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. 2014 May 8;9(5):e96922.
doi: 10.1371/journal.pone.0096922. eCollection 2014.

Involvement of microtubular network and its motors in productive endocytic trafficking of mouse polyomavirus

Vojtech Zila  1 , Francesco Difato  1 , Lucie Klimova  1 , Sandra Huerfano  1 , Jitka Forstova  1
Affiliations

Involvement of microtubular network and its motors in productive endocytic trafficking of mouse polyomavirus

Vojtech Zila et al. PLoS One. .

Abstract

Infection of non-enveloped polyomaviruses depends on an intact microtubular network. Here we focus on mouse polyomavirus (MPyV). We show that the dynamics of MPyV cytoplasmic transport reflects the characteristics of microtubular motor-driven transport with bi-directional saltatory movements. In cells treated with microtubule-disrupting agents, localization of MPyV was significantly perturbed, the virus was retained at the cell periphery, mostly within membrane structures resembling multicaveolar complexes, and at later times post-infection, only a fraction of the virus was found in Rab7-positive endosomes and multivesicular bodies. Inhibition of cytoplasmic dynein-based motility by overexpression of dynamitin affected perinuclear translocation of the virus, delivery of virions to the ER and substantially reduced the numbers of infected cells, while overexpression of dominant-negative form of kinesin-1 or kinesin-2 had no significant impact on virus localization and infectivity. We also found that transport along microtubules was important for MPyV-containing endosome sequential acquisition of Rab5, Rab7 and Rab11 GTPases. However, in contrast to dominant-negative mutant of Rab7 (T22N), overexpression of dominant-negative mutant Rab11 (S25N) did not affect the virus infectivity. Altogether, our study revealed that MPyV cytoplasmic trafficking leading to productive infection bypasses recycling endosomes, does not require the function of kinesin-1 and kinesin-2, but depends on functional dynein-mediated transport along microtubules for translocation of the virions from peripheral, often caveolin-positive compartments to late endosomes and ER - a prerequisite for efficient delivery of the viral genome to the nucleus.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Tracking of MPyV in living cells expressing EGFP-fused tubulin.
3T6 cells expressing EGFP-tubulin (green) were infected with Alexa Fluor 546-labeled MPyV (red) (MOI of 102 to 103 virus particles per cell) at 37°C and scanned with ΔT = 3 s. Virions were transported to both directions: to the nuclear periphery (upper panel) and to the cell periphery (lower panel). Selected frames from two different cells at 1 h p.i. are shown in detail (see Movie S1). Arrowheads point to MPyV virions. Bars, 10 µm. Cells were examined with an Olympus IX81 CellR microscope equipped with an MT20 illumination system.
Figure 2
Figure 2. Single particle tracking analysis of MPyV transport.
3T6 cells were infected with Alexa Fluor 546-labeled MPyV (MOI of 102 to 103 virus particles per cell) at 37°C and scanned with ΔT = 6 s. (A) Complex trajectories marked in white tracking curves in four selected cells shown in transmission light. Bars, 5 µm. (B–D) Dynamics of three independent single particle trackings (upper graphs) representing three main types of virion transport velocities with single particle position tracking in x-y coordinates (middle graphs) and course of movement velocities (lower graphs) measured at time intervals of 6 s (mean velocity was averaged from each three following time steps). (E) Frequency of virion transport velocity rates, counted from more than 200 different tracking experiments in 3T6 cells, with a distinct peak at 0.6 µm/s (x-coordinate was cropped to cut off the high frequency of short-range movements at rates <0.3 µm/s). (F) Histogram of fast movement distances counted from 109 single fast movements and sampled into 0.5 µm step intervals, with a maximum at 1.5 µm and the average distance of 2.5 µm. (G) Time span frequency of fast (≥0.6 µm/s) movements with maximum counts at 3.3 and 7.2 s.
Figure 3
Figure 3. Role of microtubular motors in MPyV productive trafficking.
