scholarly journals Membrane Binding of the Rous Sarcoma Virus Gag Protein Is Cooperative and Dependent on the Spacer Peptide Assembly Domain

2015 ◽  
Vol 90 (5) ◽  
pp. 2473-2485 ◽  
Author(s):  
Robert A. Dick ◽  
Marilia Barros ◽  
Danni Jin ◽  
Mathias Lösche ◽  
Volker M. Vogt
Keyword(s):  
Plasma Membrane ◽  
Nucleic Acid ◽  
Rous Sarcoma Virus ◽  
Membrane Binding ◽  
Membrane Interaction ◽  
Gag Proteins ◽  
Peptide Assembly ◽  
Rous Sarcoma ◽  
Sarcoma Virus

ABSTRACTThe principles underlying membrane binding and assembly of retroviral Gag proteins into a lattice are understood. However, little is known about how these processes are related. Using purified Rous sarcoma virus Gag and Gag truncations, we studied the interrelation of Gag-Gag interaction and Gag-membrane interaction. Both by liposome binding and by surface plasmon resonance on a supported bilayer, Gag bound to membranes much more tightly than did matrix (MA), the isolated membrane binding domain. In principle, this difference could be explained either by protein-protein interactions leading to cooperativity in membrane binding or by the simultaneous interaction of the N-terminal MA and the C-terminal nucleocapsid (NC) of Gag with the bilayer, since both are highly basic. However, we found that NC was not required for strong membrane binding. Instead, the spacer peptide assembly domain (SPA), a putative 24-residue helical sequence comprising the 12-residue SP segment of Gag and overlapping the capsid (CA) C terminus and the NC N terminus, was required. SPA is known to be critical for proper assembly of the immature Gag lattice. A single amino acid mutation in SPA that abrogates assemblyin vitrodramatically reduced binding of Gag to liposomes.In vivo, plasma membrane localization was dependent on SPA. Disulfide cross-linking based on ectopic Cys residues showed that the contacts between Gag proteins on the membrane are similar to the known contacts in virus-like particles. Taken together, we interpret these results to mean that Gag membrane interaction is cooperative in that it depends on the ability of Gag to multimerize.IMPORTANCEThe retroviral structural protein Gag has three major domains. The N-terminal MA domain interacts directly with the plasma membrane (PM) of cells. The central CA domain, together with immediately adjoining sequences, facilitates the assembly of thousands of Gag molecules into a lattice. The C-terminal NC domain interacts with the genome, resulting in packaging of viral RNA. For assemblyin vitrowith purified Gag, in the absence of membranes, binding of NC to nucleic acid somehow facilitates further Gag-Gag interactions that lead to formation of the Gag lattice. The contributions of MA-mediated membrane binding to virus particle assembly are not well understood. Here, we report that in the absence of nucleic acid, membranes provide a platform that facilitates Gag-Gag interactions. This study demonstrates that the binding of Gag, but not of MA, to membranes is cooperative and identifies SPA as a major factor that controls this cooperativity.

scholarly journals Hydrodynamic and Membrane Binding Properties of Purified Rous Sarcoma Virus Gag Protein

2015 ◽  
Vol 89 (20) ◽  
pp. 10371-10382 ◽  
Author(s):  
Robert A. Dick ◽  
Siddhartha A. K. Datta ◽  
Hirsh Nanda ◽  
Xianyang Fang ◽  
Yi Wen ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Rous Sarcoma Virus ◽  
Acyl Chain ◽  
Membrane Binding ◽  
Membrane Association ◽  
Gag Protein ◽  
Rous Sarcoma ◽  
Sarcoma Virus ◽  
Hiv 1

ABSTRACTPreviously, 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 interactionsin vitro. RSV Gag is unusual in that it is not naturally myristoylated. From its ability to assemble into virus-like particlesin 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 interactionin vitro, either by directly contacting acidic lipids or by promoting Gag multimerization.IMPORTANCERetroviruses 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.

scholarly journals Characterization of Rous Sarcoma Virus Gag Particles Assembled In Vitro

2001 ◽  
Vol 75 (6) ◽  
pp. 2753-2764 ◽  
Author(s):  
Fang Yu ◽  
Swati M. Joshi ◽  
Yu May Ma ◽  
Richard L. Kingston ◽  
Martha N. Simon ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Rous Sarcoma Virus ◽  
Radial Distance ◽  
Binding Motif ◽  
Gag Protein ◽  
Binding Motifs ◽  
Rous Sarcoma ◽  
Sarcoma Virus ◽  
The Mean

