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. 2009 Apr;5(4):e1000394.
doi: 10.1371/journal.ppat.1000394. Epub 2009 Apr 24.

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

Affiliations

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

David K Cureton et al. PLoS Pathog. 2009 Apr.

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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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. 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).
Figure 2
Figure 2. 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.
Figure 3
Figure 3. Enhanced dynamin recruitment during viral internalization.
(A) A tile view of images from a BSC-1 cell co-expressing dyn2-eGFP (green) and tom-LCa (red) cropped from a time-lapse movie (Video S3), showing appearance of VSV at the cell surface (t = −8 s) relative to detection of clathrin (t = 0 s). (B) A graph of the kinetics of dyn2 and clathrin recruitment to the CCPs shown in panel A. The fluorescence was expressed as a % of the average maximum clathrin measured in all pits lacking virus, and a weighted average was plotted as described in Materials and Methods. (C) A graph of the average kinetics of LCa and dyn2 recruitment to CCP containing (right) or lacking (left) 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. Viral events were from 5 cells, and events lacking virus were analyzed in 3 of the same cells. (D) A graph of the maximum dynamin recruitment relative to pit lifetime for CCVs containing (blue) or lacking (open circles) virus. Dynamin fluorescence is expressed as a % of the average peak observed for pits lacking virus. The average lifetime of pits containing virus was 148+/−71 s (5 cells), and was statistically distinct (Student's t-test: p = 7e-4) to that for pits lacking virus (55+/−21 s from 3 of same cells). The peak dynamin fluorescence intensities in pits containing and lacking virus were 548+/−221 and 100+/−45, respectively. The difference between these values is statistically significant (Student's t-test: p = 2e-5). (E) A kymograph view of BSC-1 cells expressing σ2-eGFP and tom-LCa. Note the lack of CCP internalization (arrows) and virus (blue) uptake in cells treated with 80 µM dynasore. Video S4 depicts inhibition of VSV internalization following dynasore treatment. (F) A graph of the % of bound VSV particles that were internalized in the presence (n = 4 cells) and absence (n = 5 cells) of dynasore. (G) Graphs of the effect of dynasore on VSV entry and gene expression. Cells were infected with rVSV-LUC (MOI = 0.5), and exposed to the indicated concentration of dynasore 1–4 h p.i. (left) or prior to infection −0.5–4 h p.i. (right). Luminescence values were measured at 5 h p.i. and are the average+/−standard deviation of triplicate samples.
Figure 4
Figure 4. Auxilin recruitment during uncoating of vesicles containing VSV.
(A) Kymograph views of VSV internalization by a BSC-1 cell expressing tom-LCa and eGFP-aux1 (left), along with a graph of the kinetics of auxilin (aux1) and clathrin (LCa) recruitment (right). The internalization event shown is depicted in Video S5. (B) Graphs of the average kinetics of clathrin and auxilin recruitment to CCP containing (right) or lacking (left) 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. Viral events were from 2 independent cells, and events lacking virus were analyzed in 1 of these cells. The difference between the maximum aux1 fluorescence in pits containing and lacking VSV is not statistically significant (Student's t-test: p = 0.1).
Figure 5
Figure 5. 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.
Figure 6
Figure 6. Chemical inhibition of actin polymerization reduces the efficiency of VSV internalization.
(A–D) Where indicated, BSC-1 cells were treated with 20 µM cytochalasin D (cytoD), or 6.3 µM latrunculin B (latB) for 10 min. prior to inoculation with VSV and acquisition of time-lapse images. Video S9 depicts the effect of cytoD treatment on VSV internalization. The % of attached particles captured by a coated pit (A), and subsequently internalized (B), along with the % of CCP lacking virus that complete within 80 s (C), were plotted to generate the graphs shown. Data in (A) and (B) are from 28 independent control cells, 5 cells treated with cytoD, and 2 cells treated with latB. Data in (C) are from 2 independent cells in each condition. (D) The graphs show clathrin fluorescence vs. CCV lifetime for complete (circles) or incomplete (triangles) internalization events, for pits containing (blue) or lacking (open circles) VSV. Clathrin fluorescence is expressed relative to its intensity in untreated pits lacking virus, which were set to 100. Internalization was analyzed from 4 cells in the absence of cytoD (16 complete VSV internalizations, 23 complete non viral events) or 3 cells (8 complete and 11 incomplete VSV events, 24 complete non viral events) in the presence of cytoD. The peak clathrin fluorescence in viral events was not statistically significantly different in the presence and absence of cytoD (Student's t-test p = 0.2 for complete and incomplete events). (E) Effect of cytoD on VSV entry and gene expression. Cells were infected with rVSV-LUC (MOI = 0.5) and exposed to the indicated concentration of cytochalasin D 2–5 h p.i. (left) or prior to infection −0.5–5 h p.i. (right). Luminescence values were measured at 5 h p.i. as described in Materials and Methods. The values shown are the mean of triplicates+/−standard deviation. (F) Effect of latB on VSV entry and gene expression. Cells were treated as in (E) except latB was substituted for cytoD.
Figure 7
Figure 7. Kinetics of VSV diffusion and capture by clathrin-coated pits.
(A) A graph of the diffusion (mean squared displacement) of 4 single particles relative to the time of clathrin detection (t = 0) is shown. Note the break in the Y axis to emphasize the difference between rapidly (open circles) and slowly (blue) diffusing particles. (B) A graph of the average rate of diffusion (mean square displacement) for 13 particles (n = 4 cells) tracked for 60 s prior to clathrin detection (t = 0) is shown. (C) A histogram of the time between VSV attachment and the appearance of clathrin or adaptor is shown for 98 particles (left; n = 28 cells), and compared with the simulated kinetics of random capture of VSV (middle) or LDL (right) by CCPs. The mean times to cargo capture are shown in the upper right corner of each panel.
Figure 8
Figure 8. 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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