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2.
Figure 5

Figure 5. Negative stain EM of WT and cytosol-localized SV40.. From: A Large and Intact Viral Particle Penetrates the Endoplasmic Reticulum Membrane to Reach the Cytosol.

(A) Buffer, WT SV40, S1 derived from mock-infected cells (i.e. mock-infected S1), and S1 derived from SV40-infected cells for 12 hrs (i.e. SV40-infected S1) were immunoprecipitated with VP1-specific antibodies, and the immunoprecipitate captured by magnetic beads. Samples were subjected to SDS-PAGE and silver stained. (B) WT SV40 immunoprecipitate was subjected to negative stain EM. Bar represents 50 nm. (C-D) SV40-infected S1 immunoprecipitate was subjected to negative stain EM. Bar represents 50 nm.

Takamasa Inoue, et al. PLoS Pathog. 2011 May;7(5):e1002037.
3.
Figure 2

Figure 2. ER-localized SV40 is released into the ER lumen and undergoes sequential disulfide bond disruption.. From: A Large and Intact Viral Particle Penetrates the Endoplasmic Reticulum Membrane to Reach the Cytosol.

(A) Cells treated with or without BFA at the indicated time points were infected with SV40 for varying amounts of time, harvested, and analyzed according to Figure 1C. Samples were immunoblotted with antibodies against VP1, PDI, or calnexin. (B) Cells were intoxicated with CTB for 90 min, and processed according to Figure 1C. Samples were immunoblotted with antibodies against PDI, calnexin, and CTB. (C) Cells were infected with SV40 and harvested at 12 h.p.i. S1, S2, and P2 were prepared as in A and analyzed by non-reducing and reducing SDS-PAGE, followed by immnoblotting with antibodies against VP1. (D) Cells were infected with SV40 for the indicated times, and S1 subjected to non-reducing SDS-PAGE followed by immunoblotting against VP1.

Takamasa Inoue, et al. PLoS Pathog. 2011 May;7(5):e1002037.
4.
Figure 3

Figure 3. Immunoprecipitation of ER- and cytosol-localized SV40 using conformation-specific antibodies.. From: A Large and Intact Viral Particle Penetrates the Endoplasmic Reticulum Membrane to Reach the Cytosol.

(A) Cells were infected with SV40 for 12 hrs, harvested, and processed according to Figure 1C to obtain S1 and S2. These fractions were incubated with the indicated VP1 antibody concentration, or a control antibody. The precipitated samples were subjected to SDS-PAGE followed by immunoblotting against VP1. 5% input is shown. (B) S2 in A and WT SV40 were subjected to immunoprecipitation using either VP1 or a control antibody, and the precipitated sample subjected to SDS-PAGE and immunoblotted with antibodies against VP2/VP3 or VP1. 5% input is shown. (C) The inputs and immunoprecipitates in B were subjected to PCR to amplify a region of SV40 genome. 5% input is shown. (D) As in B, except the S1 was used for immunoprecipitation. (E) As in C, except the S1 was used as the starting material.

Takamasa Inoue, et al. PLoS Pathog. 2011 May;7(5):e1002037.
5.
Figure 4

Figure 4. ER-localized SV40 is large, while cytosol-localized SV40 is large and small.. From: A Large and Intact Viral Particle Penetrates the Endoplasmic Reticulum Membrane to Reach the Cytosol.

(A) S1 and S2 derived from cells infected with SV40 for 12 hrs, and purified SV40, were subjected to gel filtration. Fractions were analyzed by SDS-PAGE, followed by immunoblotting with antibodies against VP1. VP1 was detected in two peak fractions, “large” (fractions 13–15) and “small” (fractions 22–23). (B) S1, S2, and purified SV40 in A were subjected to continuous sucrose gradient centrifugation and analyzed as in A. VP1 was mainly detected in two peak fractions, “small” (fractions 1–3) and “large” (fractions 5–9). (C) S1 in A was subjected to sucrose gradient centrifugation. Fractions were analyzed as in A and subjected to PCR to amplify a part of SV40 genome. (D) As in B, except samples were layered over a 20% sucrose cushion, centrifuged, and the sedimented material (large) and material near the top of the cushion (small) was analyzed by immunoblotting. (E) Cells were transfected with an SV40 genome and processed as in B, except the pellet was subjected to repeated freeze-thaw to extract the nuclear-localized virus.

Takamasa Inoue, et al. PLoS Pathog. 2011 May;7(5):e1002037.
6.
Figure 7

Figure 7. Release of SV40 into the cytosol depends on the host proteasome.. From: A Large and Intact Viral Particle Penetrates the Endoplasmic Reticulum Membrane to Reach the Cytosol.

