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Volker M Vogt - One of the best experts on this subject based on the ideXlab platform.
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the structure of Immature Virus like rous sarcoma Virus gag particles reveals a structural role for the p10 domain in assembly
Journal of Virology, 2015Co-Authors: Florian K. M. Schur, Wim J. H. Hagen, Robert A Dick, Volker M Vogt, John A. G. BriggsAbstract:ABSTRACT The polyprotein Gag is the primary structural component of retroViruses. Gag consists of independently folded domains connected by flexible linkers. Interactions between the conserved capsid (CA) domains of Gag mediate formation of hexameric protein lattices that drive assembly of Immature Virus particles. Proteolytic cleavage of Gag by the viral protease (PR) is required for maturation of retroViruses from an Immature form into an infectious form. Within the assembled Gag lattices of HIV-1 and Mason-Pfizer monkey Virus (M-PMV), the C-terminal domain of CA adopts similar quaternary arrangements, while the N-terminal domain of CA is packed in very different manners. Here, we have used cryo-electron tomography and subtomogram averaging to study in vitro -assembled, Immature Virus-like Rous sarcoma Virus (RSV) Gag particles and have determined the structure of CA and the surrounding regions to a resolution of ∼8 A. We found that the C-terminal domain of RSV CA is arranged similarly to HIV-1 and M-PMV, whereas the N-terminal domain of CA adopts a novel arrangement in which the upstream p10 domain folds back into the CA lattice. In this position the cleavage site between CA and p10 appears to be inaccessible to PR. Below CA, an extended density is consistent with the presence of a six-helix bundle formed by the spacer-peptide region. We have also assessed the affect of lattice assembly on proteolytic processing by exogenous PR. The cleavage between p10 and CA is indeed inhibited in the assembled lattice, a finding consistent with structural regulation of proteolytic maturation. IMPORTANCE RetroViruses first assemble into Immature Virus particles, requiring interactions between Gag proteins that form a protein layer under the viral membrane. Subsequently, Gag is cleaved by the viral protease enzyme into separate domains, leading to rearrangement of the Virus into its infectious form. It is important to understand how Gag is arranged within Immature retroViruses, in order to understand how Virus assembly occurs, and how maturation takes place. We used the techniques cryo-electron tomography and subtomogram averaging to obtain a detailed structural picture of the CA domains in Immature assembled Rous sarcoma Virus Gag particles. We found that part of Gag next to CA, called p10, folds back and interacts with CA when Gag assembles. This arrangement is different from that seen in HIV-1 and Mason-Pfizer monkey Virus, illustrating further structural diversity of retroviral structures. The structure provides new information on how the Virus assembles and undergoes maturation.
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nmr relaxation studies of an rna binding segment of the rous sarcoma Virus gag polyprotein in free and bound states a model for autoinhibition of assembly
Biochemistry, 2010Co-Authors: Gwen M Taylor, Volker M Vogt, Carol Beth PostAbstract:Assembly of retroVirus particles is promoted by interaction of the Gag polyprotein with RNA. Nonspecific RNA association with the nucleocapsid domain (NC) of Gag induces the dimerization of Gag through protein−protein contacts in the capsid domain (CA), followed by higher order assembly to form the Immature Virus particle. NMR relaxation studies were conducted to investigate the initial steps of Rous sarcoma Virus (RSV) assembly by examining the association with nucleic acid of a fragment of Gag comprising the C-terminal domain of CA (CTD) postulated to mediate Gag dimerization, the spacer region between CA and NC (SP), and NC. This fragment, CTD-SP-NC (residues 394−577), spans the critical SP region and allows assessment of this key Gag−nucleic acid interaction in the context of the Gag polyprotein rather than the isolated domains. Main-chain amide relaxation of CTD-SP-NC was measured in the absence and presence of (GT)4, an 8-mer DNA oligonucleotide that binds tightly to the polyprotein but is too short...