(A and C) 3T6 cells were transfected with: (A) plasmid DNA for transient expression of EGFP (pEGFP-N1) or dynamitin-EGFP (inhibiting the dynein motor function) or (C) plasmid DNA for transient expression of RFP-DTC protein (pRFP-DTC), RFP-fused C-terminal fragment of kinesin-1 (pRFP-KHCct; inhibiting kinesin-1 motor function), EGFP (pEGFP-N1) or EGFP-fused dominant-negative subunits of kinesin-2 (pEGFP-KIF3A-HL or pEGFP-KAP3-CT). Cells expressing constructs were infected with MPyV, incubated until 24 h p.i., fixed and immunostained for MPyV LT antigen. The efficiency of infection was determined by levels (%) of LT antigen-positive cells normalized to that obtained in control, mock-transfected cells. During the experiment, more than 500 cells were counted for each sample. Data in the graphs represent mean values ± s.d. from three independent experiments. (B and D) Control, mock-transfected cells, or cells expressing dynamitin-EGFP, RFP-KHCct or EGFP-KIF3A-HL, infected with MPyV (MOI of 103 virus particles per cell), fixed 5 h p.i. and immunostained for MPyV VP1 capsid protein. DNA in nuclei was stained with DAPI (blue). Confocal sections of representative cells with corresponding signal in green or red channel and differential interference contrast (DIC) images are presented (the virus localization during EGFP-KIF3A-CT expression is not presented as it was similar to that in cells expressing the -HL form of kinesin-2). Arrowhead point to the virus at nuclear periphery. Arrows point to the virions at cell periphery. Bars, 10 µm.
Figure 4
Figure 4. Dynein motor is required for trafficking of MPyV to the ER.
3T6 cells were transfected with plasmid DNA for expression of dynamitin-EGFP, infected with MPyV (MOI of 5×102 virus particles per cell) and fixed 5 h p.i. Cells were immunostained for MPyV VP1 capsid protein (red) and BiP (GRP78) marker of ER (blue). (A) Confocal sections of representative control (mock-transfected) cells and dynamitin-EGFP expressing cells at 5 h p.i. with enlarged details. Arrowheads point to selected MPyV virions co-localized with BiP protein. Arrows point to selected MPyV that did not co-localize with BiP protein. Bars, 10 µm. (B) Quantification of co-localization of MPyV virions with BiP at 5 h p.i. The percentage of co-localizing virions was calculated from images such as shown in panel A and levels (%) of co-localizing virions in dynamitin-expressing cells were normalized to that in control. During the experiment, more than 600 virions in at least 10 different cells were evaluated for each sample. Data in the graph represent mean values ± s.d. from three independent experiments; Student’s t-test was used.
Figure 5
Figure 5. Effect of cytoskeleton-disrupting drugs on subcellular localization of MPyV.
Non-transfected 3T6 cells (A and E) or cells transiently expressing EGFP-tagged marker of interest (B–D) were infected with MPyV (MOI of 5×102 virus particles per cell) and fixed 5 h p.i. Cells were immunostained for MPyV VP1 capsid protein (red) and for a second marker of interest (caveolin-1, BiP) if not fused with EGFP (green). Confocal sections of cells with enlarged details are shown. Arrowheads point to selected MPyV virions co-localized with the marker of interest. Arrows point to selected MPyV that did not co-localize with the marker of interest. Bars, 10 µm. (F) Quantification of co-localization of MPyV virions with indicated markers at 5 h p.i. in non-treated cells (control) or cells pre-treated (1 h) and infected in the presence of nocodazole (Noc) or latrunculin A (LatA). (G) Quantification of co-localization of MPyV virions with EGFP-Rab5 at 1.5 h p.i., in control, Noc- or LatA-treated cells. The percentage of co-localizing virions was calculated from images such as those showed in A–E. During the experiment, more than 600 virions in at least 10 different cells were evaluated for each sample. Data in the graph represent mean values ± s.d. from three independent experiments.
Figure 6
Figure 6. Localization of MPyV in nocodazole-treated cells.
3T6 cells were pre-treated (1 h) with nocodazole, infected with MPyV in the presence of the drug and fixed 5 h p.i. (A) Cells immunostained for MPyV VP1 (red) and caveolin-1 (green). (B) Cells expressing EGFP-fused Rab7 GTPase (green) immunostained for VP1 protein (red). Confocal sections of cells are shown. Arrowheads point to selected MPyV virions co-localizing with indicated marker. Bars, 10 µm. (C and D) Immunolabeling of thawed cryosections of cells with anti-caveolin-1 antibody, followed by immunolabeling with secondary antibody conjugated with 10 nm gold particles (seen as darkly stained dots). Arrowheads point to selected virions. Empty arrowheads point to flask-shape “empty” caveolar structures. Bars, 100 nm. Pm, plasma membrane; MVBs, multivesicular bodies.
