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 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 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 Genetic Studies of the β-Hairpin Loop of Rous Sarcoma Virus Capsid Protein

2006 ◽  
Vol 81 (3) ◽  
pp. 1288-1296 ◽  
Author(s):  
Jared L. Spidel ◽  
Carol B. Wilson ◽  
Rebecca C. Craven ◽  
John W. Wills
Keyword(s):  
Binding Site ◽  
Rous Sarcoma Virus ◽  
Critical Role ◽  
Strong Support ◽  
Genome Replication ◽  
Consensus Binding Site ◽  
Rous Sarcoma ◽  
Capsid Shell ◽  
Sarcoma Virus

ABSTRACT The first few residues of the Rous sarcoma virus (RSV) CA protein comprise a structurally dynamic region that forms part of a Gag-Gag interface in immature virus particles. Dissociation of this interaction during maturation allows refolding and formation of a β-hairpin structure important for assembly of CA monomers into the mature capsid shell. A consensus binding site for the cellular Ubc9 protein was previously identified within this region, suggesting that binding of Ubc9 and subsequent small ubiquitin-like modifier protein 1 (SUMO-1) modification of CA may play a role either in regulating the assembly activity of CA in immature particles or mature cores or in controlling postentry function(s) during the establishment of infection. In the present study, mutations designed to eliminate the consensus binding site were used to dissect the potentially overlapping functions of these residues. The resulting replication defects could not be traced to a failure to form particles of normal composition but, rather, to a deficit in genome replication. Genetic suppressors of two detrimental β-hairpin mutations improved infectivity without restoring the consensus site or creating a novel one elsewhere. Optimal restoration of infectivity to a Lys-to-Arg mutant required a combination of secondary changes, one on the surface of each domain of CA. Rather than arguing for a critical role of Ubc9 and SUMO in RSV replication, these findings provide strong support for a structural role of the N-terminal residues and a particularly striking example of long-range interactions between regions of CA in achieving a functional core competent for genome replication.

scholarly journals Changes in Rous Sarcoma Virus RNA Secondary Structure near the Primer Binding Site upon tRNATrpPrimer Annealing

1999 ◽  
Vol 73 (8) ◽  
pp. 6307-6318 ◽  
Author(s):  
Shannon Morris ◽  
Jonathan Leis
Keyword(s):  
Binding Site ◽  
Rous Sarcoma Virus ◽  
Primer Binding Site ◽  
Wild Type ◽  
Stem Loop ◽  
Rous Sarcoma ◽  
Sarcoma Virus ◽  
Primer Annealing ◽  
Nuclease Mapping

ABSTRACT Predicted secondary-structure elements encompassing the primer binding site in the 5′ untranslated region of Rous sarcoma virus (RSV) RNA play an integral role in multiple viral replications steps including reverse transcription, DNA integration, and RNA packaging (A. Aiyar, D. Cobrinik, Z. Ge, H. J. Kung, and J. Leis, J. Virol. 66:2464–2472, 1992; D. Cobrinik, A. Aiyar, Z. Ge, M. Katzman, H. Huang, and J. Leis, J. Virol. 65:3864–3872, 1991; J. T. Miller, Z. Ge, S. Morris, K. Das, and J. Leis, J. Virol. 71:7648–7656, 1997). These elements include the U5-Leader stem, U5-IR stem-loop, and U5-TΨC interaction region. Limited digestion of the 5′ untranslated region of wild-type and mutant RSV RNAs with structure- and/or sequence-specific RNases detects the presence of the U5-Leader stem and the U5-IR stem-loop. When a tRNATrp primer is annealed to wild-type RNAs in vitro, limited nuclease mapping indicates that the U5-IR stem becomes partially unwound. This is not observed when mutant RNAs with altered U5-IR stem-loop structures are substituted for wild-type RNAs. The U5-Leader stem also becomes destabilized when the tRNA primer is annealed to either wild-type or mutant RNA fragments. Nuclease mapping studies of tRNATrp, as well as the viral RNA, indicate that the U5-TΨC helix does form in vitro upon primer annealing. Collectively, these data suggest that the various structural elements near the RSV primer binding site undergo significant changes during the process of primer annealing.