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cryo electron microscopy reveals conserved and divergent features of gag packing in Immature particles of rous sarcoma Virus and human immunodeficiency Virus
Journal of Molecular Biology, 2006Co-Authors: John A. G. Briggs, Martha N Simon, Stephen D Fuller, Marc C Johnson, Volker M VogtAbstract:RetroVirus assembly proceeds via multimerisation of the major structural protein, Gag, into a tightly packed, spherical particle that buds from the membrane of the host cell. The lateral packing arrangement of the human immunodeficiency Virus type 1 (HIV-1) Gag CA (capsid) domain in the Immature Virus has been described. Here we have used cryo-electron microscopy (cryo-EM) and image processing to determine the lateral and radial arrangement of Gag in in vivo and in vitro assembled Rous sarcoma Virus (RSV) particles and to compare these features with those of HIV-1. We found that the lateral packing arrangement in the vicinity of the inner sub-domain of CA is conserved between these retroViruses. The curvature of the lattice, however, is different. RSV Gag protein adopts a more tightly curved lattice than is seen in HIV-1, and the virions therefore contain fewer copies of Gag. In addition, consideration of the relationship between the radial position of different Gag domains and their lateral spacings in particles of different diameters, suggests that the N-terminal MA (matrix) domain does not form a single, regular lattice in Immature retroVirus particles.
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The stoichiometry of Gag protein in HIV-1
Nature Structural & Molecular Biology, 2004Co-Authors: John A. G. Briggs, Hans-georg Kräusslich, Volker M Vogt, Martha N Simon, Ingolf Gross, Stephen D Fuller, Marc C JohnsonAbstract:The major structural components of HIV-1 are encoded as a single polyprotein, Gag, which is sufficient for Virus particle assembly. Initially, Gag forms an approximately spherical shell underlying the membrane of the Immature particle. After proteolytic maturation of Gag, the capsid (CA) domain of Gag reforms into a conical shell enclosing the RNA genome. This mature shell contains 1,000–1,500 CA proteins assembled into a hexameric lattice with a spacing of 10 nm. By contrast, little is known about the structure of the Immature Virus. We used cryo-EM and scanning transmission EM to determine that an average (145 nm diameter) complete Immature HIV particle contains ∼5,000 structural (Gag) proteins, more than twice the number from previous estimates. In the Immature Virus, Gag forms a hexameric lattice with a spacing of 8.0 nm. Thus, less than half of the CA proteins form the mature core.
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the stoichiometry of gag protein in hiv 1
Nature Structural & Molecular Biology, 2004Co-Authors: John A. G. Briggs, Hans-georg Kräusslich, Volker M Vogt, Martha N Simon, Ingolf Gross, Stephen D Fuller, Marc C JohnsonAbstract:The major structural components of HIV-1 are encoded as a single polyprotein, Gag, which is sufficient for Virus particle assembly. Initially, Gag forms an approximately spherical shell underlying the membrane of the Immature particle. After proteolytic maturation of Gag, the capsid (CA) domain of Gag reforms into a conical shell enclosing the RNA genome. This mature shell contains 1,000-1,500 CA proteins assembled into a hexameric lattice with a spacing of 10 nm. By contrast, little is known about the structure of the Immature Virus. We used cryo-EM and scanning transmission EM to determine that an average (145 nm diameter) complete Immature HIV particle contains approximately 5,000 structural (Gag) proteins, more than twice the number from previous estimates. In the Immature Virus, Gag forms a hexameric lattice with a spacing of 8.0 nm. Thus, less than half of the CA proteins form the mature core.
John A. G. Briggs - One of the best experts on this subject based on the ideXlab platform.
