QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 21 August 2007
doi: 10.1242/jcs.03483

This Article Summary Full Text Full Text (PDF) Supplementary Material Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Beske, O. Articles by Andino, R. Search for Related Content PubMed PubMed Citation Articles by Beske, O. Articles by Andino, R.

Poliovirus infection blocks ERGIC-to-Golgi trafficking and induces microtubule-dependent disruption of the Golgi complex

Oren Beske^1,*, Mike Reichelt^2,*, Matthew P. Taylor², Karla Kirkegaard² and Raul Andino^1,

¹ Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA 94143, USA
² Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA 94305, USA

View larger version (40K): [in this window] [in a new window]   Fig. 1. Transport of VSVG-ts045-GFP protein during poliovirus infection. HeLa cells were transfected with VSVG-ts045-GFP-expressing plasmid and incubated at 40°C overnight to accumulate VSVG-ts045-GFP protein in the ER. (A) Uninfected HeLa cells were either maintained at 40°C (A, left image) or shifted to 32°C for 30 minutes as indicated. The cells were subsequently fixed and mounted for fluorescence microscopy. (B) VSVG-ts045-GFP transfected cells were infected with poliovirus at an MOI of 0.5 PFU/cell at 40°C for 3.5 hours then shifted to 32°C for 30 minutes Cells were then fixed and analyzed for GFP fluorescence or the presence of poliovirus 2C protein by indirect immunofluorescence as indicated to distinguish between uninfected cells and infected cells in the same image. Arrowheads indicate Golgi complexes, arrows indicate ERGIC puncta.

View larger version (71K): [in this window] [in a new window]   Fig. 2. Confocal microscopy of VSVG-ts045-GFP protein transport during poliovirus infection. Huh7 cells were transiently transfected with VSVG-ts045-GFP for 16 hours at 40°C. (A) Uninfected cells were shifted to 32°C for 0, 10 or 20 minutes as indicated, fixed and visualized for GFP fluorescence by confocal microscopy. (B) Transfected cells were infected at an MOI of 30 PFU/cell, incubated at 40°C for 3 hours and shifted to 32°C for 0, 10 minutes or 20 minutes as indicated, fixed and visualized for GFP fluorescence by confocal microscopy. For each panel, an image was selected to represent most abundant class of expression pattern for that condition and time point. Arrows identify VSVG-ts045-GFP puncta and arrowheads identify Golgi-like patterns. (C) The numbers of cells with ER-like, ER with puncta, and Golgi-like patterns were determined for both infected and uninfected cells at 0, 5, 10 and 20 minutes after temperature shift. Three cover slips per time point were evaluated and the percentage of cells with each pattern category was determined. At least 50 cells per coverslip were evaluated. For each type of pattern, the percentage of cells is plotted as a function of time for uninfected () and infected () cells.

View larger version (92K): [in this window] [in a new window]   Fig. 3. Colocalization of punctate VSVG-ts045-GFP staining with ERGIC p53. (A-F) Huh7 cells were transfected with VSVG-ts045-GFP-expressing plasmid and infected with poliovirus as in Fig. 2. After 3 hours of infection at 40°C, the cells were shifted to 32°C and incubation continued for either 20 minutes (A-C) or 60 minutes (D-F). After fixation, cells were labeled with an anti-ERGIC p53 antibody and visualized by confocal microscopy for indirect immunofluorescence of ERGICp53 (A,D) and GFP fluorescence (B,E). (C,F) Merged images.

View larger version (132K): [in this window] [in a new window]   Fig. 4. EM of accumulated secreted alkaline phosphatase (SEAP) activity in uninfected and poliovirus-infected cells. (A-E) Huh7 cells were transiently transfected with SEAP-expressing plasmid pGeneGripTM for 16 hours and either (A) mock-infected or (B-E) infected with poliovirus for 4 hours at an MOI of 30 PFU/cell. Cells were fixed and incubated with a solution containing glycerol phosphate and lead citrate, so that alkaline phosphatase activity would lead to the formation of electron-dense lead citrate. Samples were either processed for (A-D) conventional embedding in epoxyresin or (E) cryosectioning. Thawed cryosections were immunogold-labelled with an affinity-purified polyclonal rabbit anti-ERGICp53 antibody followed by a 15-nm gold-conjugated protein-A incubation. Ultrathin sections (60-80 nm) of all samples were analyzed by EM. Low magnification confirmed that cells shown in B-E were infected, as evidenced by condensed nuclear morphology and the presence of numerous virus-induced vesicular structures (data not shown). Empty arrowheads mark budding structures at ER membranes filled with electron-dense precipitate. Empty arrows identify small (80 nm) vesicles filled with precipitate. Stars mark precipitate-filled tubular-vesicular structures. (E) Solid arrowheads identify 15-nm gold particles; solid arrows identify precipitate-filled tubular-vesicular structures. ER, structures morphologically consistent with endoplasmic reticulum; dmv, double-membraned vesicle.

View larger version (105K): [in this window] [in a new window]   Fig. 5. Dynamics of the Golgi complex in poliovirus-infected cells. COS-7 cells were transfected with plasmid (GT-GFP) that expressed Golgi resident protein galactosyl transferase fused to GFP and incubated for 16 hours at 37°C. Before mock infection (A,C) or infection with poliovirus at an MOI of 10 PFU/cell (B,D), cells were pretreated for 1.5 hours in medium alone or in medium containing 5 µg/ml nocodazole as indicated. Cells were placed on a heated 37°C stage and images were obtained by fluorescence microscopy every 1.5 minutes for up to 5 hours, the images shown were taken 90 minutes apart. A complete set of images is provided as supplementary material Movies 1-4.