ABSTRACT Purified retrovirus Gag proteins or Gag protein fragments are able to assemble into virus-like particles (VLPs) in vitro in the presence of RNA. We have examined the role of nucleic acid and of the NC domain in assembly of VLPs from a Rous sarcoma virus (RSV) Gag protein and have characterized these VLPs using transmission electron microscopy (TEM), scanning TEM (STEM), and cryoelectron microscopy (cryo-EM). RNAs of diverse sizes, single-stranded DNA oligonucleotides as small as 22 nucleotides, double-stranded DNA, and heparin all promoted efficient assembly. The percentages of nucleic acid by mass, in the VLPs varied from 5 to 8%. The mean mass of VLPs, as determined by STEM, was 6.5 × 107 Da for both RNA-containing and DNA oligonucleotide-containing particles, corresponding to a stoichiometry of about 1,200 protein molecules per VLP, slightly lower than the 1,500 Gag molecules estimated previously for infectious RSV. By cryo-EM, the VLPs showed the characteristic morphology of immature retroviruses, with discernible regions of high density corresponding to the two domains of the CA protein. In spherically averaged density distributions, the mean radial distance to the density corresponding to the C-terminal domain of CA was 33 nm, considerably smaller than that of equivalent human immunodeficiency virus type 1 particles. Deletions of the distal portion of NC, including the second Zn-binding motif, had little effect on assembly, but deletions including the charged residues between the two Zn-binding motifs abrogated assembly. Mutation of the cysteine and histidine residues in the first Zn-binding motif to alanine did not affect assembly, but mutation of the basic residues between the two Zn-binding motifs, or of the basic residues in the N-terminal portion of NC, abrogated assembly. Together, these findings establish VLPs as a good model for immature virions and establish a foundation for dissection of the interactions that lead to assembly.

scholarly journals Nucleic Acid Binding-Induced Gag Dimerization in the Assembly of Rous Sarcoma Virus Particles In Vitro

2004 ◽  
Vol 78 (1) ◽  
pp. 52-60 ◽  
Author(s):  
Yu May Ma ◽  
Volker M. Vogt
Keyword(s):  
Nucleic Acid ◽  
Protein Interactions ◽  
Rous Sarcoma Virus ◽  
Minimum Length ◽  
Protein Protein Interactions ◽  
Ph Change ◽  
Rous Sarcoma ◽  
Sarcoma Virus

ABSTRACT As also found for other retroviruses, the Rous sarcoma virus structural protein Gag is necessary and sufficient for formation of virus-like particles (VLPs). Purified polypeptide fragments comprising most of Gag spontaneously assemble in vitro at pH 6.5 into VLPs lacking a membrane, a process that requires nucleic acid. We showed previously that the minimum length of a DNA oligonucleotide that can support efficient assembly is 16 nucleotides (nt), twice the protein's binding site size. This observation suggests that the essential role of nucleic acid in assembly is to promote the formation of Gag dimers. In order to gain further insight into the role of dimerization, we have studied the assembly properties of two proteins, a nearly full-length Gag (ΔMBDΔPR) capable of proper in vitro assembly and a smaller Gag fragment (CTD-NC) capable of forming only irregular aggregates but with the same pH and oligonucleotide length requirements as for assembly with the larger protein. In analyses by sedimentation velocity and by cross-linking, both proteins remained monomeric in the absence of oligonucleotides or in the presence of an oligonucleotide of length 8 nt (GT8). At pH 8, which does not support assembly, binding to GT16 induced the formation of dimers of ΔMBDΔPR but not of CTD-NC, implying that dimerization requires the N-terminal domain of the capsid moiety of Gag. Assembly of VLPs was induced by shifting the pH of dimeric complexes of ΔMBDΔPR and GT16 from 8 to 6.5. An analogue of GT16 with a ribonucleotide linkage in the middle also supported dimer formation at pH 8. Even after quantitative cleavage of the oligonucleotide by treatment of the complex with RNase, these dimers could be triggered to undergo assembly by pH change. This result implies that protein-protein interactions stabilize the dimer. We propose that binding of two adjacent Gag molecules on a stretch of nucleic acid leads to protein-protein interactions that create a Gag dimer and that this species has an exposed surface not present in monomers which allows polymerization of the dimers into a spherical shell.

scholarly journals Characterization of pp60src phosphorylation in vitro in Rous sarcoma virus-transformed cell membranes.