Figure 7
Figure 7. Immuno-electron microscopy of 3T6 cells at 5 h post-infection with MPyV.
Thawed cryosections were immunolabeled with anti-caveolin-1 antibody followed by incubation with secondary antibody conjugated with 10 nm gold particles. Arrowheads point to selected virions. Bars, 100 nm. Pm, plasma membrane; MVBs, multivesicular bodies; MLB, multilamellar body; Nu, nucleus.
Figure 8
Figure 8. Internalization and infectivity of MPyV in nocodazole-treated cells.
(A) 3T6 cells were pre-treated in culture medium alone or in medium supplemented with nocodazole for 1 h at 37°C and incubated with MPyV (MOI of 5×102 virus particles/cell) for 15 or 90 min at 37°C, also in the presence or absence of the drug. After that, immunofluorescence analysis was performed using an anti-MPyV VP1 antibody added to live cells, followed by fixation. Confocal sections of representative cells are shown. Bars, 10 µm. (B) Immunolabeling of thawed cryosections of cells pre-treated and infected with MPyV in the presence of nocodazole. Cells were immunolabeled with anti-caveolin-1 antibody, followed by immunolabeling with secondary antibody conjugated with 10 nm gold particles (seen as darkly stained dots). Arrowheads point to selected virions. Empty arrowhead points to caveolar invagination. Asterisk indicates virion internalizing to an invagination lacking caveolin-1. Arrow points to virion internalizing via a region at the plasma membrane enriched for caveolin-1. Pm, plasma membrane. Bars, 50 nm. (C) 3T6 cells were pre-treated (1 h) with nocodazole and infected with MPyV. The drug was washed out at 7 h p.i. and cells were further incubated until 24 h p.i. (middle bar) or for additional 24 h after washing (right bar). As a control, cells were infected in the absence of the drug and fixed 24 h p.i. Cells were immunostained for MPyV LT antigen and the efficiency of infection was determined by the levels (%) of LT antigen-positive cells, normalized to that in control. During the experiment, at least 500 cells of each sample were counted. Data in the graph represent mean values ± s.d. from three independent experiments. Immunofluorescent staining of microtubules (anti-α-tubulin antibody; panel on the right) shows the morphology of microtubular network at the time of washing (7 h p.i.) in control or nocodazole-treated cells. Bars, 10 µm.
Figure 9
Figure 9. Rab11 GTPase is not required for MPyV infection.
3T6 cells were transfected with plasmid DNA for transient expression of (A) wild-type EGFP-Rab11 (wt), dominant-negative EGFP-Rab11 (DN) or constitutively active EGFP-Rab11 (CA), or (B) wild-type EGFP-Rab7 (wt), dominant-negative EGFP-Rab7 (DN) or constitutively active EGFP-Rab7 (CA). After 24 h, cells were infected with MPyV, incubated until 24 h p.i., fixed and immunostained for MPyV LT antigen. The efficiency of infection was determined by the levels (%) of LT antigen-positive cells from that expressing EGFP-fused version of the Rab11 or Rab7 GTPase normalized to that obtained in cells expressing its wild-type version. During the experiment, more than 500 cells were counted for each sample or control. Data in the graph represent mean values ± s.d. from three independent experiments.
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References

    1. Gilbert JM, Benjamin TL (2000) Early steps of polyomavirus entry into cells. J Virol 74: 8582–8588. - PMC - PubMed
    1. Gilbert JM, Goldberg IG, Benjamin TL (2003) Cell penetration and trafficking of polyomavirus. J Virol 77: 2615–2622. - PMC - PubMed
    1. Liebl D, Difato F, Hornikova L, Mannova P, Stokrova J, et al. (2006) Mouse polyomavirus enters early endosomes, requires their acidic pH for productive infection, and meets transferrin cargo in Rab11-positive endosomes. J Virol 80: 4610–4622. - PMC - PubMed
    1. Tsai B, Gilbert JM, Stehle T, Lencer W, Benjamin TL, et al. (2003) Gangliosides are receptors for murine polyoma virus and SV40. EMBO J 22: 4346–4355. - PMC - PubMed
    1. Gilbert J, Benjamin T (2004) Uptake pathway of polyomavirus via ganglioside GD1a. J Virol 78: 12259–12267. - PMC - PubMed

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