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Hydrodynamic and Membrane Binding Properties of Purified Rous Sarcoma Virus Gag Protein.
J Virol. 2015 Oct; 89(20):10371-82.JV

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-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.

IMPORTANCE

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. These findings shed light on Gag assembly and membrane binding, critical steps in the viral life cycle and an untapped target for antiretroviral drugs.

Authors+Show Affiliations

Department of MolecularBiology and Genetics, Cornell University, Ithaca, New York, USA rad82@cornell.edu dattasi@mail.nih.gov.HIV Dynamics and Replication Program, Center for Cancer Research, National Cancer Institute, Frederick, Maryland, USA rad82@cornell.edu dattasi@mail.nih.gov.NIST Center for Neutron Research, National Institute of Standards and Technology, Gaithersburg, Maryland, USA Department of Physics, Carnegie Mellon University, Pittsburgh, Pennsylvania, USA.Protein-Nucleic Acid Interaction Section, Structural Biophysics Laboratory, National Cancer Institute, National Institutes of Health, Frederick, Maryland, USA.Department of MolecularBiology and Genetics, Cornell University, Ithaca, New York, USA.Department of Physics, Carnegie Mellon University, Pittsburgh, Pennsylvania, USA.Protein-Nucleic Acid Interaction Section, Structural Biophysics Laboratory, National Cancer Institute, National Institutes of Health, Frederick, Maryland, USA.HIV Dynamics and Replication Program, Center for Cancer Research, National Cancer Institute, Frederick, Maryland, USA.Department of MolecularBiology and Genetics, Cornell University, Ithaca, New York, USA.

Pub Type(s)

Journal Article
Research Support, N.I.H., Extramural
Research Support, N.I.H., Intramural
Research Support, U.S. Gov't, Non-P.H.S.

Language

eng

PubMed ID

26246573

Citation

Dick, Robert A., et al. "Hydrodynamic and Membrane Binding Properties of Purified Rous Sarcoma Virus Gag Protein." Journal of Virology, vol. 89, no. 20, 2015, pp. 10371-82.
Dick RA, Datta SA, Nanda H, et al. Hydrodynamic and Membrane Binding Properties of Purified Rous Sarcoma Virus Gag Protein. J Virol. 2015;89(20):10371-82.
Dick, R. A., Datta, S. A., Nanda, H., Fang, X., Wen, Y., Barros, M., Wang, Y. X., Rein, A., & Vogt, V. M. (2015). Hydrodynamic and Membrane Binding Properties of Purified Rous Sarcoma Virus Gag Protein. Journal of Virology, 89(20), 10371-82. https://doi.org/10.1128/JVI.01628-15
Dick RA, et al. Hydrodynamic and Membrane Binding Properties of Purified Rous Sarcoma Virus Gag Protein. J Virol. 2015;89(20):10371-82. PubMed PMID: 26246573.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Hydrodynamic and Membrane Binding Properties of Purified Rous Sarcoma Virus Gag Protein. AU - Dick,Robert A, AU - Datta,Siddhartha A K, AU - Nanda,Hirsh, AU - Fang,Xianyang, AU - Wen,Yi, AU - Barros,Marilia, AU - Wang,Yun-Xing, AU - Rein,Alan, AU - Vogt,Volker M, Y1 - 2015/08/05/ PY - 2015/06/23/received PY - 2015/07/28/accepted PY - 2015/8/7/entrez PY - 2015/8/8/pubmed PY - 2015/12/19/medline SP - 10371 EP - 82 JF - Journal of virology JO - J. Virol. VL - 89 IS - 20 N2 - UNLABELLED: 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. IMPORTANCE: 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. These findings shed light on Gag assembly and membrane binding, critical steps in the viral life cycle and an untapped target for antiretroviral drugs. SN - 1098-5514 UR - https://www.unboundmedicine.com/medline/citation/26246573/Hydrodynamic_and_Membrane_Binding_Properties_of_Purified_Rous_Sarcoma_Virus_Gag_Protein_ L2 - http://jvi.asm.org/cgi/pmidlookup?view=long&pmid=26246573 DB - PRIME DP - Unbound Medicine ER -