1985 ◽  
Vol 5 (5) ◽  
pp. 916-922 ◽  
Author(s):  
M D Resh ◽  
R L Erikson
Keyword(s):  
Plasma Membrane ◽  
Rous Sarcoma Virus ◽  
Membrane Vesicles ◽  
Terminal Region ◽  
Transformed Cell ◽  
Amino Terminal ◽  
Rous Sarcoma ◽  
Sarcoma Virus ◽  
Carboxy Terminal

Phosphorylation of the src gene product pp60v-src was studied in plasma membrane fractions prepared from Rous sarcoma virus-transformed vole cells. Upon addition of [gamma-32P]ATP to isolated membrane vesicles, phosphate was incorporated into a 60,000-dalton polypeptide identified as pp60v-src. In the presence of vanadate, pp60v-src phosphorylation was stimulated ca. 30-fold. At low concentrations of ATP (1 microM), this reaction occurred almost exclusively on the carboxy-terminal 26,000-dalton region of pp60v-src. However, at higher ATP concentrations (100 microM), additional sites of phosphorylation were evident in the amino-terminal 34,000-dalton region. Kinetic analyses, performed under conditions in which ATP hydrolysis was minimal, revealed that the phosphorylation reaction at the carboxy terminus exhibited a higher Vmax and a lower Km for ATP than those occurring at the amino terminus. In addition, the amino-terminal region of pp60v-src was more rapidly dephosphorylated than the carboxy-terminal region. These results indicate that interaction of pp60v-src with the plasma membrane may limit the extent of amino-terminal phosphorylation by lowering the rate of the reaction and the affinity for the substrate while increasing its susceptibility to phosphoprotein phosphatases. We suggest that the use of transformed-cell membrane preparations provides a model system for studying the possible regulatory roles of phosphorylation and dephosphorylation on pp60v-src function.

scholarly journals An infectious Rous Sarcoma Virus Gag mutant that is defective in nuclear cycling

2021 ◽  
Author(s):  
Clifton L Ricana ◽  
Marc C Johnson
Keyword(s):  
Plasma Membrane ◽  
Rous Sarcoma Virus ◽  
Nuclear Export ◽  
Membrane Binding ◽  
Viral Proteins ◽  
Cis Acting ◽  
Rous Sarcoma ◽  
Virion Release ◽  
Sarcoma Virus ◽  
Plasma Membrane Binding

During retroviral replication, unspliced viral genomic RNA (gRNA) must escape the nucleus for translation into viral proteins and packaging into virions. 'Complex' retroviruses such as Human Immunodeficiency Virus (HIV) use cis-acting elements on the unspliced gRNA in conjunction with trans-acting viral proteins to facilitate this escape. 'Simple' retroviruses such as Mason-Pfizer Monkey Virus (MPMV) and Murine Leukemia Virus (MLV) exclusively use cis-acting elements on the gRNA in conjunction with host nuclear export proteins for nuclear escape. Uniquely, the simple retrovirus Rous Sarcoma Virus (RSV) has a Gag structural protein that cycles through the nucleus prior to plasma membrane binding. This trafficking has been implicated in facilitating gRNA nuclear export and is thought to be a required mechanism. Previously described mutants that abolish nuclear cycling displayed enhanced plasma membrane binding, enhanced virion release, and a significant loss in genome incorporation resulting in loss of infectivity. Here, we describe a nuclear cycling deficient RSV Gag mutant that has similar plasma membrane binding and genome incorporation to WT virus and surprisingly, is replication competent albeit with a slower rate of spread compared to WT. This mutant suggests that RSV Gag nuclear cycling is not strictly required for RSV replication.

scholarly journals An infectious Rous Sarcoma Virus Gag mutant that is defective in nuclear cycling

2021 ◽  
Author(s):  
Clifton L Ricaña ◽  
Marc C. Johnson
Keyword(s):  
Plasma Membrane ◽  
Rous Sarcoma Virus ◽  
Nuclear Export ◽  
Membrane Binding ◽  
Viral Proteins ◽  
Cis Acting ◽  
Rous Sarcoma ◽  
Virion Release ◽  
Sarcoma Virus ◽  
Plasma Membrane Binding

