Limits...
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 is indispensable for SV40 cytosol arrival.A. CV-1 cells transfected with the indicated siRNAs for 24 h were incubated with SV40 (MOI ~5), harvested 12 hpi, and processed according to the semi-permeabilized cytosol arrival assay (see Methods). Hsp90 and PDI serve as markers for the cytosol and membrane fraction, respectively. B. Relative VP1 band intensities in the cytosol fraction in (A) were quantified. Data are normalized to ctrl siRNA. Values represent the mean ± SD (n = 3). C. Membrane fraction in (A) was solubilized in a buffer containing 1% Triton X-100. After centrifugation, the extracted material containing ER-localized SV40 was analyzed by immunoblotting with VP1 antibodies (top panel). Relative VP1 band intensities in the ER-localized fraction were quantified as in (B) (bottom panel). D. As in (A), except cells were treated with 10 nM cholera toxin for 90 min before harvesting and analyzing using the indicated antibodies. E. CV-1 cells were transfected with either ctrl or Hsp105 siRNA #1. After 24 h, cells were infected with SV40 (MOI ~20) for 16 h. Cells were then fixed, stained, and analyzed by immunofluorescence microscopy. The experimental set-up is depicted on the left side of the figure. The insert shows a 2x enlarged area of the dotted box. Scale bar, 20 μm. F. The siRNA-transfected cells were scored for the presence of at least one BAP31-positive focus in each cell, and the values normalized to the ctrl siRNA. The bar graphs represent mean values ± SD (n≥3). ** p <0.01. G. Results in (F) were further assessed by quantifying the number of BAP31 foci/cell under control and knockdown conditions, or the size of the BAP31 foci based on the measured area (in pixels) using the ImageJ software. * p <0.05, ** p <0.01, *** p <0.001.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4526233&req=5

ppat.1005086.g003: Hsp105 is indispensable for SV40 cytosol arrival.A. CV-1 cells transfected with the indicated siRNAs for 24 h were incubated with SV40 (MOI ~5), harvested 12 hpi, and processed according to the semi-permeabilized cytosol arrival assay (see Methods). Hsp90 and PDI serve as markers for the cytosol and membrane fraction, respectively. B. Relative VP1 band intensities in the cytosol fraction in (A) were quantified. Data are normalized to ctrl siRNA. Values represent the mean ± SD (n = 3). C. Membrane fraction in (A) was solubilized in a buffer containing 1% Triton X-100. After centrifugation, the extracted material containing ER-localized SV40 was analyzed by immunoblotting with VP1 antibodies (top panel). Relative VP1 band intensities in the ER-localized fraction were quantified as in (B) (bottom panel). D. As in (A), except cells were treated with 10 nM cholera toxin for 90 min before harvesting and analyzing using the indicated antibodies. E. CV-1 cells were transfected with either ctrl or Hsp105 siRNA #1. After 24 h, cells were infected with SV40 (MOI ~20) for 16 h. Cells were then fixed, stained, and analyzed by immunofluorescence microscopy. The experimental set-up is depicted on the left side of the figure. The insert shows a 2x enlarged area of the dotted box. Scale bar, 20 μm. F. The siRNA-transfected cells were scored for the presence of at least one BAP31-positive focus in each cell, and the values normalized to the ctrl siRNA. The bar graphs represent mean values ± SD (n≥3). ** p <0.01. G. Results in (F) were further assessed by quantifying the number of BAP31 foci/cell under control and knockdown conditions, or the size of the BAP31 foci based on the measured area (in pixels) using the ImageJ software. * p <0.05, ** p <0.01, *** p <0.001.

Mentions: As Hsp105 localizes to the ER membrane and is important in SV40 infection, we hypothesized that it might promote the extraction of SV40 into the cytosol from the ER membrane. To test this, we monitored arrival of SV40 into the cytosol from the ER membrane using a semi-permeabilized cytosol arrival assay established previously [37,39]. In this assay, siRNA transfected CV-1 cells were harvested post-infection and treated with a low concentration of digitonin to semi-permeabilize the plasma membrane without affecting internal membranes. Subsequent centrifugation generates two fractions, a supernatant fraction that harbors cytosolic proteins and virus that reaches the cytosol (referred as “cytosolic” fraction), and a pellet fraction that contains membranes including the ER, as well as associated viral particles (referred as “membrane” fraction). The cytosolic Hsp90 was found predominantly in the cytosolic fraction (Fig 3A, second panel), while the ER marker protein disulfide isomerase (PDI) was found exclusively in the membrane fraction (Fig 3A, seventh panel), verifying the integrity of the fractionation procedure. Importantly, using this assay, silencing Hsp105 by either siRNA (Fig 3A, fourth panel) markedly decreased the VP1 level in the cytosol (Fig 3A, first panel, compare lanes 2 and 3 to 1; the VP1 band intensity is quantified in Fig 3B). These data indicate that Hsp105 exerts an important role in promoting cytosol arrival of SV40 from the ER. The residual VP1 observed in the cytosol fraction when Hsp105 is knocked down is consistent with the low infection observed when this chaperone is down-regulated (Fig 2B).


