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A Non-enveloped Virus Hijacks Host Disaggregation Machinery to Translocate across the Endoplasmic Reticulum Membrane.

Ravindran MS, Bagchi P, Inoue T, Tsai B - PLoS Pathog. (2015)

Bottom Line: Here we uncover a novel role of this machinery in driving membrane translocation during viral entry.Combining biochemical, cell-based, and imaging approaches, we find that the Hsp110 family member Hsp105 associates with the ER membrane J-protein B14.Hence the energy provided by the Hsc70-dependent Hsp105 disaggregation machinery can be harnessed to catalyze a membrane translocation event.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, Michigan, United States of America.

ABSTRACT
Mammalian cytosolic Hsp110 family, in concert with the Hsc70:J-protein complex, functions as a disaggregation machinery to rectify protein misfolding problems. Here we uncover a novel role of this machinery in driving membrane translocation during viral entry. The non-enveloped virus SV40 penetrates the endoplasmic reticulum (ER) membrane to reach the cytosol, a critical infection step. Combining biochemical, cell-based, and imaging approaches, we find that the Hsp110 family member Hsp105 associates with the ER membrane J-protein B14. Here Hsp105 cooperates with Hsc70 and extracts the membrane-penetrating SV40 into the cytosol, potentially by disassembling the membrane-embedded virus. Hence the energy provided by the Hsc70-dependent Hsp105 disaggregation machinery can be harnessed to catalyze a membrane translocation event.

No MeSH data available.


Related in: MedlinePlus

Hsp105 overexpression enhances SV40 extraction into the cytosol.A. COS-7 cells expressing the indicated S-tagged construct were infected with SV40 (MOI ~5). 12 hpi, cells were processed as in Fig 3A, and the samples immunoblotted using the indicated antibodies. B. VP1 band intensities of the cytosol-localized SV40 in (A) were quantified and normalized against the Hsp90 band intensity. Values represent the mean ± SD (n≥3). * p <0.05. C. Cells expressing the indicated F- or S-tagged construct are scored for the TAg expression after 24 hpi (MOI ~0.5), and the values normalized to GFP. The bar graphs represent mean values ± SD (n≥3). D. CV-1 cells expressing GFP-S or Hsp105 WT-S were infected with SV40 (MOI ~30) for 16 h. Cells were fixed, stained with BAP31 and VP1 antibodies, and imaged as in Fig 3E. The experimental set-up is depicted on the left side of the figure. In the first column, a representative cell expressing GFP-S (top row) or Hsp105 WT-S (second row) amongst cells not expressing this protein is shown. The insert shows a 2x enlarged area of the dotted box. Scale bar, 20 μm. E. Cells expressing the indicated tagged construct are scored for the presence of at least one BAP31-positive focus in each cell, and the values normalized to GFP. The bar graphs represent mean values ± SD (n≥3). F. Cells transfected with either ctrl or Hsp105 siRNA #1 were subsequently transfected with the indicated tagged construct, and BAP31 foci were quantified as in (E). * p <0.05.
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ppat.1005086.g004: Hsp105 overexpression enhances SV40 extraction into the cytosol.A. COS-7 cells expressing the indicated S-tagged construct were infected with SV40 (MOI ~5). 12 hpi, cells were processed as in Fig 3A, and the samples immunoblotted using the indicated antibodies. B. VP1 band intensities of the cytosol-localized SV40 in (A) were quantified and normalized against the Hsp90 band intensity. Values represent the mean ± SD (n≥3). * p <0.05. C. Cells expressing the indicated F- or S-tagged construct are scored for the TAg expression after 24 hpi (MOI ~0.5), and the values normalized to GFP. The bar graphs represent mean values ± SD (n≥3). D. CV-1 cells expressing GFP-S or Hsp105 WT-S were infected with SV40 (MOI ~30) for 16 h. Cells were fixed, stained with BAP31 and VP1 antibodies, and imaged as in Fig 3E. The experimental set-up is depicted on the left side of the figure. In the first column, a representative cell expressing GFP-S (top row) or Hsp105 WT-S (second row) amongst cells not expressing this protein is shown. The insert shows a 2x enlarged area of the dotted box. Scale bar, 20 μm. E. Cells expressing the indicated tagged construct are scored for the presence of at least one BAP31-positive focus in each cell, and the values normalized to GFP. The bar graphs represent mean values ± SD (n≥3). F. Cells transfected with either ctrl or Hsp105 siRNA #1 were subsequently transfected with the indicated tagged construct, and BAP31 foci were quantified as in (E). * p <0.05.

Mentions: We used a gain-of-function strategy to assess Hsp105’s role in extracting SV40 into the cytosol. The CV-1 derived COS-7 cells were transfected with GFP-S, HspBP1-S, or Hsp105 WT-S, and subjected to the cytosol arrival assay as described above. COS-7 cells were used because of their ability to support a high DNA transfection efficiency required for this experiment. We found that over-expressing Hsp105 WT-S (Fig 4A, fourth panel, compare lane 3 to 2 and 1) but not HspBP1-S stimulated SV40 arrival to the cytosol (Fig 4A, first panel; the VP1 band intensity is quantified in Fig 4B). These findings demonstrate that Hsp105 stimulates SV40 extraction into the cytosol. Not surprisingly, Hsp105 WT overexpression also enhanced SV40 infection (Fig 4C, compare second to first bar). This stimulation requires Hsp105’s nucleotide exchange activity as overexpressing Hsp105 NE*-F or G*-F did not robustly enhance infection (Fig 4C, compare third and fourth bars to second bar), and is specific because overexpressing neither Hsc70, SGTA, nor HspBP1 stimulated infection.