(A) MG132 and DMSO were added to CV-1 cells at the indicated post-infection time points and at 0 h.p.i., respectively. Cells were harvested at 12 h.p.i. and subjected to the ER-to-cytosol membrane penetration assay. S1 was analyzed. (B) The VP1 band intensities in A were quantified with ImageJ (NIH). Data represent the mean +/− SD of at least 3 independent experiments. (C) As in A except where indicated, cells were treated with epoxomicin or DMSO at 0 h.p.i. (D) Cells were infected with SV40 and treated with MG132 or DMSO as in A, and analyzed as in Figure 1B. In a field of view, 320/359 cells scored TAg-positive in DMSO-treated cells. In MG132-treated cells, 114/205 cells scored TAg-positive at 0 h.p.i., 111/188 cells at 3.5 h.p.i., 155/221 cells at 7 h.p.i., 193/279 cells at 9 h.p.i., and 250/333 cells at 11 h.p.i. (E) Cells treated with or without BFA or MG132 were infected with SV40 for 12 hrs, processed to obtain the S2 and P2, and the sample subjected to non-reducing SDS-PAGE followed by immunoblotting against VP1.

Takamasa Inoue, et al. PLoS Pathog. 2011 May;7(5):e1002037.
7.
Figure 6

Figure 6. Role of VP3 and viral genome in SV40 ER-to-cytosol membrane transport.. From: A Large and Intact Viral Particle Penetrates the Endoplasmic Reticulum Membrane to Reach the Cytosol.

(A) WT and mutant viruses lacking VP2, VP3, or both were analyzed by immunoblotting with antibodies against VP1 and VP2/VP3. (B) Cells were infected with the indicated virus for 6 hrs, harvested, and subjected to the semi-permeabilized assay to obtain S2. S2 was subjected to non-reducing SDS-PAGE followed by immunoblotting against VP1. (C) As in B, except both S2 and P2 were prepared. Both fractions were subjected to reducing SDS-PAGE and immunoblotted with a VP1 antibody. (D) Cells were infected with the indicated virus for either 8 or 12 hrs, harvested, and subjected to the ER-to-cytosol membrane penetration assay. S1 (60% of total) and P1 (5% of total) were analyzed by SDS-PAGE, followed by immunoblotting with the indicated antibodies. (E) Cells were infected with the indicated virus and analyzed as in Figure 1B. In a field of view, 467/471 cells scored TAg-positive using WT SV40, 26/387 cells using SV40 (-VP2), 0/375 using SV40 (-VP3), and 0/421 cells using SV40 (-VP2/-VP3). (F) The extent of TAg expression induced by WT and SV40 (-genome) was analyzed as in E. In a field of view, 205/359 cells scored TAg-positive using WT SV40, and 17/379 cells using SV40 (-genome). m.o.i. = 5 was used. (G) Cells were infected with either WT SV40 or SV40 (-genome) for 12 hrs and analyzed as in D. (H) Cells were infected with either WT SV40 or SV40 (-genome) for 12 hrs, and processed to obtain S2 and P2. S2 was subjected to both non-reducing and reducing SDS-PAGE, and P2 was subjected to reducing SDS-PAGE.

Takamasa Inoue, et al. PLoS Pathog. 2011 May;7(5):e1002037.
8.
Figure 1

Figure 1. Establishment of a cell-based ER-to-cytosol membrane penetration assay for SV40.. From: A Large and Intact Viral Particle Penetrates the Endoplasmic Reticulum Membrane to Reach the Cytosol.

(A) SV40-infected cells were treated with or without BFA at infection (0 h.p.i.), and infection allowed for 12 hrs. WCE was prepared and analyzed by non-reducing and reducing SDS-PAGE, and immunoblotted with an antibody against VP1. (B) Large T antigen (TAg)-positive cells were counted in SV40-infected cells treated with or without BFA at infection (0 h.p.i.), and the results reported as the % of TAg expressing cells. Data represent the mean +/− SD of at least 3 independent experiments. In a field of view, 345/378 cells were scored TAg-positive in control cells, while 0/344 cells were scored TAg-positive for BFA-treated cells. (C) A schematic diagram of the ER-to-cytosol transport assay and the ensuing fractionation strategy. (D) Cells treated with or without BFA were incubated with SV40 for the indicated amount of time and processed according to Figure 1C. 10% of P1 and 20% of S1 were loaded. (E) As in D, except BFA was added to cells 4 h.p.i. (F) As in D, except nocodazole was added to cells 0 h.p.i. and the cells harvested 12 h.p.i. (G) Cells transfected with either a scrambled or ERp57 siRNA were infected with SV40 for 12 hrs and processed as in D. (H) SV40-infected cells were harvested at the indicated post-infection time points, lysed in SDS sample buffer and analyzed by immunoblotting with antibodies against TAg, VP1, and Hsp90. Arrow head indicates the initiation time point of TAg synthesis, while arrow indicates the initiation time point of VP1 synthesis. (I) Cells treated with or without BFA at infection were infected with SV40 for 12 hrs and processed as in D. In addition, the S1 was subjected to PCR to amplify a part of the SV40 genome. (J) As in I except where indicated, a VP2/VP3 antibody was used.

Takamasa Inoue, et al. PLoS Pathog. 2011 May;7(5):e1002037.

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