During retroviral replication, unspliced viral genomic RNA (gRNA) must escape the nucleus for translation into viral proteins and packaging into virions. “Complex” retroviruses such as Human Immunodeficiency Virus (HIV) use cis-acting elements on the unspliced gRNA in conjunction with trans-acting viral proteins to facilitate this escape. “Simple” retroviruses such as Mason-Pfizer Monkey Virus (MPMV) and Murine Leukemia Virus (MLV) exclusively use cis-acting elements on the gRNA in conjunction with host nuclear export proteins for nuclear escape. Uniquely, the simple retrovirus Rous Sarcoma Virus (RSV) has a Gag structural protein that cycles through the nucleus prior to plasma membrane binding. This trafficking has been implicated in facilitating gRNA nuclear export and is thought to be a required mechanism. Previously described mutants that abolish nuclear cycling displayed enhanced plasma membrane binding, enhanced virion release, and a significant loss in genome incorporation resulting in loss of infectivity. Here, we describe a nuclear cycling deficient RSV Gag mutant that has similar plasma membrane binding and genome incorporation to WT virus and surprisingly, is replication competent albeit with a slower rate of spread compared to WT. This mutant suggests that RSV Gag nuclear cycling is not strictly required for RSV replication. Importance While mechanisms for retroviral Gag assembly at the plasma membrane are beginning to be characterized, characterization of intermediate trafficking locales remain elusive. This is in part due to the difficulty of tracking individual proteins from translation to plasma membrane binding. RSV Gag nuclear cycling is a unique phenotype that may provide comparative insight to viral trafficking evolution and may present a model intermediate to cis- and trans-acting mechanisms for gRNA export.

scholarly journals Repositioning Basic Residues in the M Domain of the Rous Sarcoma Virus Gag Protein

2000 ◽  
Vol 74 (23) ◽  
pp. 11222-11229 ◽  
Author(s):  
Eric M. Callahan ◽  
John W. Wills
Keyword(s):  
Plasma Membrane ◽  
Rous Sarcoma Virus ◽  
Electrostatic Interactions ◽  
Particle Production ◽  
Gag Protein ◽  
Particle Release ◽  
Gag Proteins ◽  
Rous Sarcoma ◽  
Sarcoma Virus ◽  
Acidic Phospholipids

ABSTRACT The first 86 residues of the Rous sarcoma virus (RSV) Gag protein form a membrane-binding (M) domain that directs Gag to the plasma membrane during budding. Unlike other retroviral Gag proteins, RSV Gag is not myristylated; however, the RSV M domain does contain 11 basic residues that could potentially interact with acidic phospholipids in the plasma membrane. To investigate this possibility, we analyzed mutants in which basic residues in the M domain were replaced with asparagines or glutamines. The data show that neutralizing as few as two basic residues in the M domain blocked particle release and prevented Gag from localizing to the plasma membrane. Though not as severe, single neutralizations also diminished budding and, when expressed in the context of proviral clones, reduced the ability of RSV to spread in cell cultures. To further explore the role of basic residues in particle production, we added lysines to new positions in the M domain. Using this approach, we found that the budding efficiency of RSV Gag can be improved by adding pairs of lysines and that the basic residues in the M domain can be repositioned without affecting particle release. These data provide the first gain-of-function evidence for the importance of basic residues in a retroviral M domain and support a model in which RSV Gag binds to the plasma membrane via electrostatic interactions.

scholarly journals Rous Sarcoma Virus Gag Protein-Oligonucleotide Interaction Suggests a Critical Role for Protein Dimer Formation in Assembly

2002 ◽  
Vol 76 (11) ◽  
pp. 5452-5462 ◽  
Author(s):  
Yu May Ma ◽  
Volker M. Vogt
Keyword(s):  
Nucleic Acid ◽  
Binding Site ◽  
Rous Sarcoma Virus ◽  
Free Form ◽  
Zinc Ions ◽  
Gag Protein ◽  
Rous Sarcoma ◽  
Sarcoma Virus

ABSTRACT The structural protein Gag is the only viral product required for retrovirus assembly. Purified Gag proteins or fragments of Gag are able in vitro to spontaneously form particles resembling immature virions, but this process requires nucleic acid, as well as the nucleocapsid domain of Gag. To examine the role of nucleic acid in the assembly in vitro, we used a purified, slightly truncated version of the Rous sarcoma virus Gag protein, ΔMBDΔPR, and DNA oligonucleotides composed of the simple repeating sequence GT. Apparent binding constants were determined for oligonucleotides of different lengths, and from these values the binding site size of the protein on the DNA was calculated. The ability of the oligonucleotides to promote assembly in vitro was assessed with a quantitative assay based on electron microscopy. We found that excess zinc or magnesium ion inhibited the formation of virus-like particles without interfering with protein-DNA binding, implying that interaction with nucleic acid is necessary but not sufficient for assembly in vitro. The binding site size of the ΔMBDΔPR protein, purified in the presence of EDTA to remove zinc ions at the two cysteine-histidine motifs, was estimated to be 11 nucleotides (nt). This value decreased to 8 nt when the protein was purified in the presence of low concentrations of zinc ions. The minimum length of DNA oligonucleotide that promoted efficient assembly in vitro was 22 nt for the zinc-free form of the protein and 16 nt for the zinc-bound form. To account for this striking 1:2 ratio between binding site size and oligonucleotide length requirement, we propose a model in which the role of nucleic acid in assembly is to promote formation of a species of Gag dimer, which itself is a critical intermediate in the polymerizaton of Gag to form the protein shell of the immature virion.