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 is indispensable for SV40 cytosol arrival.A. CV-1 cells transfected with the indicated siRNAs for 24 h were incubated with SV40 (MOI ~5), harvested 12 hpi, and processed according to the semi-permeabilized cytosol arrival assay (see Methods). Hsp90 and PDI serve as markers for the cytosol and membrane fraction, respectively. B. Relative VP1 band intensities in the cytosol fraction in (A) were quantified. Data are normalized to ctrl siRNA. Values represent the mean ± SD (n = 3). C. Membrane fraction in (A) was solubilized in a buffer containing 1% Triton X-100. After centrifugation, the extracted material containing ER-localized SV40 was analyzed by immunoblotting with VP1 antibodies (top panel). Relative VP1 band intensities in the ER-localized fraction were quantified as in (B) (bottom panel). D. As in (A), except cells were treated with 10 nM cholera toxin for 90 min before harvesting and analyzing using the indicated antibodies. E. CV-1 cells were transfected with either ctrl or Hsp105 siRNA #1. After 24 h, cells were infected with SV40 (MOI ~20) for 16 h. Cells were then fixed, stained, and analyzed by immunofluorescence microscopy. The experimental set-up is depicted on the left side of the figure. The insert shows a 2x enlarged area of the dotted box. Scale bar, 20 μm. F. The siRNA-transfected cells were scored for the presence of at least one BAP31-positive focus in each cell, and the values normalized to the ctrl siRNA. The bar graphs represent mean values ± SD (n≥3). ** p <0.01. G. Results in (F) were further assessed by quantifying the number of BAP31 foci/cell under control and knockdown conditions, or the size of the BAP31 foci based on the measured area (in pixels) using the ImageJ software. * p <0.05, ** p <0.01, *** p <0.001.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4526233&req=5

ppat.1005086.g003: Hsp105 is indispensable for SV40 cytosol arrival.A. CV-1 cells transfected with the indicated siRNAs for 24 h were incubated with SV40 (MOI ~5), harvested 12 hpi, and processed according to the semi-permeabilized cytosol arrival assay (see Methods). Hsp90 and PDI serve as markers for the cytosol and membrane fraction, respectively. B. Relative VP1 band intensities in the cytosol fraction in (A) were quantified. Data are normalized to ctrl siRNA. Values represent the mean ± SD (n = 3). C. Membrane fraction in (A) was solubilized in a buffer containing 1% Triton X-100. After centrifugation, the extracted material containing ER-localized SV40 was analyzed by immunoblotting with VP1 antibodies (top panel). Relative VP1 band intensities in the ER-localized fraction were quantified as in (B) (bottom panel). D. As in (A), except cells were treated with 10 nM cholera toxin for 90 min before harvesting and analyzing using the indicated antibodies. E. CV-1 cells were transfected with either ctrl or Hsp105 siRNA #1. After 24 h, cells were infected with SV40 (MOI ~20) for 16 h. Cells were then fixed, stained, and analyzed by immunofluorescence microscopy. The experimental set-up is depicted on the left side of the figure. The insert shows a 2x enlarged area of the dotted box. Scale bar, 20 μm. F. The siRNA-transfected cells were scored for the presence of at least one BAP31-positive focus in each cell, and the values normalized to the ctrl siRNA. The bar graphs represent mean values ± SD (n≥3). ** p <0.01. G. Results in (F) were further assessed by quantifying the number of BAP31 foci/cell under control and knockdown conditions, or the size of the BAP31 foci based on the measured area (in pixels) using the ImageJ software. * p <0.05, ** p <0.01, *** p <0.001.
Mentions: As Hsp105 localizes to the ER membrane and is important in SV40 infection, we hypothesized that it might promote the extraction of SV40 into the cytosol from the ER membrane. To test this, we monitored arrival of SV40 into the cytosol from the ER membrane using a semi-permeabilized cytosol arrival assay established previously [37,39]. In this assay, siRNA transfected CV-1 cells were harvested post-infection and treated with a low concentration of digitonin to semi-permeabilize the plasma membrane without affecting internal membranes. Subsequent centrifugation generates two fractions, a supernatant fraction that harbors cytosolic proteins and virus that reaches the cytosol (referred as “cytosolic” fraction), and a pellet fraction that contains membranes including the ER, as well as associated viral particles (referred as “membrane” fraction). The cytosolic Hsp90 was found predominantly in the cytosolic fraction (Fig 3A, second panel), while the ER marker protein disulfide isomerase (PDI) was found exclusively in the membrane fraction (Fig 3A, seventh panel), verifying the integrity of the fractionation procedure. Importantly, using this assay, silencing Hsp105 by either siRNA (Fig 3A, fourth panel) markedly decreased the VP1 level in the cytosol (Fig 3A, first panel, compare lanes 2 and 3 to 1; the VP1 band intensity is quantified in Fig 3B). These data indicate that Hsp105 exerts an important role in promoting cytosol arrival of SV40 from the ER. The residual VP1 observed in the cytosol fraction when Hsp105 is knocked down is consistent with the low infection observed when this chaperone is down-regulated (Fig 2B).

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