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Results: 7

1.
Figure 7

Figure 7. Model for sorting of Py from the plasma membrane to the ER.. From: A Lipid Receptor Sorts Polyomavirus from the Endolysosome to the Endoplasmic Reticulum to Cause Infection.

Polyomavirus binds to ganglioside GD1a or non-ganglioside receptors at the plasma membrane and is transport to the endolysosome. The low pH in this environment induces a conformational change on the virus that facilitates its subsequent structural alteration in the ER. Py that is bound to GD1a in the endolyosome is sorted to the ER where it undergoes an ERp29-dependent structural change [5] that initiates viral penetration across the ER membrane.

Mengding Qian, et al. PLoS Pathog. 2009 June;5(6):e1000465.
2.
Figure 2

Figure 2. Expression of Rab5- and Rab7-interfering mutants affects Py infection.. From: A Lipid Receptor Sorts Polyomavirus from the Endolysosome to the Endoplasmic Reticulum to Cause Infection.

NIH 3T3 cells expressing (A) CFP control, wild-type CFP-Rab5, dominant-negative CFP-Rab5 (DN), or constitutively active CFP-Rab5 (CA) or (B) YFP control, wild-type YFP-Rab7, or dominant-negative Rab7 (DN) were incubated with Py. 48 hrs later, the extent of infection in transfected cells was assayed as in Figure 1B. Data represent the mean+/−SD of at least four independent experiments. In Figure 2A, 27/262 cells expressed T antigen in the CFP-expressing cells. In Figure 2B, 75/464 cells expressed T antigen in the YFP-expressing cells. A two-tailed t test was used.

Mengding Qian, et al. PLoS Pathog. 2009 June;5(6):e1000465.
3.
Figure 6

Figure 6. Quantum-dot coated with a GD1a antibody is transported to the ER.. From: A Lipid Receptor Sorts Polyomavirus from the Endolysosome to the Endoplasmic Reticulum to Cause Infection.

(A) Q-dot GD1a Ab (high) was incubated with liposomes or liposomes containing GD1a. Samples were floated in a sucrose gradient, fractionated, subjected to SDS-PAGE, and immunoblotted for the GD1a antibody heavy chain. (B) Q-dot:Myc Ab (high), Q-dot:TfR Ab (high), Q-dot:GD1a Ab (low), Q-dot:GD1a Ab (middle), and Q-dot:GD1a Ab (high) were incubated with GD1a-supplemented cells at 4°C, washed to remove unbound Q-dots, and imaged. Left panels, representative images. Yellow lines, edge of cells. Scale bars, 10 µm for bright field image, and 2 µm for Q-dot image. Right panel, quantification of the indicated Q-dot binding to the plasma membrane from at least 3 cells. Data are mean+/−SD. (C) Co-localization of Q-dot:GD1a Ab (high) with CFP-HO2 in GD1a-supplemented NIH 3T3 cells. Left panel, representative images (ER image processed as in Figure 5A). Scale bar, 2 µm. Right panel, quantification of the indicated Q-dot co-localizing with the ER at various conditions from at least 3 different cells. Data are the mean+/−SD. A two-tailed t test was used.

Mengding Qian, et al. PLoS Pathog. 2009 June;5(6):e1000465.
4.
Figure 5

Figure 5. Increased co-localization of Py with the ER in GD1a-supplemented cells.. From: A Lipid Receptor Sorts Polyomavirus from the Endolysosome to the Endoplasmic Reticulum to Cause Infection.

(A) Live cell imaging of labeled Py co-localization with the ER. NIH 3T3 cells co-expressing CFP-HO2 and YFP-Rab7 were infected with labeled Py and the extent of co-localization of Py with the ER was analyzed 4–6 hrs post-infection. The images of the ER were subjected to filtering (see Figure S5) to more clearly define the ER tubule boundaries. Scale bar, 1 µm. (B) Quantification of Py and ER co-localization in GD1a (4°C, 37°C, BFA+37°C) and GD1a-supplemented cells. More than 300 viral particles were analyzed from at least 5 different cells. (C) Py and ER co-localization in control and GD1a-supplemented cells using immunofluorescence staining. Scale bar, 2 µm. (below) Quantification of the extent of co-localization, normalized to control cells. Arrowhead, Py that co-localized with the ER. Arrow, Py that did not co-localize with the ER. (D) Quantification of Py and ER co-localization in cells expressing either wild-type YFP-Rab7 (WT) or dominant-negative YFP-Rab7 (DN) using live cell tracking, as in A. The extent of co-localization was normalized to wild-type Rab7 expressing cells. (E) Quantification of CTB and ER co-localization in cells expressing either wild-type YFP-Rab7 or dominant-negative YFP-Rab7. Data are the mean+/−SD. A two-tailed t test was used.

