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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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Actin cytoskeletal dynamics during clathrin-dependent uptake of VSV.(A, C, and E). VSV internalization events in BSC-1 cells co-expressing tom-LCa and eGFP-actin (A), arp3-eGFP (C), or cortactin-eGFP (E) are shown as kymographs and in Videos S6, S7, S8 respectively. The fluorescence over time (left to right) of the actin cytoskeletal component (top), tom-LCa (middle), and a merge of these traces with the virus are shown (bottom) and are represented graphically at the right using the same approach as in Figure 1D. (B, D, and F) Graphs of the average kinetics of clathrin and actin (B), arp3 (D) and cortactin (F) recruitment to vesicles lacking (left) or containing (right) virus are shown. 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. Virus internalization data was collected from 5, 3, and 2 cells for panels B, D and F respectively, and compared with events lacking virus from 2, 2, and 3 of the same cells, respectively. The differences between the maximum actin, Arp3, and cortactin fluorescence values in pits containing and lacking VSV are statistically significant. Student's t-test: actin p = 2e-5; Arp3 p = 4e-4; cortactin p = 0.003.
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ppat-1000394-g005: Actin cytoskeletal dynamics during clathrin-dependent uptake of VSV.(A, C, and E). VSV internalization events in BSC-1 cells co-expressing tom-LCa and eGFP-actin (A), arp3-eGFP (C), or cortactin-eGFP (E) are shown as kymographs and in Videos S6, S7, S8 respectively. The fluorescence over time (left to right) of the actin cytoskeletal component (top), tom-LCa (middle), and a merge of these traces with the virus are shown (bottom) and are represented graphically at the right using the same approach as in Figure 1D. (B, D, and F) Graphs of the average kinetics of clathrin and actin (B), arp3 (D) and cortactin (F) recruitment to vesicles lacking (left) or containing (right) virus are shown. 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. Virus internalization data was collected from 5, 3, and 2 cells for panels B, D and F respectively, and compared with events lacking virus from 2, 2, and 3 of the same cells, respectively. The differences between the maximum actin, Arp3, and cortactin fluorescence values in pits containing and lacking VSV are statistically significant. Student's t-test: actin p = 2e-5; Arp3 p = 4e-4; cortactin p = 0.003.

Mentions: The effects of VSV on clathrin-coated pit formation suggest parallels with internalization of large and more stable clathrin-coated structures (Saffarian and Kirchhausen, submitted) and the invasive bacteria Listeria monocytogenes [11]. Actin polymerization has a role in the internalization of such structures, although it is not required for internalization of conventional clathrin-coated pits [12] and Saffarian and Kirchhausen, submitted. We therefore tested whether actin, an actin nucleation factor, Arp3, or cortactin, an activator of the Arp2/3 complex, is recruited to coated pits. We used for this purpose, cells expressing tom-LCa and eGFP fused to each of these proteins. During growth of virus-containing pits, the levels of actin, Arp3, and cortactin increased steadily (Figure 5B, D and F). Moreover, for both Arp3 and cortactin, there was a peak of fluorescence just prior to the onset of vesicle uncoating (Figure 5A–F, Videos S6, S7, S8). In contrast, pits that lacked VSV had low and variable levels of actin and Arp3 fluorescence throughout the growth phase that were barely above the background (Figure 5B and D). Cortactin recruitment was approximately 4-fold lower than observed in virus-containing pits (Figure 5F). We conclude that actin assembly is associated with VSV entry through a clathrin pathway.


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)

Actin cytoskeletal dynamics during clathrin-dependent uptake of VSV.(A, C, and E). VSV internalization events in BSC-1 cells co-expressing tom-LCa and eGFP-actin (A), arp3-eGFP (C), or cortactin-eGFP (E) are shown as kymographs and in Videos S6, S7, S8 respectively. The fluorescence over time (left to right) of the actin cytoskeletal component (top), tom-LCa (middle), and a merge of these traces with the virus are shown (bottom) and are represented graphically at the right using the same approach as in Figure 1D. (B, D, and F) Graphs of the average kinetics of clathrin and actin (B), arp3 (D) and cortactin (F) recruitment to vesicles lacking (left) or containing (right) virus are shown. 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. Virus internalization data was collected from 5, 3, and 2 cells for panels B, D and F respectively, and compared with events lacking virus from 2, 2, and 3 of the same cells, respectively. The differences between the maximum actin, Arp3, and cortactin fluorescence values in pits containing and lacking VSV are statistically significant. Student's t-test: actin p = 2e-5; Arp3 p = 4e-4; cortactin p = 0.003.
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Related In: Results  -  Collection

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

ppat-1000394-g005: Actin cytoskeletal dynamics during clathrin-dependent uptake of VSV.(A, C, and E). VSV internalization events in BSC-1 cells co-expressing tom-LCa and eGFP-actin (A), arp3-eGFP (C), or cortactin-eGFP (E) are shown as kymographs and in Videos S6, S7, S8 respectively. The fluorescence over time (left to right) of the actin cytoskeletal component (top), tom-LCa (middle), and a merge of these traces with the virus are shown (bottom) and are represented graphically at the right using the same approach as in Figure 1D. (B, D, and F) Graphs of the average kinetics of clathrin and actin (B), arp3 (D) and cortactin (F) recruitment to vesicles lacking (left) or containing (right) virus are shown. 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. Virus internalization data was collected from 5, 3, and 2 cells for panels B, D and F respectively, and compared with events lacking virus from 2, 2, and 3 of the same cells, respectively. The differences between the maximum actin, Arp3, and cortactin fluorescence values in pits containing and lacking VSV are statistically significant. Student's t-test: actin p = 2e-5; Arp3 p = 4e-4; cortactin p = 0.003.
Mentions: The effects of VSV on clathrin-coated pit formation suggest parallels with internalization of large and more stable clathrin-coated structures (Saffarian and Kirchhausen, submitted) and the invasive bacteria Listeria monocytogenes [11]. Actin polymerization has a role in the internalization of such structures, although it is not required for internalization of conventional clathrin-coated pits [12] and Saffarian and Kirchhausen, submitted. We therefore tested whether actin, an actin nucleation factor, Arp3, or cortactin, an activator of the Arp2/3 complex, is recruited to coated pits. We used for this purpose, cells expressing tom-LCa and eGFP fused to each of these proteins. During growth of virus-containing pits, the levels of actin, Arp3, and cortactin increased steadily (Figure 5B, D and F). Moreover, for both Arp3 and cortactin, there was a peak of fluorescence just prior to the onset of vesicle uncoating (Figure 5A–F, Videos S6, S7, S8). In contrast, pits that lacked VSV had low and variable levels of actin and Arp3 fluorescence throughout the growth phase that were barely above the background (Figure 5B and D). Cortactin recruitment was approximately 4-fold lower than observed in virus-containing pits (Figure 5F). We conclude that actin assembly is associated with VSV entry through a clathrin pathway.

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