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the structure of Immature Virus like rous sarcoma Virus gag particles reveals a structural role for the p10 domain in assembly
Journal of Virology, 2015Co-Authors: Florian K. M. Schur, Wim J. H. Hagen, Robert A Dick, Volker M Vogt, John A. G. BriggsAbstract:ABSTRACT The polyprotein Gag is the primary structural component of retroViruses. Gag consists of independently folded domains connected by flexible linkers. Interactions between the conserved capsid (CA) domains of Gag mediate formation of hexameric protein lattices that drive assembly of Immature Virus particles. Proteolytic cleavage of Gag by the viral protease (PR) is required for maturation of retroViruses from an Immature form into an infectious form. Within the assembled Gag lattices of HIV-1 and Mason-Pfizer monkey Virus (M-PMV), the C-terminal domain of CA adopts similar quaternary arrangements, while the N-terminal domain of CA is packed in very different manners. Here, we have used cryo-electron tomography and subtomogram averaging to study in vitro -assembled, Immature Virus-like Rous sarcoma Virus (RSV) Gag particles and have determined the structure of CA and the surrounding regions to a resolution of ∼8 A. We found that the C-terminal domain of RSV CA is arranged similarly to HIV-1 and M-PMV, whereas the N-terminal domain of CA adopts a novel arrangement in which the upstream p10 domain folds back into the CA lattice. In this position the cleavage site between CA and p10 appears to be inaccessible to PR. Below CA, an extended density is consistent with the presence of a six-helix bundle formed by the spacer-peptide region. We have also assessed the affect of lattice assembly on proteolytic processing by exogenous PR. The cleavage between p10 and CA is indeed inhibited in the assembled lattice, a finding consistent with structural regulation of proteolytic maturation. IMPORTANCE RetroViruses first assemble into Immature Virus particles, requiring interactions between Gag proteins that form a protein layer under the viral membrane. Subsequently, Gag is cleaved by the viral protease enzyme into separate domains, leading to rearrangement of the Virus into its infectious form. It is important to understand how Gag is arranged within Immature retroViruses, in order to understand how Virus assembly occurs, and how maturation takes place. We used the techniques cryo-electron tomography and subtomogram averaging to obtain a detailed structural picture of the CA domains in Immature assembled Rous sarcoma Virus Gag particles. We found that part of Gag next to CA, called p10, folds back and interacts with CA when Gag assembles. This arrangement is different from that seen in HIV-1 and Mason-Pfizer monkey Virus, illustrating further structural diversity of retroviral structures. The structure provides new information on how the Virus assembles and undergoes maturation.
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Cryo-electron microscopy of tubular arrays of HIV-1 Gag resolves structures essential for Immature Virus assembly
Proceedings of the National Academy of Sciences of the United States of America, 2014Co-Authors: Tanmay A.m. Bharat, Luis R. Castillo Menendez, Wim J. H. Hagen, Vanda Lux, Sébastien Igonet, Martin Schorb, Florian K. M. Schur, Hans-georg Kräusslich, John A. G. BriggsAbstract:The assembly of HIV-1 is mediated by oligomerization of the major structural polyprotein, Gag, into a hexameric protein lattice at the plasma membrane of the infected cell. This leads to budding and release of progeny Immature Virus particles. Subsequent proteolytic cleavage of Gag triggers rearrangement of the particles to form mature infectious virions. Obtaining a structural model of the assembled lattice of Gag within Immature Virus particles is necessary to understand the interactions that mediate assembly of HIV-1 particles in the infected cell, and to describe the substrate that is subsequently cleaved by the viral protease. An 8-A resolution structure of an Immature Virus-like tubular array assembled from a Gag-derived protein of the related retroVirus Mason–Pfizer monkey Virus (M-PMV) has previously been reported, and a model for the arrangement of the HIV-1 capsid (CA) domains has been generated based on homology to this structure. Here we have assembled tubular arrays of a HIV-1 Gag-derived protein with an Immature-like arrangement of the C-terminal CA domains and have solved their structure by using hybrid cryo-EM and tomography analysis. The structure reveals the arrangement of the C-terminal domain of CA within an Immature-like HIV-1 Gag lattice, and provides, to our knowledge, the first high-resolution view of the region immediately downstream of CA, which is essential for assembly, and is significantly different from the respective region in M-PMV. Our results reveal a hollow column of density for this region in HIV-1 that is compatible with the presence of a six-helix bundle at this position.
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Structural Biology of HIV Assembly
Advances in HIV-1 Assembly and Release, 2013Co-Authors: Alex De Marco, Hans-georg Kräusslich, John A. G. BriggsAbstract:During its replication cycle, HIV-1 assembles an Immature Virus particle by accumulation of viral proteins underneath the cellular membrane. Upon budding of the assembled proteins through the membrane to release an enveloped, Immature Virus particle, a set of proteolytic cleavage events promotes dramatic changes in the structural organization of the particle to form the mature, infectious virion (Fig. 1a).
