Binding of pp60v-src to membranes: evidence for multiple membrane interactions

1992 ◽  
Vol 70 (10-11) ◽  
pp. 1187-1192 ◽  
Author(s):  
Lauren Silverman ◽  
Catherine T. Sigal ◽  
Marilyn D. Resh
Keyword(s):  
Membrane Binding ◽  
Cross Linking ◽  
Binding Characteristics ◽  
Binding Domains ◽  
Rous Sarcoma ◽  
Subcellular Membrane ◽  
Sarcoma Virus ◽  
The Relationship

Membrane association of pp60v-src, the myristylated transforming protein of Rous sarcoma virus, has been shown to be a receptor-mediated process, which is inhibited by myristylated src peptides containing the N-terminal 11 amino acids of the v-src sequence (MGYsrc). By cross-linking radiolabeled MGYsrc peptide to fibroblast membranes, a 32-kilodalton membrane protein was identified as a candidate src receptor. To elucidate the potential role of p32 in binding pp60v-src, we studied the relationship between binding of MGYsrc peptide and pp60v-src polypeptide to cellular membranes. The subcellular membrane distribution of p32 was distinct from that of pp60v-src in transformed cells. Moreover, under certain defined in vitro conditions, it was possible to inhibit peptide cross-linking to p32 without significantly affecting pp60v-src membrane binding. However, when internal sequences were removed from pp60v-src, the binding characteristics of the src deletion polypeptide and MGYsrc peptide became identical. These data indicate that the presence of internal membrane binding domains influences the interaction of myristylated N-terminal src sequences with p32, and suggest that accessory binding factors might be involved in establishing stable contact between pp60v-src and the membrane phospholipid bilayer.Key words: myristylation, pp60v-src, membrane binding, cross-linking.

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 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 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.

Tridegin, a Novel Peptidic Inhibitor of Factor XIIIa from the Leech, Haementeria ghilianii, Enhances Fibrinolysis In Vitro

1997 ◽  
Vol 77 (05) ◽  
pp. 0959-0963 ◽  
Author(s):  
Lisa Seale ◽  
Sarah Finney ◽  
Roy T Sawyer ◽  
Robert B Wallis
Keyword(s):  
Potent Inhibitor ◽  
Platelet Rich Plasma ◽  
Factor Xiiia ◽  
Cross Linking ◽  
Fibrinolytic Enzymes ◽  
Small Polypeptide ◽  
Tissue Plasminogen ◽  
Protein Cross Linking

SummaryTridegin is a potent inhibitor of factor Xllla from the leech, Haementeria ghilianii, which inhibits protein cross-linking. It modifies plasmin-mediated fibrin degradation as shown by the absence of D-dimer and approximately halves the time for fibrinolysis. Plasma clots formed in the presence of Tridegin lyse more rapidly when either streptokinase, tissue plasminogen activator or hementin is added 2 h after clot formation. The effect of Tridegin is markedly increased if clots are formed from platelet-rich plasma. Platelet-rich plasma clots are lysed much more slowly by the fibrinolytic enzymes used and if Tridegin is present, the rate of lysis returns almost to that of platelet- free clots. These studies indicate the important role of platelets in conferring resistance to commonly used fibrinolytic enzymes and suggest that protein cross-linking is an important step in this effect. Moreover they indicate that Tridegin, a small polypeptide, may have potential as an adjunct to thrombolytic therapy.

Role of the mitogenic property and kinase activity of p60src in tumor formation by Rous sarcoma virus.

1984 ◽  
Vol 49 (2) ◽  
pp. 325-332 ◽  
Author(s):  
F Poirier ◽  
P Jullien ◽  
P Dezelee ◽  
G Dambrine ◽  
E Esnault ◽  
...  
Keyword(s):  
Rous Sarcoma Virus ◽  
Kinase Activity ◽  
Tumor Formation ◽  
Rous Sarcoma ◽  
Sarcoma Virus

scholarly journals RNA Helicase A Regulates the Replication of RNA Viruses

Viruses ◽  
2021 ◽  
Vol 13 (3) ◽  
pp. 361
Author(s):  
Rui-Zhu Shi ◽  
Yuan-Qing Pan ◽  
Li Xing
Keyword(s):  
Virus Replication ◽  
Rna Binding ◽  
Rna Viruses ◽  
Rna Helicase ◽  
Rna Helicase A ◽  
Double Stranded Rna ◽  
Binding Domains ◽  
N Terminus

The RNA helicase A (RHA) is a member of DExH-box helicases and characterized by two double-stranded RNA binding domains at the N-terminus. RHA unwinds double-stranded RNA in vitro and is involved in RNA metabolisms in the cell. RHA is also hijacked by a variety of RNA viruses to facilitate virus replication. Herein, this review will provide an overview of the role of RHA in the replication of RNA viruses.

scholarly journals Role of interleukins in the pathogenesis of pulmonary fibrosis

2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Yi Xin She ◽  
Qing Yang Yu ◽  
Xiao Xiao Tang
Keyword(s):  
Pulmonary Fibrosis ◽  
Therapeutic Strategy ◽  
Future Directions ◽  
Regulation Mechanisms ◽  
Underlying Mechanisms ◽  
Multiple Cells ◽  
The Relationship

AbstractInterleukins, a group of cytokines participating in inflammation and immune response, are proved to be involved in the formation and development of pulmonary fibrosis. In this article, we reviewed the relationship between interleukins and pulmonary fibrosis from the clinical, animal, as well as cellular levels, and discussed the underlying mechanisms in vivo and in vitro. Despite the effects of interleukin-targeted treatment on experimental pulmonary fibrosis, clinical applications are lacking and unsatisfactory. We conclude that intervening in one type of interleukins with similar functions in IPF may not be enough to stop the development of fibrosis as it involves a complex network of regulation mechanisms. Intervening interleukins combined with other existing therapy or targeting interleukins affecting multiple cells/with different functions at the same time may be one of the future directions. Furthermore, the intervention time is critical as some interleukins play different roles at different stages. Further elucidation on these aspects would provide new perspectives on both the pathogenesis mechanism, as well as the therapeutic strategy and drug development.

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