View larger version (36K): [in this window] [in a new window]   Fig. 6. Characteristics of Golgi dispersion during poliovirus infection. (A) Golgi-specific glycosylation of VSVG-ts045-GFP was monitored under various conditions. COS-7 cells were transfected with plasmid that expressed VSVG-ts045-GFP, incubated overnight at 40°C, pulse-labeled with [35S]-methionine, and incubated with unlabeled methionine under the conditions described below. Cells were either mock-infected and kept at 40°C (lanes 1 and 2), shifted to 32°C for 30 minutes (lanes 3 and 4), treated with 2 µg/ml BFA for 1 hour at 40°C (lanes 5 and 6) or infected with poliovirus at 40°C for 4 hours (lanes 7 and 8). Labeled VSVG-ts045 was immunoprecipitated from cell extracts and digested with Endo H or not as indicated. Protein samples were analyzed by SDS-PAGE and visualized by autoradiography. Arrow indicates Endo-H-sensitive VSVG-ts045-GFP. (B) Membrane association of Golgi marker galactosyl transferase fused to GFP (GT-GFP) in uninfected and poliovirus-infected cells. COS-7 cells were transfected with plasmid that expressed GT-GFP and then either mock-infected or infected with poliovirus at an MOI of 10 PFU/ml for 3.5 hours. Whole-cell extracts were brought to 59% sucrose, overlaid with one 2-ml layer of 52% sucrose and a second 2-ml layer of 29% sucrose and centrifuged for 90 minutes at 196,000 g in an SW41 rotor. Fractions were collected and subjected to TCA precipitation. Proteins were displayed by SDS-PAGE, and the GT-GFP protein visualized by immunoblot. Fractions that contained 29% sucrose (lanes 1, 5), the 29%/52% interface (2, 6), 52% sucrose (lanes 3, 7) and 59% sucrose (lanes 4, 8) are shown.

View larger version (49K): [in this window] [in a new window]   Fig. 7. Effect of nocodazole treatment on Golgi dispersion in poliovirus-infected cells. The localization of two different Golgi markers, GM130 and -COP, were monitored by confocal immunofluorescence microscopy. (A-L) HeLa cells were pretreated with 10 µg/ml nocodazole (+Noc) or with medium without nocodazole (–Noc) for 1.5 hours Cells were then mock-infected (uninfected) or infected with poliovirus (MOI=10 PFU/ml) in the presence or absence of 10 µg/ml nocodazole. Cells were fixed at 3.5 hours post infection. Cells grown on cover slips were processed for indirect immunofluorescence and confocal microscopy as shown; the other cells in the dish were processed for conventional EM (see Fig. 8). The Golgi complex was stained with a murine monoclonal antibody directed against GM130 (red; A-D) and an affinity-purified polyconal rabbit antibody against -COP (green, E-H) that detects COPI present on Golgi, ERGIC and COPI vesicular structures.

View larger version (115K): [in this window] [in a new window]   Fig. 8. Ultrastructure of a Golgi complex following nocodazole treatment of uninfected and poliovirus-infected cells. Cells from the experiment shown in Fig. 7 were processed for EM and ultrathin sections (60 nm) were analyzed. For both uninfected and infected cells, the arrows connect representative Golgi mini-stacks at low and high (inset) magnifications.

View larger version (28K): [in this window] [in a new window]   Fig. 9. Effect of nocodazole on SEAP secretion in uninfected and poliovirus-infected cells. COS-7 cells were transfected with a plasmid, pIRES-SEAP, that expressed (A) SEAP protein under the translational control of the IRES element from encephalomyocarditis virus (EMCV) to ensure effective translation in poliovirus-infected cells. (B) Transfected COS-7 cells were incubated for 16 hours at 37°C with or without 2 µg/ml BFA as indicated. Aliquots from the supernatants were sampled every hour from both cultures and assayed for SEAP activity. (C) Transfected COS-7 cells were either incubated with serum-free medium with or without 5 µg/ml nocodazole for 1.5 hours The medium was then replaced with fresh medium with or without nocodazole and aliquots taken every hour to determine SEAP activity. (D) Transfected COS-7 cells were mock-infected or infected with poliovirus at an MOI of 10 PFU/cell. SEAP activity was assayed in both the supernatants and the cells of mock-infected and infected cells at the time points indicated. The percentage of SEAP activity in infected cells compared with that in uninfected cells is plotted for secreted and intracellular SEAP as a function of time post infection. Experiments were performed in triplicate and the standard error is shown. (E) pIRES-SEAP-transfected cells were treated for 1.5 hours with serum-free medium with or without 5 µg/ml nocodazole and either mock-infected or infected with poliovirus at an MOI of 10 PFU/cell. Aliquots of medium were sampled every hour and assayed for SEAP activity. The percentage of extracellular and intracellular SEAP activity in infected cells compared with uninfected cells as a function of time after infection in the presence of nocodazole is shown.

View larger version (49K): [in this window] [in a new window]   Fig. 10. Dispersed of the Golgi complex in cells infected with 3A-2 poliovirus secretion mutant. (A) COS-7 cells were transfected as in the experiment of Fig. 9 with pIRES-SEAP. Transfected COS-7 cells were mock-infected or infected with wild-type (WT) or mutant 3A-2 (3A-2) poliovirus at an MOI of 10 PFU/cell. SEAP activity was assayed in the cell culture supernatants. (B) COS-7 cells were transfected with plasmid (GT-GFP) that expressed Golgi resident protein galactosyl transferase fused to GFP and incubated for 16 hours at 37°C. Cells were mock-infected or infected with wild-type or mutant 3A-2 poliovirus at an MOI of 10 PFU/cell. Cells were incubated for 3 hours at 37°C, fixed and visualized for GFP fluorescence (green) and immunoflurescence using a anti GM130 antibody (red) by confocal microscopy.