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Structure of the Immature retroviral capsid at 8 Å resolution by cryo-electron microscopy
Nature, 2012Co-Authors: Tanmay A.m. Bharat, Alex De Marco, Norman E. Davey, Pavel Ulbrich, James D. Riches, Michaela Rumlová, Carsten Sachse, Tomáš Ruml, John A. G. BriggsAbstract:A hybrid cryo-electron microscopy/tomography approach is used to solve the structure of the Immature Mason–Pfizer monkey Virus Gag lattice at a resolution of 8 A, allowing the derivation of a model for the structure of retroviral capsid in the Immature Gag shell. RetroViruses such as HIV-1 assemble as Immature Virus particles that leave the infected cell, and are then proteolytically cleaved to become mature infectious virions. Immature Virus assembly and the prevention of proteolytic cleavage are major targets for antiretrovirals. The data reported in this paper represent a significant advance in understanding retroVirus maturation. The authors use a hybrid cryo-electron microscopy/tomography approach to solve the structure of the Gag multidomain polyprotein lattice from Immature Mason–Pfizer monkey Virus (M-PMV). This structure allows the derivation of a model for the structure of the retroviral capsid in the Immature Gag shell. The assembly of retroViruses such as HIV-1 is driven by oligomerization of their major structural protein, Gag. Gag is a multidomain polyprotein including three conserved folded domains: MA (matrix), CA (capsid) and NC (nucleocapsid)1. Assembly of an infectious virion proceeds in two stages2. In the first stage, Gag oligomerization into a hexameric protein lattice leads to the formation of an incomplete, roughly spherical protein shell that buds through the plasma membrane of the infected cell to release an enveloped Immature Virus particle. In the second stage, cleavage of Gag by the viral protease leads to rearrangement of the particle interior, converting the non-infectious Immature Virus particle into a mature infectious virion. The Immature Gag shell acts as the pivotal intermediate in assembly and is a potential target for anti-retroviral drugs both in inhibiting Virus assembly and in disrupting Virus maturation3. However, detailed structural information on the Immature Gag shell has not previously been available. For this reason it is unclear what protein conformations and interfaces mediate the interactions between domains and therefore the assembly of retroVirus particles, and what structural transitions are associated with retroVirus maturation. Here we solve the structure of the Immature retroviral Gag shell from Mason–Pfizer monkey Virus by combining cryo-electron microscopy and tomography. The 8-A resolution structure permits the derivation of a pseudo-atomic model of CA in the Immature retroVirus, which defines the protein interfaces mediating retroVirus assembly. We show that transition of an Immature retroVirus into its mature infectious form involves marked rotations and translations of CA domains, that the roles of the amino-terminal and carboxy-terminal domains of CA in assembling the Immature and mature hexameric lattices are exchanged, and that the CA interactions that stabilize the Immature and mature Viruses are almost completely distinct.
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The molecular architecture of HIV.
Journal of molecular biology, 2011Co-Authors: John A. G. Briggs, Hans-georg KräusslichAbstract:Assembly of human immunodeficiency Virus type 1 is driven by oligomerization of the Gag polyprotein at the plasma membrane of an infected cell, leading to membrane envelopment and budding of an Immature Virus particle. Proteolytic cleavage of Gag at five positions subsequently causes a dramatic rearrangement of the interior virion organization to form an infectious particle. Within the mature Virus, the genome is encased within a conical capsid core. Here, we describe the molecular architecture of the Virus assembly site, the Immature Virus, the maturation intermediates and the mature Virus core and highlight recent advances in our understanding of these processes from electron microscopy and X-ray crystallography studies.
Marc C Johnson - One of the best experts on this subject based on the ideXlab platform.
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cryo electron microscopy reveals conserved and divergent features of gag packing in Immature particles of rous sarcoma Virus and human immunodeficiency Virus
Journal of Molecular Biology, 2006Co-Authors: John A. G. Briggs, Martha N Simon, Stephen D Fuller, Marc C Johnson, Volker M VogtAbstract:RetroVirus assembly proceeds via multimerisation of the major structural protein, Gag, into a tightly packed, spherical particle that buds from the membrane of the host cell. The lateral packing arrangement of the human immunodeficiency Virus type 1 (HIV-1) Gag CA (capsid) domain in the Immature Virus has been described. Here we have used cryo-electron microscopy (cryo-EM) and image processing to determine the lateral and radial arrangement of Gag in in vivo and in vitro assembled Rous sarcoma Virus (RSV) particles and to compare these features with those of HIV-1. We found that the lateral packing arrangement in the vicinity of the inner sub-domain of CA is conserved between these retroViruses. The curvature of the lattice, however, is different. RSV Gag protein adopts a more tightly curved lattice than is seen in HIV-1, and the virions therefore contain fewer copies of Gag. In addition, consideration of the relationship between the radial position of different Gag domains and their lateral spacings in particles of different diameters, suggests that the N-terminal MA (matrix) domain does not form a single, regular lattice in Immature retroVirus particles.