A Non-enveloped Virus Hijacks Host Disaggregation Machinery to Translocate across the Endoplasmic Reticulum Membrane.

Ravindran MS, Bagchi P, Inoue T, Tsai B - PLoS Pathog. (2015)

Hsp105 overexpression enhances SV40 extraction into the cytosol.A. COS-7 cells expressing the indicated S-tagged construct were infected with SV40 (MOI ~5). 12 hpi, cells were processed as in Fig 3A, and the samples immunoblotted using the indicated antibodies. B. VP1 band intensities of the cytosol-localized SV40 in (A) were quantified and normalized against the Hsp90 band intensity. Values represent the mean ± SD (n≥3). * p <0.05. C. Cells expressing the indicated F- or S-tagged construct are scored for the TAg expression after 24 hpi (MOI ~0.5), and the values normalized to GFP. The bar graphs represent mean values ± SD (n≥3). D. CV-1 cells expressing GFP-S or Hsp105 WT-S were infected with SV40 (MOI ~30) for 16 h. Cells were fixed, stained with BAP31 and VP1 antibodies, and imaged as in Fig 3E. The experimental set-up is depicted on the left side of the figure. In the first column, a representative cell expressing GFP-S (top row) or Hsp105 WT-S (second row) amongst cells not expressing this protein is shown. The insert shows a 2x enlarged area of the dotted box. Scale bar, 20 μm. E. Cells expressing the indicated tagged construct are scored for the presence of at least one BAP31-positive focus in each cell, and the values normalized to GFP. The bar graphs represent mean values ± SD (n≥3). F. Cells transfected with either ctrl or Hsp105 siRNA #1 were subsequently transfected with the indicated tagged construct, and BAP31 foci were quantified as in (E). * p <0.05.
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Related In: Results  -  Collection

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Show All Figures
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ppat.1005086.g004: Hsp105 overexpression enhances SV40 extraction into the cytosol.A. COS-7 cells expressing the indicated S-tagged construct were infected with SV40 (MOI ~5). 12 hpi, cells were processed as in Fig 3A, and the samples immunoblotted using the indicated antibodies. B. VP1 band intensities of the cytosol-localized SV40 in (A) were quantified and normalized against the Hsp90 band intensity. Values represent the mean ± SD (n≥3). * p <0.05. C. Cells expressing the indicated F- or S-tagged construct are scored for the TAg expression after 24 hpi (MOI ~0.5), and the values normalized to GFP. The bar graphs represent mean values ± SD (n≥3). D. CV-1 cells expressing GFP-S or Hsp105 WT-S were infected with SV40 (MOI ~30) for 16 h. Cells were fixed, stained with BAP31 and VP1 antibodies, and imaged as in Fig 3E. The experimental set-up is depicted on the left side of the figure. In the first column, a representative cell expressing GFP-S (top row) or Hsp105 WT-S (second row) amongst cells not expressing this protein is shown. The insert shows a 2x enlarged area of the dotted box. Scale bar, 20 μm. E. Cells expressing the indicated tagged construct are scored for the presence of at least one BAP31-positive focus in each cell, and the values normalized to GFP. The bar graphs represent mean values ± SD (n≥3). F. Cells transfected with either ctrl or Hsp105 siRNA #1 were subsequently transfected with the indicated tagged construct, and BAP31 foci were quantified as in (E). * p <0.05.
Mentions: We used a gain-of-function strategy to assess Hsp105’s role in extracting SV40 into the cytosol. The CV-1 derived COS-7 cells were transfected with GFP-S, HspBP1-S, or Hsp105 WT-S, and subjected to the cytosol arrival assay as described above. COS-7 cells were used because of their ability to support a high DNA transfection efficiency required for this experiment. We found that over-expressing Hsp105 WT-S (Fig 4A, fourth panel, compare lane 3 to 2 and 1) but not HspBP1-S stimulated SV40 arrival to the cytosol (Fig 4A, first panel; the VP1 band intensity is quantified in Fig 4B). These findings demonstrate that Hsp105 stimulates SV40 extraction into the cytosol. Not surprisingly, Hsp105 WT overexpression also enhanced SV40 infection (Fig 4C, compare second to first bar). This stimulation requires Hsp105’s nucleotide exchange activity as overexpressing Hsp105 NE*-F or G*-F did not robustly enhance infection (Fig 4C, compare third and fourth bars to second bar), and is specific because overexpressing neither Hsc70, SGTA, nor HspBP1 stimulated infection.

Bottom Line: Here we uncover a novel role of this machinery in driving membrane translocation during viral entry.Combining biochemical, cell-based, and imaging approaches, we find that the Hsp110 family member Hsp105 associates with the ER membrane J-protein B14.Hence the energy provided by the Hsc70-dependent Hsp105 disaggregation machinery can be harnessed to catalyze a membrane translocation event.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, Michigan, United States of America.

ABSTRACT
Mammalian cytosolic Hsp110 family, in concert with the Hsc70:J-protein complex, functions as a disaggregation machinery to rectify protein misfolding problems. Here we uncover a novel role of this machinery in driving membrane translocation during viral entry. The non-enveloped virus SV40 penetrates the endoplasmic reticulum (ER) membrane to reach the cytosol, a critical infection step. Combining biochemical, cell-based, and imaging approaches, we find that the Hsp110 family member Hsp105 associates with the ER membrane J-protein B14. Here Hsp105 cooperates with Hsc70 and extracts the membrane-penetrating SV40 into the cytosol, potentially by disassembling the membrane-embedded virus. Hence the energy provided by the Hsc70-dependent Hsp105 disaggregation machinery can be harnessed to catalyze a membrane translocation event.

No MeSH data available.


Related in: MedlinePlus