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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 engages ER membrane-penetrating SV40 and promotes disassembly of the virus.A. The indicated purified protein in Fig 2E was incubated with DTT/EGTA-treated SV40. SV40 was immunoprecipitated from the sample and analyzed by immunoblotting using the indicated antibodies. B. CV-1 cells expressing the indicated S-tagged protein were infected with SV40 (MOI ~10). 12 hpi, S-tagged proteins affinity purified from WCE and immunoblot was performed with the indicated antibodies. C. As in (B), except cells were infected with SV40 for the indicated time. D. Cells expressing Hsp105 WT-S were infected with SV40 for 12 h, cross-linked with DSP, and fractionated to generate a cytosol and membrane fraction. Hsp105 WT-S was affinity isolated from each fraction, and the samples analyzed by immunoblotting with the indicated antibodies. E. As in (B), except the indicated F-tagged constructs were expressed in the cells. F. Triton X-100 extracted, ER-localized SV40 was incubated with the indicated purified protein(s), and subjected to discontinuous sucrose gradient centrifugation. Fractions were collected (as shown on the left side of the figure) from the top of the gradient and analyzed for the presence of SV40 by immunoblotting using VP1 antibodies.
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ppat.1005086.g005: Hsp105 engages ER membrane-penetrating SV40 and promotes disassembly of the virus.A. The indicated purified protein in Fig 2E was incubated with DTT/EGTA-treated SV40. SV40 was immunoprecipitated from the sample and analyzed by immunoblotting using the indicated antibodies. B. CV-1 cells expressing the indicated S-tagged protein were infected with SV40 (MOI ~10). 12 hpi, S-tagged proteins affinity purified from WCE and immunoblot was performed with the indicated antibodies. C. As in (B), except cells were infected with SV40 for the indicated time. D. Cells expressing Hsp105 WT-S were infected with SV40 for 12 h, cross-linked with DSP, and fractionated to generate a cytosol and membrane fraction. Hsp105 WT-S was affinity isolated from each fraction, and the samples analyzed by immunoblotting with the indicated antibodies. E. As in (B), except the indicated F-tagged constructs were expressed in the cells. F. Triton X-100 extracted, ER-localized SV40 was incubated with the indicated purified protein(s), and subjected to discontinuous sucrose gradient centrifugation. Fractions were collected (as shown on the left side of the figure) from the top of the gradient and analyzed for the presence of SV40 by immunoblotting using VP1 antibodies.

Mentions: Mechanistically, we envision that Hsp105 binds to SV40 at the ER-cytosol interface to extract the membrane-embedded virus into the cytosol. To determine if the Hsp105-SV40 interaction is direct, we incubated purified SV40 with purified proteins (Fig 2E); SGTA-F or GFP-F serves as a positive and negative control, respectively. In this experiment, SV40 was pretreated with the reducing agent dithiothreitol (DTT) and the calcium chelator EGTA to partially mimic conformational altered virus [36,40]. Importantly, when the virus was precipitated from the sample (Fig 5A, second panel), Hsp105 WT and mutant co-precipitated (Fig 5A, first panel, lanes 3–5); as expected, SGTA-F was pulled down but not GFP-F (Fig 5A, first panel, compare lane 1 to 2). Thus Hsp105 can interact with SV40 directly; because the Hsp105 mutants also bind to SV40 in vitro, they are unlikely globally misfolded.


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 engages ER membrane-penetrating SV40 and promotes disassembly of the virus.A. The indicated purified protein in Fig 2E was incubated with DTT/EGTA-treated SV40. SV40 was immunoprecipitated from the sample and analyzed by immunoblotting using the indicated antibodies. B. CV-1 cells expressing the indicated S-tagged protein were infected with SV40 (MOI ~10). 12 hpi, S-tagged proteins affinity purified from WCE and immunoblot was performed with the indicated antibodies. C. As in (B), except cells were infected with SV40 for the indicated time. D. Cells expressing Hsp105 WT-S were infected with SV40 for 12 h, cross-linked with DSP, and fractionated to generate a cytosol and membrane fraction. Hsp105 WT-S was affinity isolated from each fraction, and the samples analyzed by immunoblotting with the indicated antibodies. E. As in (B), except the indicated F-tagged constructs were expressed in the cells. F. Triton X-100 extracted, ER-localized SV40 was incubated with the indicated purified protein(s), and subjected to discontinuous sucrose gradient centrifugation. Fractions were collected (as shown on the left side of the figure) from the top of the gradient and analyzed for the presence of SV40 by immunoblotting using VP1 antibodies.
© Copyright Policy
Related In: Results  -  Collection

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

ppat.1005086.g005: Hsp105 engages ER membrane-penetrating SV40 and promotes disassembly of the virus.A. The indicated purified protein in Fig 2E was incubated with DTT/EGTA-treated SV40. SV40 was immunoprecipitated from the sample and analyzed by immunoblotting using the indicated antibodies. B. CV-1 cells expressing the indicated S-tagged protein were infected with SV40 (MOI ~10). 12 hpi, S-tagged proteins affinity purified from WCE and immunoblot was performed with the indicated antibodies. C. As in (B), except cells were infected with SV40 for the indicated time. D. Cells expressing Hsp105 WT-S were infected with SV40 for 12 h, cross-linked with DSP, and fractionated to generate a cytosol and membrane fraction. Hsp105 WT-S was affinity isolated from each fraction, and the samples analyzed by immunoblotting with the indicated antibodies. E. As in (B), except the indicated F-tagged constructs were expressed in the cells. F. Triton X-100 extracted, ER-localized SV40 was incubated with the indicated purified protein(s), and subjected to discontinuous sucrose gradient centrifugation. Fractions were collected (as shown on the left side of the figure) from the top of the gradient and analyzed for the presence of SV40 by immunoblotting using VP1 antibodies.
Mentions: Mechanistically, we envision that Hsp105 binds to SV40 at the ER-cytosol interface to extract the membrane-embedded virus into the cytosol. To determine if the Hsp105-SV40 interaction is direct, we incubated purified SV40 with purified proteins (Fig 2E); SGTA-F or GFP-F serves as a positive and negative control, respectively. In this experiment, SV40 was pretreated with the reducing agent dithiothreitol (DTT) and the calcium chelator EGTA to partially mimic conformational altered virus [36,40]. Importantly, when the virus was precipitated from the sample (Fig 5A, second panel), Hsp105 WT and mutant co-precipitated (Fig 5A, first panel, lanes 3–5); as expected, SGTA-F was pulled down but not GFP-F (Fig 5A, first panel, compare lane 1 to 2). Thus Hsp105 can interact with SV40 directly; because the Hsp105 mutants also bind to SV40 in vitro, they are unlikely globally misfolded.

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