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The stoichiometry of Gag protein in HIV-1
Nature Structural & Molecular Biology, 2004Co-Authors: John A. G. Briggs, Hans-georg Kräusslich, Volker M Vogt, Martha N Simon, Ingolf Gross, Stephen D Fuller, Marc C JohnsonAbstract:The major structural components of HIV-1 are encoded as a single polyprotein, Gag, which is sufficient for Virus particle assembly. Initially, Gag forms an approximately spherical shell underlying the membrane of the Immature particle. After proteolytic maturation of Gag, the capsid (CA) domain of Gag reforms into a conical shell enclosing the RNA genome. This mature shell contains 1,000–1,500 CA proteins assembled into a hexameric lattice with a spacing of 10 nm. By contrast, little is known about the structure of the Immature Virus. We used cryo-EM and scanning transmission EM to determine that an average (145 nm diameter) complete Immature HIV particle contains ∼5,000 structural (Gag) proteins, more than twice the number from previous estimates. In the Immature Virus, Gag forms a hexameric lattice with a spacing of 8.0 nm. Thus, less than half of the CA proteins form the mature core.
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the stoichiometry of gag protein in hiv 1
Nature Structural & Molecular Biology, 2004Co-Authors: John A. G. Briggs, Hans-georg Kräusslich, Volker M Vogt, Martha N Simon, Ingolf Gross, Stephen D Fuller, Marc C JohnsonAbstract:The major structural components of HIV-1 are encoded as a single polyprotein, Gag, which is sufficient for Virus particle assembly. Initially, Gag forms an approximately spherical shell underlying the membrane of the Immature particle. After proteolytic maturation of Gag, the capsid (CA) domain of Gag reforms into a conical shell enclosing the RNA genome. This mature shell contains 1,000-1,500 CA proteins assembled into a hexameric lattice with a spacing of 10 nm. By contrast, little is known about the structure of the Immature Virus. We used cryo-EM and scanning transmission EM to determine that an average (145 nm diameter) complete Immature HIV particle contains approximately 5,000 structural (Gag) proteins, more than twice the number from previous estimates. In the Immature Virus, Gag forms a hexameric lattice with a spacing of 8.0 nm. Thus, less than half of the CA proteins form the mature core.
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dimeric rous sarcoma Virus capsid protein structure relevant to Immature gag assembly
Journal of Molecular Biology, 2004Co-Authors: Narayanasamy Nandhagopal, Michael G. Rossmann, Marc C Johnson, Alan A Simpson, Adam B Francisco, Gisela W Schatz, Volker M VogtAbstract:The structure of the N-terminal domain (NTD) of Rous sarcoma Virus (RSV) capsid protein (CA), with an upstream 25 amino acid residue extension corresponding to the C-terminal portion of the Gag p10 protein, has been determined by X-ray crystallography. Purified Gag proteins of retroViruses can assemble in vitro into Virus-like particles closely resembling in vivo-assembled Immature Virus particles, but without a membrane. When the 25 amino acid residues upstream of CA are deleted, Gag assembles into tubular particles. The same phenotype is observed in vivo. Thus, these residues act as a “shape determinant” promoting spherical assembly, when they are present, or tubular assembly, when they are absent. We show that, unlike the NTD on its own, the extended NTD protein has no b-hairpin loop at the N terminus of CA and that the molecule forms a dimer in which the amino-terminal extension forms the interface between monomers. Since dimerization of Gag has been inferred to be a critical step in assembly of spherical, Immature Gag particles, the dimer interface may represent a structural feature that is essential in retroVirus assembly.
