title: The Structure of Immature Virus-Like Rous Sarcoma Virus Gag Particles Reveals a Structural Role for the p10 Domain in Assembly.
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 Å. 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.
  
  
  OBJECTIVE
  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.
authors.affiliation: Structural and Computational Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany Molecular Medicine Partnership Unit, Heidelberg, Germany.
authors.forename: Florian K M
authors.initials: FK
authors.lastname: Schur
authors.affiliation: Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York, USA.
authors.forename: Robert A
authors.initials: RA
authors.lastname: Dick
authors.affiliation: Structural and Computational Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany.
authors.forename: Wim J H
authors.initials: WJ
authors.lastname: Hagen
authors.affiliation: Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York, USA vmv1@cornell.edu john.briggs@embl.de.
authors.forename: Volker M
authors.initials: VM
authors.lastname: Vogt
authors.affiliation: Structural and Computational Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany Molecular Medicine Partnership Unit, Heidelberg, Germany vmv1@cornell.edu john.briggs@embl.de.
authors.forename: John A G
authors.initials: JA
authors.lastname: Briggs
mesh_terms: D002213:Capsid / Q000737:chemistry / Q000648:ultrastructure*; D020285:Cryoelectron Microscopy; D018360:Crystallography, X-Ray; D055032:Electron Microscope Tomography; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D015683:Gene Products, gag / Q000737:chemistry* / Q000235:genetics; D015497:HIV-1 / Q000737:chemistry / Q000648:ultrastructure; D016093:Mason-Pfizer monkey virus / Q000737:chemistry / Q000648:ultrastructure; D008958:Models, Molecular; D010447:Peptide Hydrolases / Q000737:chemistry / Q000302:isolation & purification; D055550:Protein Stability; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D059748:Proteolysis; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D052736:Rous sarcoma virus / Q000737:chemistry / Q000648:ultrastructure*; D014764:Viral Proteins / Q000737:chemistry / Q000302:isolation & purification; D019065:Virus Assembly / Q000502:physiology
chemical_list: D015683:Gene Products, gag; D011994:Recombinant Proteins; D014764:Viral Proteins; D010447:Peptide Hydrolases