title: RNA and Nucleocapsid Are Dispensable for Mature HIV-1 Capsid Assembly.
abstract: Human immunodeficiency virus type 1 (HIV-1) is released from infected cells in an immature, noninfectious form in which the structural polyprotein Gag is arranged in a hexameric lattice, forming an incomplete spherical shell. Maturation to the infectious form is mediated by the viral protease, which cleaves Gag at five sites, releasing the CA (capsid) protein, which forms a conical capsid encasing the condensed RNA genome. The pathway of this structural rearrangement is currently not understood, and it is unclear how cone assembly is initiated. RNA represents an integral structural component of retroviruses, and the viral nucleoprotein core has previously been proposed to nucleate mature capsid assembly. We addressed this hypothesis by replacing the RNA-binding NC (nucleocapsid) domain of HIV-1 Gag and the adjacent spacer peptide 2 (SP2) by a leucine zipper (LZ) protein-protein interaction domain [Gag(LZ)] in the viral context. We found that Gag(LZ)-carrying virus [HIV(LZ)] was efficiently released and viral polyproteins were proteolytically processed, though with reduced efficiency. Cryo-electron tomography revealed that the particles lacked a condensed nucleoprotein and contained an increased proportion of aberrant core morphologies caused either by the absence of RNA or by altered Gag processing. Nevertheless, a significant proportion of HIV(LZ) particles contained mature capsids with the wild-type morphology. These results clearly demonstrate that the nucleoprotein complex is dispensable as a nucleator for mature HIV-1 capsid assembly in the viral context.
  
  
  OBJECTIVE
  Formation of a closed conical capsid encasing the viral RNA genome is essential for HIV-1 infectivity. It is currently unclear what viral components initiate and regulate the formation of the capsid during virus morphogenesis, but it has been proposed that the ribonucleoprotein complex plays a role. To test this, we prepared virus-like particles lacking the viral nucleocapsid protein and RNA and analyzed their three-dimensional structure by cryo-electron tomography. While most virions displayed an abnormal morphology under these conditions, some particles showed a normal mature morphology with closed conical capsids. These data demonstrate that the presence of RNA and the nucleocapsid protein is not required for the formation of a mature, cone-shaped HIV-1 capsid.
authors.affiliation: Structural and Computational Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany Molecular Medicine Partnership Unit, Heidelberg, Germany.
authors.forename: Simone
authors.initials: S
authors.lastname: Mattei
authors.affiliation: Department of Infectious Diseases, Virology, University Hospital Heidelberg, Heidelberg, Germany.
authors.forename: Annica
authors.initials: A
authors.lastname: Flemming
authors.affiliation: Department of Infectious Diseases, Virology, University Hospital Heidelberg, Heidelberg, Germany.
authors.forename: Maria
authors.initials: M
authors.lastname: Anders-Össwein
authors.affiliation: Department of Infectious Diseases, Virology, University Hospital Heidelberg, Heidelberg, Germany Molecular Medicine Partnership Unit, Heidelberg, Germany.
authors.forename: Hans-Georg
authors.initials: HG
authors.lastname: Kräusslich
authors.affiliation: Structural and Computational Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany Molecular Medicine Partnership Unit, Heidelberg, Germany john.briggs@embl.de Barbara_Mueller@med.uni-heidelberg.de.
authors.forename: John A G
authors.initials: JA
authors.lastname: Briggs
authors.affiliation: Department of Infectious Diseases, Virology, University Hospital Heidelberg, Heidelberg, Germany Molecular Medicine Partnership Unit, Heidelberg, Germany john.briggs@embl.de Barbara_Mueller@med.uni-heidelberg.de.
authors.forename: Barbara
authors.initials: B
authors.lastname: Müller
mesh_terms: D050976:Basic-Leucine Zipper Transcription Factors / Q000235:genetics; D002213:Capsid / Q000502:physiology*; D020285:Cryoelectron Microscopy; D057809:HEK293 Cells; D016655:HIV Core Protein p24 / Q000235:genetics / Q000378:metabolism; D015497:HIV-1 / Q000235:genetics* / Q000502:physiology; D006801:Humans; D015151:Immunoblotting; D016350:Leucine Zippers / Q000235:genetics; D019251:Nucleocapsid / Q000378:metabolism*; D012367:RNA, Viral / Q000378:metabolism*; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics; D019065:Virus Assembly / Q000502:physiology*
chemical_list: D050976:Basic-Leucine Zipper Transcription Factors; C501391:GCN4 protein, S cerevisiae; D016655:HIV Core Protein p24; D012367:RNA, Viral; D029701:Saccharomyces cerevisiae Proteins; C514799:p24 protein, Human Immunodeficiency Virus Type 1