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Title: Hydrodynamic and Membrane Binding Properties of Purified Rous Sarcoma Virus Gag Protein

Abstract

Previously, no retroviral Gag protein has been highly purified in milligram quantities and in a biologically relevant and active form. We have purified Rous sarcoma virus (RSV) Gag protein and in parallel several truncation mutants of Gag and have studied their biophysical properties and membrane interactions in vitro. RSV Gag is unusual in that it is not naturally myristoylated. From its ability to assemble into virus-like particles in vitro, we infer that RSV Gag is biologically active. By size exclusion chromatography and small-angle X-ray scattering, Gag in solution appears extended and flexible, in contrast to previous reports on unmyristoylated HIV-1 Gag, which is compact. However, by neutron reflectometry measurements of RSV Gag bound to a supported bilayer, the protein appears to adopt a more compact, folded-over conformation. At physiological ionic strength, purified Gag binds strongly to liposomes containing acidic lipids. This interaction is stimulated by physiological levels of phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P2] and by cholesterol. However, unlike HIV-1 Gag, RSV Gag shows no sensitivity to acyl chain saturation. In contrast with full-length RSV Gag, the purified MA domain of Gag binds to liposomes only weakly. Similarly, both an N-terminally truncated version of Gag that is missing the MA domain and a C-terminallymore » truncated version that is missing the NC domain bind only weakly. These results imply that NC contributes to membrane interaction in vitro, either by directly contacting acidic lipids or by promoting Gag multimerization. Retroviruses like HIV assemble at and bud from the plasma membrane of cells. Assembly requires the interaction between thousands of Gag molecules to form a lattice. Previous work indicated that lattice formation at the plasma membrane is influenced by the conformation of monomeric HIV. We have extended this work to the more tractable RSV Gag. Our results show that RSV Gag is highly flexible and can adopt a folded-over conformation on a lipid bilayer, implicating both the N and C termini in membrane binding. In addition, binding of Gag to membranes is diminished when either terminal domain is truncated. RSV Gag membrane association is significantly less sensitive than HIV Gag membrane association to lipid acyl chain saturation. Lastly, these findings shed light on Gag assembly and membrane binding, critical steps in the viral life cycle and an untapped target for antiretroviral drugs.« less

Authors:
 [1];  [2];  [3];  [4];  [1];  [5];  [4];  [2];  [1]
+ Show Author Affiliations
  1. Cornell Univ., Ithaca, NY (United States)
  2. National Cancer Inst., Frederick, MD (United States)
  3. National Inst. of Standards and Technology (NIST), Gaithersburg, MD (United States); Carnegie Mellon Univ., Pittsburgh, PA (United States)
  4. National Inst. of Health, Frederick, MD (United States)
  5. Carnegie Mellon Univ., Pittsburgh, PA (United States)
Publication Date:
Research Org.:
Argonne National Laboratory (ANL), Argonne, IL (United States)
Sponsoring Org.:
National Institutes of Health (NIH)
OSTI Identifier:
1249235
Grant/Contract Number:  
R01GM107013; RO1GM101647; 70NANB13H009
Resource Type:
Accepted Manuscript
Journal Name:
Journal of Virology
Additional Journal Information:
Journal Volume: 89; Journal Issue: 20; Journal ID: ISSN 0022-538X
Publisher:
American Society for Microbiology
Country of Publication:
United States
Language:
ENGLISH
Subject:
59 BASIC BIOLOGICAL SCIENCES

