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

Journal Article · · Journal of Virology
DOI:https://doi.org/10.1128/JVI.01628-15· OSTI ID:1249235
 [1];  [2];  [3];  [4];  [1];  [5];  [4];  [2];  [1]
  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)
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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.

Research Organization:
Argonne National Laboratory (ANL), Argonne, IL (United States)
Sponsoring Organization:
National Institutes of Health (NIH)
Grant/Contract Number:
R01GM107013; RO1GM101647; 70NANB13H009
OSTI ID:
1249235
Journal Information:
Journal of Virology, Vol. 89, Issue 20; ISSN 0022-538X
Publisher:
American Society for MicrobiologyCopyright Statement
Country of Publication:
United States
Language:
ENGLISH
Citation Metrics:
Cited by: 13 works
Citation information provided by
Web of Science

References (66)

A New Lipid Anchor for Sparsely Tethered Bilayer Lipid Membranes † journal April 2009
Phosphatidylinositol (4,5) bisphosphate regulates HIV-1 Gag targeting to the plasma membrane journal October 2004
Electrostatic Interactions Drive Membrane Association of the Human Immunodeficiency Virus Type 1 Gag MA Domain journal March 2007
Preparation of Recombinant HIV-1 Gag Protein and Assembly of Virus-Like Particles In Vitro book January 2009
Determination of the regularization parameter in indirect-transform methods using perceptual criteria journal August 1992
Comparison of Three Ternary Lipid Bilayer Mixtures: FRET and ESR Reveal Nanodomains journal November 2010
HIV-1 Gag protein can sense the cholesterol and acyl chain environment in model membranes journal September 2012
Solution Structure of the Human Immunodeficiency Virus Type 1 p6 Protein journal October 2005
Small-Angle X-Ray Scattering- and Nuclear Magnetic Resonance-Derived Conformational Ensemble of the Highly Flexible Antitoxin PaaA2 journal June 2014
Opposing mechanisms involving RNA and lipids regulate HIV-1 Gag membrane binding through the highly basic region of the matrix domain journal January 2010
PRIMUS: a Windows PC-based system for small-angle scattering data analysis journal September 2003
NADPH oxidase activator p67phox behaves in solution as a multidomain protein with semi-flexible linkers journal January 2010
Role of the Rous Sarcoma Virus p10 Domain in Shape Determination of Gag Virus-Like Particles Assembled In Vitro and within Escherichia coli journal November 2000
Retroviral Matrix Domains Share Electrostatic Homology: Models for Membrane Binding Function throughout the Viral Life Cycle journal October 2005
In Vitro Assembly of Retroviruses journal November 2014
Membrane lipids: where they are and how they behave journal February 2008
PIP 2 and Proteins: Interactions, Organization, and Information Flow journal June 2002
The Retroviral Capsid Domain Dictates Virion Size, Morphology, and Coassembly of Gag into Virus-Like Particles journal October 2005
An assembly domain of the Rous sarcoma virus Gag protein required late in budding. journal January 1994
RNA Dimerization Defect in a Rous Sarcoma Virus Matrix Mutant journal January 2000
X-ray solution scattering (SAXS) combined with crystallography and computation: defining accurate macromolecular structures, conformations and assemblies in solution journal August 2007
The NH2-terminal sequence of the avian oncovirus gag precursor polyprotein (Pr76gag) journal December 1978
The Structure of Immature Virus-Like Rous Sarcoma Virus Gag Particles Reveals a Structural Role for the p10 Domain in Assembly journal July 2015
Positionally independent and exchangeable late budding functions of the Rous sarcoma virus and human immunodeficiency virus Gag proteins. journal January 1995
HIV-1 Assembly, Budding, and Maturation journal March 2012
Dimension, Shape, and Conformational Flexibility of a Two Domain Fungal Cellulase in Solution Probed by Small Angle X-ray Scattering journal August 2002
How Random are Intrinsically Disordered Proteins? A Small Angle Scattering Perspective journal February 2012
Multimerizable HIV Gag derivative binds to the liquid-disordered phase in model membranes: HIV Gag derivative binds to L d phase journal December 2012
Membrane interaction of retroviral Gag proteins journal April 2014
Biochemical Characterization of Rous Sarcoma Virus MA Protein Interaction with Membranes journal April 2005
Binding of Human Immunodeficiency Virus Type 1 Gag to Membrane: Role of the Matrix Amino Terminus journal May 1999
Higher-Order Structure of the Rous Sarcoma Virus SP Assembly Domain journal March 2014