scholarly journals Late Domain Function Identified in the Vesicular Stomatitis Virus M Protein by Use of Rhabdovirus-Retrovirus Chimeras

1999 ◽  
Vol 73 (4) ◽  
pp. 3359-3365 ◽  
Author(s):  
Rebecca C. Craven ◽  
Ronald N. Harty ◽  
Jason Paragas ◽  
Peter Palese ◽  
John W. Wills
Keyword(s):  
Vesicular Stomatitis Virus ◽  
Rous Sarcoma Virus ◽  
M Protein ◽  
Enveloped Viruses ◽  
Gag Proteins ◽  
Rous Sarcoma ◽  
M Proteins ◽  
Sarcoma Virus ◽  
Vesicular Stomatitis

ABSTRACT Little is known about the mechanisms used by enveloped viruses to separate themselves from the cell surface at the final step of budding. However, small sequences in the Gag proteins of several retroviruses (L domains) have been implicated in this process. A sequence has been identified in the M proteins of rhabdoviruses that closely resembles the PPPPY motif in the L domain of Rous sarcoma virus (RSV), an avian retrovirus. To evaluate whether the PPPY sequence in vesicular stomatitis virus (VSV) M protein has an activity analogous to that of the retroviral sequence, M-Gag chimeras were characterized. The N-terminal 74 amino acids of the VSV (Indiana) M protein, including the PPPY motif, was able to replace the L domain of RSV Gag and allow the assembly and release of virus-like particles. Alanine substitutions in the VSV PPPY motif severely compromised the budding activity of this hybrid protein but not that of another chimera which also contained the RSV PPPPY sequence. We conclude that this VSV sequence is functionally homologous to the RSV L domain in promoting virus particle release, making this the first example of such an activity in a virus other than a retrovirus. Both the RSV and VSV motifs have been shown to interact in vitro with certain cellular proteins that contain a WW interaction module, suggesting that the L domains are sites of interaction with unknown host machinery involved in virus release.

Characterization of pp60src phosphorylation in vitro in Rous sarcoma virus-transformed cell membranes

1985 ◽  
Vol 5 (5) ◽  
pp. 916-922
Author(s):  
M D Resh ◽  
R L Erikson
Keyword(s):  
Plasma Membrane ◽  
Rous Sarcoma Virus ◽  
Membrane Vesicles ◽  
Terminal Region ◽  
Transformed Cell ◽  
Amino Terminal ◽  
Rous Sarcoma ◽  
Sarcoma Virus ◽  
Carboxy Terminal

Phosphorylation of the src gene product pp60v-src was studied in plasma membrane fractions prepared from Rous sarcoma virus-transformed vole cells. Upon addition of [gamma-32P]ATP to isolated membrane vesicles, phosphate was incorporated into a 60,000-dalton polypeptide identified as pp60v-src. In the presence of vanadate, pp60v-src phosphorylation was stimulated ca. 30-fold. At low concentrations of ATP (1 microM), this reaction occurred almost exclusively on the carboxy-terminal 26,000-dalton region of pp60v-src. However, at higher ATP concentrations (100 microM), additional sites of phosphorylation were evident in the amino-terminal 34,000-dalton region. Kinetic analyses, performed under conditions in which ATP hydrolysis was minimal, revealed that the phosphorylation reaction at the carboxy terminus exhibited a higher Vmax and a lower Km for ATP than those occurring at the amino terminus. In addition, the amino-terminal region of pp60v-src was more rapidly dephosphorylated than the carboxy-terminal region. These results indicate that interaction of pp60v-src with the plasma membrane may limit the extent of amino-terminal phosphorylation by lowering the rate of the reaction and the affinity for the substrate while increasing its susceptibility to phosphoprotein phosphatases. We suggest that the use of transformed-cell membrane preparations provides a model system for studying the possible regulatory roles of phosphorylation and dephosphorylation on pp60v-src function.

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