Citation Formats

Dick, Robert A., Datta, Siddhartha A. K., Nanda, Hirsh, Fang, Xianyang, Wen, Yi, Barros, Marilia, Wang, Yun-Xing, Rein, Alan, and Vogt, Volker M. Hydrodynamic and Membrane Binding Properties of Purified Rous Sarcoma Virus Gag Protein. United States: N. p., 2015. Web. doi:10.1128/JVI.01628-15.
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Dick, Robert A., Datta, Siddhartha A. K., Nanda, Hirsh, Fang, Xianyang, Wen, Yi, Barros, Marilia, Wang, Yun-Xing, Rein, Alan, & Vogt, Volker M. Hydrodynamic and Membrane Binding Properties of Purified Rous Sarcoma Virus Gag Protein. United States. https://doi.org/10.1128/JVI.01628-15
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Dick, Robert A., Datta, Siddhartha A. K., Nanda, Hirsh, Fang, Xianyang, Wen, Yi, Barros, Marilia, Wang, Yun-Xing, Rein, Alan, and Vogt, Volker M. Tue . "Hydrodynamic and Membrane Binding Properties of Purified Rous Sarcoma Virus Gag Protein". United States. https://doi.org/10.1128/JVI.01628-15. https://www.osti.gov/servlets/purl/1249235.
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@article{osti_1249235,
title = {Hydrodynamic and Membrane Binding Properties of Purified Rous Sarcoma Virus Gag Protein},
author = {Dick, Robert A. and Datta, Siddhartha A. K. and Nanda, Hirsh and Fang, Xianyang and Wen, Yi and Barros, Marilia and Wang, Yun-Xing and Rein, Alan and Vogt, Volker M.},
abstractNote = {Previously, no retroviral Gag protein has been highly purified in milligram quantities and in a biologically relevant and active form. We have purified Rous sarcoma virus (RSV) Gag protein and in parallel several truncation mutants of Gag and have studied their biophysical properties and membrane interactions in vitro. RSV Gag is unusual in that it is not naturally myristoylated. From its ability to assemble into virus-like particles in vitro, we infer that RSV Gag is biologically active. By size exclusion chromatography and small-angle X-ray scattering, Gag in solution appears extended and flexible, in contrast to previous reports on unmyristoylated HIV-1 Gag, which is compact. However, by neutron reflectometry measurements of RSV Gag bound to a supported bilayer, the protein appears to adopt a more compact, folded-over conformation. At physiological ionic strength, purified Gag binds strongly to liposomes containing acidic lipids. This interaction is stimulated by physiological levels of phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P2] and by cholesterol. However, unlike HIV-1 Gag, RSV Gag shows no sensitivity to acyl chain saturation. In contrast with full-length RSV Gag, the purified MA domain of Gag binds to liposomes only weakly. Similarly, both an N-terminally truncated version of Gag that is missing the MA domain and a C-terminally truncated version that is missing the NC domain bind only weakly. These results imply that NC contributes to membrane interaction in vitro, either by directly contacting acidic lipids or by promoting Gag multimerization. Retroviruses like HIV assemble at and bud from the plasma membrane of cells. Assembly requires the interaction between thousands of Gag molecules to form a lattice. Previous work indicated that lattice formation at the plasma membrane is influenced by the conformation of monomeric HIV. We have extended this work to the more tractable RSV Gag. Our results show that RSV Gag is highly flexible and can adopt a folded-over conformation on a lipid bilayer, implicating both the N and C termini in membrane binding. In addition, binding of Gag to membranes is diminished when either terminal domain is truncated. RSV Gag membrane association is significantly less sensitive than HIV Gag membrane association to lipid acyl chain saturation. Lastly, these findings shed light on Gag assembly and membrane binding, critical steps in the viral life cycle and an untapped target for antiretroviral drugs.},
doi = {10.1128/JVI.01628-15},
journal = {Journal of Virology},
number = 20,
volume = 89,
place = {United States},
year = {Tue Sep 22 00:00:00 EDT 2015},
month = {Tue Sep 22 00:00:00 EDT 2015}
}
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https://doi.org/10.1128/JVI.01628-15
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    Works referencing / citing this record:

    The matrix domain of the Gag protein from avian sarcoma virus contains a PI(4,5)P 2 -binding site that targets Gag to the cell periphery
    journal, October 2018

    • Watanabe, Susan M.; Medina, Gisselle N.; Eastep, Gunnar N.
    • Journal of Biological Chemistry, Vol. 293, Issue 49
    • DOI: 10.1074/jbc.ra118.003947

    Structural basis for targeting avian sarcoma virus Gag polyprotein to the plasma membrane for virus assembly
    journal, October 2018

    • Vlach, Jiri; Eastep, Gunnar N.; Ghanam, Ruba H.
    • Journal of Biological Chemistry, Vol. 293, Issue 49
    • DOI: 10.1074/jbc.ra118.003944
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