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dimeric rous sarcoma Virus capsid protein structure relevant to Immature gag assembly
Journal of Molecular Biology, 2004Co-Authors: Narayanasamy Nandhagopal, Michael G. Rossmann, Marc C Johnson, Alan A Simpson, Adam B Francisco, Gisela W Schatz, Volker M VogtAbstract:The structure of the N-terminal domain (NTD) of Rous sarcoma Virus (RSV) capsid protein (CA), with an upstream 25 amino acid residue extension corresponding to the C-terminal portion of the Gag p10 protein, has been determined by X-ray crystallography. Purified Gag proteins of retroViruses can assemble in vitro into Virus-like particles closely resembling in vivo-assembled Immature Virus particles, but without a membrane. When the 25 amino acid residues upstream of CA are deleted, Gag assembles into tubular particles. The same phenotype is observed in vivo. Thus, these residues act as a "shape determinant" promoting spherical assembly, when they are present, or tubular assembly, when they are absent. We show that, unlike the NTD on its own, the extended NTD protein has no beta-hairpin loop at the N terminus of CA and that the molecule forms a dimer in which the amino-terminal extension forms the interface between monomers. Since dimerization of Gag has been inferred to be a critical step in assembly of spherical, Immature Gag particles, the dimer interface may represent a structural feature that is essential in retroVirus assembly.
Hans-georg Kräusslich - One of the best experts on this subject based on the ideXlab platform.
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Cryo-electron microscopy of tubular arrays of HIV-1 Gag resolves structures essential for Immature Virus assembly
Proceedings of the National Academy of Sciences of the United States of America, 2014Co-Authors: Tanmay A.m. Bharat, Luis R. Castillo Menendez, Wim J. H. Hagen, Vanda Lux, Sébastien Igonet, Martin Schorb, Florian K. M. Schur, Hans-georg Kräusslich, John A. G. BriggsAbstract:The assembly of HIV-1 is mediated by oligomerization of the major structural polyprotein, Gag, into a hexameric protein lattice at the plasma membrane of the infected cell. This leads to budding and release of progeny Immature Virus particles. Subsequent proteolytic cleavage of Gag triggers rearrangement of the particles to form mature infectious virions. Obtaining a structural model of the assembled lattice of Gag within Immature Virus particles is necessary to understand the interactions that mediate assembly of HIV-1 particles in the infected cell, and to describe the substrate that is subsequently cleaved by the viral protease. An 8-A resolution structure of an Immature Virus-like tubular array assembled from a Gag-derived protein of the related retroVirus Mason–Pfizer monkey Virus (M-PMV) has previously been reported, and a model for the arrangement of the HIV-1 capsid (CA) domains has been generated based on homology to this structure. Here we have assembled tubular arrays of a HIV-1 Gag-derived protein with an Immature-like arrangement of the C-terminal CA domains and have solved their structure by using hybrid cryo-EM and tomography analysis. The structure reveals the arrangement of the C-terminal domain of CA within an Immature-like HIV-1 Gag lattice, and provides, to our knowledge, the first high-resolution view of the region immediately downstream of CA, which is essential for assembly, and is significantly different from the respective region in M-PMV. Our results reveal a hollow column of density for this region in HIV-1 that is compatible with the presence of a six-helix bundle at this position.
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Structural Biology of HIV Assembly
Advances in HIV-1 Assembly and Release, 2013Co-Authors: Alex De Marco, Hans-georg Kräusslich, John A. G. BriggsAbstract:During its replication cycle, HIV-1 assembles an Immature Virus particle by accumulation of viral proteins underneath the cellular membrane. Upon budding of the assembled proteins through the membrane to release an enveloped, Immature Virus particle, a set of proteolytic cleavage events promotes dramatic changes in the structural organization of the particle to form the mature, infectious virion (Fig. 1a).
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The molecular architecture of HIV.
Journal of molecular biology, 2011Co-Authors: John A. G. Briggs, Hans-georg KräusslichAbstract:Assembly of human immunodeficiency Virus type 1 is driven by oligomerization of the Gag polyprotein at the plasma membrane of an infected cell, leading to membrane envelopment and budding of an Immature Virus particle. Proteolytic cleavage of Gag at five positions subsequently causes a dramatic rearrangement of the interior virion organization to form an infectious particle. Within the mature Virus, the genome is encased within a conical capsid core. Here, we describe the molecular architecture of the Virus assembly site, the Immature Virus, the maturation intermediates and the mature Virus core and highlight recent advances in our understanding of these processes from electron microscopy and X-ray crystallography studies.