In vitro reconstitution of the ordered assembly of the endosomal sorting complex required for transport at membrane-bound HIV-1 Gag clusters journal October 2012
Continuous distribution model for the investigation of complex molecular architectures near interfaces with scattering techniques journal November 2011
A large region within the Rous sarcoma virus matrix protein is dispensable for budding and infectivity. journal January 1996
Crystal Structure of an Intact Type II DNA Topoisomerase: Insights into DNA Transfer Mechanisms journal March 2008
Conformation of the HIV-1 Gag Protein in Solution journal January 2007
HIV-1 Gag Extension: Conformational Changes Require Simultaneous Interaction with Membrane and Nucleic Acid journal February 2011
Conformational landscape of cytochrome c folding studied by microsecond-resolved small-angle x-ray scattering journal January 2002
Rous Sarcoma Virus Gag Has No Specific Requirement for Phosphatidylinositol-(4,5)-Bisphosphate for Plasma Membrane Association In Vivo or for Liposome Interaction In Vitro journal August 2011
Phase studies of model biomembranes: Complex behavior of DSPC/DOPC/Cholesterol journal November 2007
Solution structure and dynamics of the bioactive retroviral M domain from rous sarcoma virus journal June 1998
Gag Localization and Virus-Like Particle Release Mediated by the Matrix Domain of Human T-Lymphotropic Virus Type 1 Gag Are Less Dependent on Phosphatidylinositol-(4,5)-Bisphosphate than Those Mediated by the Matrix Domain of HIV-1 Gag journal February 2011
An LYPSL Late Domain in the Gag Protein Contributes to the Efficient Release and Replication of Rous Sarcoma Virus journal April 2010
Modulation of HIV-like particle assembly in vitro by inositol phosphates journal August 2001
Repositioning Basic Residues in the M Domain of the Rous Sarcoma Virus Gag Protein journal December 2000
Alterations in the MA and NC Domains Modulate Phosphoinositide-Dependent Plasma Membrane Localization of the Rous Sarcoma Virus Gag Protein journal January 2013
The HIV-1 Nucleocapsid Protein Recruits Negatively Charged Lipids To Ensure Its Optimal Binding to Lipid Membranes journal November 2014
The synthesis of a perdeuterated phospholipid: 1,2-dimyristoyl-sn-glycero-3-phosphocholine-d72 journal May 1979
Rous Sarcoma Virus Gag Protein-Oligonucleotide Interaction Suggests a Critical Role for Protein Dimer Formation in Assembly journal June 2002
Nucleic Acid Binding-Induced Gag Dimerization in the Assembly of Rous Sarcoma Virus Particles In Vitro journal December 2003
In vitro assembly of virus-like particles with Rous sarcoma virus Gag deletion mutants: identification of the p10 domain as a morphological determinant in the formation of spherical particles. journal January 1997
Determination of the molecular weight of proteins in solution from a single small-angle X-ray scattering measurement on a relative scale journal December 2009
PR Domain of Rous Sarcoma Virus Gag Causes an Assembly/Budding Defect in Insect Cells journal May 2001
Structure of the immature HIV-1 capsid in intact virus particles at 8.8 Å resolution journal November 2014
GUV preparation and imaging: Minimizing artifacts journal July 2010
Elements in HIV-1 Gag contributing to virus particle assembly journal February 2013
Phase diagram of a 4-component lipid mixture: DSPC/DOPC/POPC/chol journal September 2013
The p12 Domain Is Unstructured in a Murine Leukemia Virus p12-CAN Gag Construct journal April 2008
A molecular switch required for retrovirus assembly participates in the hexagonal immature lattice journal April 2008
Functional Redundancy in HIV-1 Viral Particle Assembly journal September 2012
Determination of molecular weights and frictional ratios of proteins in impure systems by use of gel filtration and density gradient centrifugation. Application to crude preparations of sulfite and hydroxylamine reductases journal February 1966
Lipid mixing in dimyristoyl phosphatidylcholine-dimyristoyl glycerol dispersions: spin label ESR studies journal January 1997
SAXS Study of the PIR Domain from the Grb14 Molecular Adaptor: A Natively Unfolded Protein with a Transient Structure Primer? journal December 2004
Solution Properties of Murine Leukemia Virus Gag Protein: Differences from HIV-1 Gag journal September 2011
Self-assembly in vitro of purified CA-NC proteins from Rous sarcoma virus and human immunodeficiency virus type 1. journal January 1995

Cited By (2)

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

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