosolic side of the plasma membrane, and early stages of clathrin assembly are indistinguishable. Conventional pits then adopt a constricted U shape, while virus-containing structures form elongated clathrin-coated tubes. Pits lacking virus become fully coated and pinch off from the membrane as spherical vesicles, but viral pits mature into much larger, partially uncoated structures. In the indicated samples, cells were treated with 20 µM cytoD for 10 min., and incubated with rVSV at an M.O.I. of 5000 for 15 min. Cells were fixed and stained as described in Materials and Methods. (B) Model of VSV internalization by the clathrin and actin machinery. Conventional clathrin-coated vesicles (top) constitutively nucleate on the cell surface and grow by addition of clathrin and adaptor proteins until a fully-coated, constricted pit is formed. Pits are severed from the plasma membrane in a dynamin-dependent, but actin-independent manner, and the clathrin coat is rapidly disassembled by Hsc70 and auxilin. During clathrin-dependent internalization of VSV, pits preferentially form in close proximity to virus particles. The growing clathrin lattice imparts a curvature to the tip of the pit, but coat assembly ceases when the constricted edge of the pit meets the enclosed particle. The actin cytoskeletal machinery is then recruited by dynamin to drive further invagination of the particle. Dynamin, possibly in conjunction with actin, mediates fission of the virus-containing pit, and clathrin is uncoated.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1000394-g008: Visual comparison of CCV formation and VSV entry.(A) Electron micrographs depicting successive stages of CCV formation (top) and VSV entry (bottom). Clathrin appears as an electron dense coat on the cytosolic side of the plasma membrane, and early stages of clathrin assembly are indistinguishable. Conventional pits then adopt a constricted U shape, while virus-containing structures form elongated clathrin-coated tubes. Pits lacking virus become fully coated and pinch off from the membrane as spherical vesicles, but viral pits mature into much larger, partially uncoated structures. In the indicated samples, cells were treated with 20 µM cytoD for 10 min., and incubated with rVSV at an M.O.I. of 5000 for 15 min. Cells were fixed and stained as described in Materials and Methods. (B) Model of VSV internalization by the clathrin and actin machinery. Conventional clathrin-coated vesicles (top) constitutively nucleate on the cell surface and grow by addition of clathrin and adaptor proteins until a fully-coated, constricted pit is formed. Pits are severed from the plasma membrane in a dynamin-dependent, but actin-independent manner, and the clathrin coat is rapidly disassembled by Hsc70 and auxilin. During clathrin-dependent internalization of VSV, pits preferentially form in close proximity to virus particles. The growing clathrin lattice imparts a curvature to the tip of the pit, but coat assembly ceases when the constricted edge of the pit meets the enclosed particle. The actin cytoskeletal machinery is then recruited by dynamin to drive further invagination of the particle. Dynamin, possibly in conjunction with actin, mediates fission of the virus-containing pit, and clathrin is uncoated.
Mentions: We previously established the direct relationship between coat size and clathrin or AP-2 fluorescent intensity associated with endocytic coated pits and vesicles [1]. We compared the amount of clathrin associated with vesicles containing and lacking VSV. On average, virus-containing vesicles contained 1.5-fold more clathrin, with a maximum of 2.5-fold more, and in some instances, less clathrin than vesicles lacking virus (Figure 1F). Although pits internalizing VSV tend to have more clathrin, the increase is insufficient to coat fully a vesicle containing a viral particle. The size of VSV particles is 180×70 nm, which could fit within a spherical vesicle with an internal radius of 90 nm, or within a prolate spheroid with a polar radius of 90 nm and an equatorial radius of 35 nm. The surface area of such vesicles is respectively 9 and 3 times greater than that of a conventional clathrin-coated vesicle with an internal radius of 30 nm. Thus to be completely coated with clathrin, virus-containing vesicles would require at least 3 times the amount of clathrin observed on a conventional vesicle. Our data are not consistent with this level of clathrin on vesicles internalizing VSV, which leads us to conclude that clathrin does not fully coat such vesicles. Further support for this conclusion is provided by electron micrographs that show VSV present within clathrin-coated structures that contain an elongated tubular neck that lacks the characteristic clathrin density (Figure 8A). These images are also consistent with earlier electron micrographs that show VSV entering cells where clathrin is apparent on only a portion of the vesicle [15],[16],[17]. However, it was uncertain from this earlier work as to whether the static EM images represent an intermediate stage of the clathrin assembly process during virus internalization. By examining this process in real time in live cells, our data provides compelling evidence that virus enters cells in vesicles that lack a full clathrin coat.

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