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Structure and assembly of Immature HIV
Proceedings of the National Academy of Sciences of the United States of America, 2009Co-Authors: John A. G. Briggs, James D. Riches, Bärbel Glass, V. Bartonova, Giulia Zanetti, Hans-georg KräusslichAbstract:The major structural components of HIV are synthesized as a 55-kDa polyprotein, Gag. Particle formation is driven by the self-assembly of Gag into a curved hexameric lattice, the structure of which is poorly understood. We used cryoelectron tomography and contrast-transfer-function corrected subtomogram averaging to study the structure of the assembled Immature Gag lattice to approximate to 17-angstrom resolution. Gag is arranged in the Immature Virus as a single, continuous, but incomplete hexameric lattice whose curvature is mediated without a requirement for pentameric defects. The resolution of the structure allows positioning of individual protein domains. High-resolution crystal structures were fitted into the reconstruction to locate protein-protein interfaces involved in Gag assembly, and to identify the structural transformations associated with Virus maturation. The results of this study suggest a concept for the formation of nonsymmetrical enveloped Viruses of variable sizes.
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The stoichiometry of Gag protein in HIV-1
Nature Structural & Molecular Biology, 2004Co-Authors: John A. G. Briggs, Hans-georg Kräusslich, Volker M Vogt, Martha N Simon, Ingolf Gross, Stephen D Fuller, Marc C JohnsonAbstract:The major structural components of HIV-1 are encoded as a single polyprotein, Gag, which is sufficient for Virus particle assembly. Initially, Gag forms an approximately spherical shell underlying the membrane of the Immature particle. After proteolytic maturation of Gag, the capsid (CA) domain of Gag reforms into a conical shell enclosing the RNA genome. This mature shell contains 1,000–1,500 CA proteins assembled into a hexameric lattice with a spacing of 10 nm. By contrast, little is known about the structure of the Immature Virus. We used cryo-EM and scanning transmission EM to determine that an average (145 nm diameter) complete Immature HIV particle contains ∼5,000 structural (Gag) proteins, more than twice the number from previous estimates. In the Immature Virus, Gag forms a hexameric lattice with a spacing of 8.0 nm. Thus, less than half of the CA proteins form the mature core.
Gareth Griffiths - One of the best experts on this subject based on the ideXlab platform.
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In vitro reconstitution of an intermediate assembly stage of vaccinia Virus.
Virology, 1997Co-Authors: Maria Ericsson, Beate Sodeik, Jacomine Krijnse Locker, Gareth GriffithsAbstract:A novel method is described which facilitates the in vitro assembly of one step in the life cycle of vaccinia Virus, the formation of the spherical Immature Virus (IV). For this, advantage was taken of the ability of rifampicin to reversibly block the assembly of the IV. Rifampicin-treated, vaccinia Virus-infected HeLa cells were permeabilized with streptolysin O (SLO) and the endogenous cytosol was allowed to exit the cells at 4 degrees . Subsequently, exogenous cytosol from infected or uninfected HeLa cells as well as an ATP-regenerating system were added and the cells were incubated for different times at 37 degrees in the absence of rifampicin. The preparations were then evaluated by thin section EM. Our data show that in the presence of infected or uninfected cell cytosol and ATP a significant fraction of cells could reconstitute IV assembly in vitro. Under no conditions were we able to reconstitute any later stages of assembly. The potential of this system for the in vitro reconstitution of viral assembly in general is discussed.