scholarly journals In Vivo Interference of Rous Sarcoma Virus Budding by cis Expression of a WW Domain

2002 ◽  
Vol 76 (6) ◽  
pp. 2789-2795 ◽  
Author(s):  
Akash Patnaik ◽  
John W. Wills
Keyword(s):  
Plasma Membrane ◽  
Rous Sarcoma Virus ◽  
Gag Protein ◽  
Ww Domain ◽  
Particle Release ◽  
Rous Sarcoma ◽  
Sarcoma Virus ◽  
Membrane Budding

ABSTRACT For all enveloped viruses, the actual mechanism by which nascent virus particles separate or “pinch off” from the cell surface is largely unknown. In the case of retroviruses, the Gag protein drives the budding process, and the virus release step is directed by the late (L) assembly domain within Gag. A PPPPY motif within the L domain of Rous sarcoma virus (RSV) was previously characterized as being critical for the release of virions and shown to interact in vitro with the WW domain of Yes-associated protein (Yap). To determine whether WW domain-L domain interactions can occur in vivo, we attempted to interfere with the host cell machinery normally recruited to the site of budding by inserting this WW domain in different locations within Gag. At a C-terminal location, the WWYap domain had no effect on budding, suggesting that the intervening I domains (which provide the major region of Gag-Gag interaction) prevent its access to the L domain. When positioned on the other side of the I domains closer to the L domain, the WWYap domain resulted in a dramatic interference of particle release, and confocal microscopy revealed a block to budding on the plasma membrane. Budding was restored by attachment of the heterologous L domain of human immunodeficiency virus type 1 Gag, which does not bind WWYap. These findings suggest that cis expression of WW domains can interfere with RSV particle release in vivo via specific, high-affinity interactions at the site of assembly on the plasma membrane, thus preventing host factor accessibility to the L domain and subsequent virus-cell separation. In addition, they suggest that L domain-specific host factors function after Gag proteins begin to interact.

scholarly journals Role of the Rous Sarcoma Virus p10 Domain in Shape Determination of Gag Virus-Like Particles Assembled In Vitro and within Escherichia coli

2000 ◽  
Vol 74 (21) ◽  
pp. 10260-10268 ◽  
Author(s):  
Swati M. Joshi ◽  
Volker M. Vogt
Keyword(s):  
Escherichia Coli ◽  
Rous Sarcoma Virus ◽  
Gag Protein ◽  
Virus Like Particles ◽  
Gag Proteins ◽  
Rous Sarcoma ◽  
Crude Extracts ◽  
Sarcoma Virus ◽  
Shape Determination

ABSTRACT Purified retrovirus Gag proteins can assemble in vitro into virus-like particles (VLPs) in the presence of RNA. It was shown previously that a Rous sarcoma virus Gag protein missing only the protease domain forms spherical particles resembling immature virions lacking a membrane but that a similar protein missing the p10 domain forms tubular particles. Thus, p10 plays a role in spherical particle formation. To further study this shape-determining function, we dissected the p10 domain by mutagenesis and examined VLPs assembled within Escherichia coli or assembled in vitro from purified proteins. The results identified a minimal contiguous segment of 25 amino acid residues at the C terminus of p10 that is sufficient to restore efficient spherical assembly to a p10 deletion mutant. Random and site-directed mutations were introduced into this segment of polypeptide, and the shapes of particles formed in E. coliwere examined in crude extracts by electron microscopy. Three phenotypes were observed: tubular morphology, spherical morphology, or no regular structure. While the particle morphology visualized in crude extracts generally was the same as that visualized for purified proteins, some tubular mutants scored as spherical when tested as purified proteins, suggesting that a cellular factor may also play a role in shape determination. We also examined the assembly properties of smaller Gag proteins consisting of the capsid protein-nucleocapsid protein (CA-NC) domains with short N-terminal extensions or deletions. Addition of one or three residues allowed CA-NC to form spheres instead of tubes in vitro, but the efficiency of assembly was extremely low. Deletion of the N-terminal residue(s) abrogated assembly. Taken together, these results imply that the N terminus of CA and the adjacent upstream 25 residues play an important role in the polymerization of the Gag protein.

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