Mengding Qian, et al. PLoS Pathog. 2009 June;5(6):e1000465.
5.
Figure 1

Figure 1. Time-dependent transport of Py through the endolysosome.. From: A Lipid Receptor Sorts Polyomavirus from the Endolysosome to the Endoplasmic Reticulum to Cause Infection.

(A) NIH 3T3 cells were incubated with Texas-Red-labeled purified Py for 3 hrs at 37°C, washed, and subjected to immunofluorescence using a VP1 antibody followed by FITC-labeled secondary antibody. White line, edge of cell. Scale bar, 5 µm. (B) Cells were incubated with labeled or non-labeled Py for 48 hrs, and the extent of infection assessed by immunofluorescence using a Py large T antigen antibody. Cells stained with large T antigen in the nucleus were scored as positive for infection. Scale bars, 30 µm. (C–E) Live cell imaging of labeled Py in cells expressing (C) CFP-Rab5, (D) YFP-Rab7, or (E) LAMP1-YFP. C, D, and E are images taken at the 4–6 hrs post-infection time point. Yellow lines, edge of cells. Scale bars, 10 µm (whole cell) and 1 µm (inset). (F) Quantification of the extent of co-localization between labeled Py and the respective markers at the indicated post-infection time points. For each time point, at least 90 viral particles were analyzed from 3 cells. Data are the mean+/−SD.

Mengding Qian, et al. PLoS Pathog. 2009 June;5(6):e1000465.
6.
Figure 4

Figure 4. Decreased co-localization of Py with the late endosome and lysosome in GD1a-supplemented cells.. From: A Lipid Receptor Sorts Polyomavirus from the Endolysosome to the Endoplasmic Reticulum to Cause Infection.

(A) NIH 3T3 cells were incubated with purified GM1 or GD1a, washed, infected with Py, and the extent of infection was assessed as in Figure 1B. Results were normalized to non-supplemented cells (control cells). In the control cells, 43/984 cells expressed large T antigen. (B) Untreated (control) or GD1a-supplemented NIH 3T3 cells were incubated with Py at 4°C to allow viral binding and then treated with proteinase K where indicated (top panel) or incubated at 37°C for 1 hr before proteinase K treatment to determine viral entry (bottom panel). (C, D) The extent of co-localization of labeled Py with (C) Rab5-containing vesicles or (D) Rab7-containing vesicles at the early (0.5–2 hrs) and late (4–6 hrs) time points in both control and GD1a-supplemented NIH 3T3 cells. (E) Co-localization of labeled Py with Rab7-containing vesicles at 1–2 hrs post-infection in the ganglioside-deficient C6 cells. (F) Co-localization of labeled Py with LAMP1-containing vesicles 4–6 hrs post-infection in NIH 3T3 cells. (G) The extent of Py infection in GD1a-supplemented cells expressing wild type YFP-Rab7 or dominant negative YFP-Rab7 (DN). At least 220 transfected cells were analyzed from three independent experiments. All data are the mean+/−SD. A two-tailed t test was used.

Mengding Qian, et al. PLoS Pathog. 2009 June;5(6):e1000465.
7.
Figure 3

Figure 3. Effect of low pH on Py infection and conformational change.. From: A Lipid Receptor Sorts Polyomavirus from the Endolysosome to the Endoplasmic Reticulum to Cause Infection.

(A) NIH 3T3 cells were treated with bafilomycin A1 2 hrs before, at the same time, or 3 hrs after infection with non-labeled Py, and washed to remove the drug. 48 hrs later, the extent of large T antigen expression was determined as in Figure 1B. Infection efficiency was normalized to the control cells. 37/1021 cells expressed large T antigen in the control cells. A two-tailed t test was used. Data are the mean+/−SD. (B) Cells expressing YFP-Rab7 (top) or CFP-HO2 (bottom) were infected with non-labeled Py and treated with bafilomycin A1 simultaneously. 4.5 hrs post-infection, cells were fixed and stained with an antibody against Py VP1, followed by addition of a fluorescently tagged secondary antibody; the extent of co-localization between this fluorescent signal and the fluorescence from YFP-Rab7 or CFP-HO2 were assessed. Data are the mean+/−SD. (C) Py was incubated at pH 7.5, 6.0, or 5.0, neutralized to pH 7.5, and then treated with a low concentration of proteinase K (2.5 ng/ml). The samples were immunoblotted with an antibody against VP1. (D) Py pretreated at pH 7.5 (top and bottom panels) or at pH 5 (bottom panel) was incubated with DTT, EGTA, and either BSA or an ER lumenal extract, and then treated with a low trypsin (0.25 mg/ml) concentration. Appearance of the ER-induced VP1b fragment was analyzed by immunoblotting with an antibody against VP1. Graph on the right represents quantification of the relative VP1b level generated from Py pretreated at pH 7.5 or 5. A two-tailed t test was used. Data are the mean+/−SD.

Mengding Qian, et al. PLoS Pathog. 2009 June;5(6):e1000465.

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