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The Role of a 21-kDa Viral Membrane Protein in the Assembly of Vaccinia Virus from the Intermediate Compartment
The Journal of biological chemistry, 1996Co-Authors: Jacomine Krijnse-locker, Sibylle Schleich, Dolores Rodríguez, Bruno Goud, Eric J. Snijder, Gareth GriffithsAbstract:We have recently provided morphological evidence that a key event in the assembly of vaccinia Virus is the formation of a novel cisternal domain of the intermediate compartment (IC) between the endoplasmic reticulum and the Golgi complex (Sodeik, B., Doms, R. W., Ericsson, M., Hiller, G., Machamer, C. E., van't Hof, W., van Meer, G., Moss, B., and Griffiths, G. (1993) J. Cell Biol. 121, 521-541). This tightly apposed cisternal domain incompletely surrounds the spherical Immature Virus that matures into the first of the two distinct infectious forms of vaccinia, the intracellular mature Virus (IMV). In this study we describe the characterization of an abundant membrane protein of the IMV, the gene product of A17L, a 21-kDa protein that has recently been shown to be essential for the formation of the viral membranes (Rodriguez, D., Esteban, M., and Rodriguez, J. R. (1995) J. Virol. 69, 4640-4648). Upon translation in vitro, p21 associated with rough microsomal membranes in a co-translational manner. Using NH2- and COOH-terminal specific antibodies, we show that both in vitro as well as in vivo, p21 adopts a topology where the NH2 and COOH termini are cytoplasmically orientated. Immunocytochemical experiments demonstrated that p21 is a component of the inner of the two cisternal membranes of the Immature Virus as well as of membranes of the IC, identified using antibodies against Rab1. Taken together, these data provide the first molecular evidence in support of our assembly model; they show that an essential membrane protein of the IMV inserts into the rough endoplasmic reticulum, but gets efficiently targeted to the IC and membranes of the viral factory.
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A novel immunogold cryoelectron microscopic approach to investigate the structure of the intracellular and extracellular forms of vaccinia Virus.
The EMBO journal, 1996Co-Authors: Norbert Roos, Jacomine Krijnse-locker, Marek Cyrklaff, Sally Cudmore, R Blasco, Gareth GriffithsAbstract:We introduce a novel approach for combining immunogold labelling with cryoelectron microscopy of thin vitrified specimens. The method takes advantage of the observation that particles in suspension are concentrated at the air-water interface and remain there during the subsequent immunogold labelling procedure. Subsequently, a thin aqueous film can be formed that is vitrified and observed by cryoelectron microscopy. In our view, a key early step in the assembly of vaccinia Virus, the formation of the spherical Immature Virus, involves the formation of a specialized cisternal domain of the intermediate compartment between the endoplasmic reticulum and the Golgi. Using this novel cryoelectron microscopy approach, we show that in the intracellular mature Virus (IMV) the core remains surrounded by a membrane cisterna that comes off the viral core upon treatment with dithiothreitol, exposing an antigen on the surface of the viral core. Complementary protease studies suggest that the IMV may be sealed not by membrane fusion but by a proteinaceous structure that interrupts the outer membrane. We also describe the structure and membrane topology of the second infectious form of vaccinia, the extracellular enveloped Virus, and confirm that this form possesses an extra membrane overlying the IMV.
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Assembly of vaccinia Virus: incorporation of p14 and p32 into the membrane of the intracellular mature Virus.
Journal of virology, 1995Co-Authors: Beate Sodeik, Maria Ericsson, Sally Cudmore, M Esteban, E G Niles, Gareth GriffithsAbstract:The cytoplasmic assembly of vaccinia Virus begins with the transformation of a two-membraned cisterna derived from the intermediate compartment between the endoplasmic reticulum and the Golgi complex. This cisterna develops into a viral crescent which eventually forms a spherical Immature Virus (IV) that matures into the intracellular mature Virus (IMV). Using immunoelectron microscopy, we determined the subcellular localization of p32 and p14, two membrane-associated proteins of vaccinia Virus. p32 was associated with vaccinia Virus membranes at all stages of virion assembly, starting with the viral crescents, as well as with the membranes which accumulated during the inhibition of assembly by rifampin. There was also low but significant labelling of membranes of some cellular compartments, especially those in the vicinity of the Golgi complex. In contrast, anti-p14 labelled neither the crescents nor the IV but gave strong labelling of an intermediate form between IV and IMV and was then associated with all later viral forms. This protein was also not significantly detected on identifiable cellular membranes. Both p32 and p14 were abundantly expressed on the surface of intact IMV. Our data are consistent with a model whereby p32 would become inserted into cellular membranes before being incorporated into the crescents whereas p14 would be posttranslationally associated with the viral outer membrane at a specific later stage of the viral life cycle.
