Cryo-electron Microscopy Reveals Conserved and Divergent Features of Gag Packing in Immature Particles of Rous Sarcoma Virus and Human Immunodeficiency Virus

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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.

Introduction

The major structural components of retrovirus virions are synthesised as a single polyprotein, Gag,1 which is sufficient for the production of virus-like particles (VLPs) from cells.2 Coincident with or following Gag multimerisation, Gag induces membrane curvature and budding with the assistance of cellular components.3 The immature particles are approximately spherical, but with variable diameters.4, 5, 6, 7, 8, 9 Gag is arranged in a radial fashion with the N-terminal matrix (MA) domain interacting with the viral membrane, followed by the two capsid (CA) sub-domains and the nucleocapsid (NC) domain pointing inwards towards the centre of the virion.8 The structure of the immature particle is primarily maintained by three layers of interaction: NC–RNA interactions towards the centre, CA–CA interactions in the middle of the Gag layer, and MA–membrane interactions at the inner surface of the lipid bilayer. These interactions are sufficiently strong to promote the localisation and assembly of Gag, while retaining sufficient inherent flexibility to produce budding particles accommodating a variable number of proteins with no global symmetry.6, 9

During or shortly after budding, Gag is cleaved by the viral protease into its constituent components, leading to a dramatic morphological change. This cleavage leads to maturation of the virus into its infectious form. The NC domain, as part of the ribonucleoprotein complex, becomes condensed in the centre of the virion, the CA domain forms a core around the ribonucleoprotein, and MA remains associated with the viral membrane. In preparations of human immunodeficiency virus type 1 (HIV-1) and of some other retroviruses, a small number of virions with immature morphology are evident, indicating that some virus particles fail to undergo maturation. Large numbers of immature virus particles can be produced by inhibiting maturation using a protease inhibitor. Immature HIV-1 virions have been described by cryo-electron microscopy (cryo-EM).6, 8, 10 The radially arranged Gag proteins are visible as a striated layer underneath the viral membrane. We recently used image analysis of cryo-electron micrographs of immature HIV-1 to show that Gag adopts a hexagonal lattice with a unit-cell spacing of 8 nm at the outer CA sub-domain.10 In contrast to HIV-1,5 immature virus particles are not seen in preparations of Rous sarcoma virus (RSV), an avian alpharetrovirus.7 Immature virus-like RSV particles can be produced using a Baculovirus expression system from a Gag construct with the PR domain deleted11 or from vertebrate cells expressing protease defective or deleted forms of Gag.

The morphologies of mature RSV and HIV-1 have been described by cryo-EM.5, 7, 8 HIV-1 has a characteristic cone-shaped core, whereas the RSV core appears isometric. In both virions the core is heterogeneous in both size and shape and MA can be observed as a third density layer below the lipid bilayer.

Like other large, flexible, multi-domain proteins, Gag has not proved amenable to structural analysis in its entirety. This complicates efforts to understand the Gag–Gag interactions involved in multimerisation. High-resolution structures are, however, available for the constituent domains. The structures of the membrane binding portion of the MA domains of RSV12 and HIV-113, 14 Gag show that, despite significant sequence divergence, MA adopts the same fold in both viruses. Surface features other than the membrane-binding region do not exhibit the same conservation. RSV MA differs from HIV-1 MA and most other retroviral MA proteins in that it lacks a myristate modification. MA domains from SIV and HIV-1 trimerise in three-dimensional crystals,14, 15 and MA trimerisation may play a role in virus assembly, mediated in part by the myristate.16 Based upon observed trimerisation16 and dimerisation17 of retroviral MA, and upon the expectation that MA will pack laterally in the assembling virion, a number of models have been proposed for the structure of two-dimensional arrays of MA domains.15, 17, 18

The structures of CA from RSV and HIV-1 are strikingly similar.19, 20, 21, 22, 23, 24, 25 In both cases the N-terminal sub-domain (NTD) is composed of seven alpha helices arranged to form a tapered globular core, and the C-terminal sub-domain (CTD) is a smaller domain composed of four alpha helices. For both viruses CA provides the main protein–protein interactions contributing to assembly of the immature retroviral Gag lattice.22, 26, 27, 28, 29, 30

RSV Gag and HIV-1 Gag differ by the presence and location of extra domains. Between MA and CA of RSV Gag is a stretch of 84 residues called p2A, p2B, and p10. Every RSV Gag molecule includes PR at a position distal to NC. In contrast, in HIV-1, as in most other retroviruses, only about 5% of Gag molecules are extended C-terminally by frameshifting to include the PR domain. Distal to NC in HIV-1 Gag is p6, an unstructured domain that harbours sequences required during budding for the release of the virus particle from the cell.

The assembly pathways of RSV and HIV-1 Gag resemble one another, and are likely to proceed via RNA-bound oligomeric intermediates,31, 32, 33 with bulk assembly believed to take place at the plasma membrane. The assembly of both viruses has been extensively studied in vitro. In both cases Gag constructs containing CA and NC, but lacking MA, p10 and PR, assemble into tubes in the presence of RNA.34 An N-terminal extension upstream of CA leads to the assembly of spherical particles.35, 36, 37, 38 The morphology of the in vitro assembled particles has been described by cryo-EM and by standard negative staining. The in vitro assembled RSV Gag particles39 are slightly smaller than those formed by HIV-1 Gag.10, 37, 40, 41 Three concentric rings of density are observed, and the outer two rings of density exhibit track-like striations. The innermost of these peaks was assigned to NC, and the outer two peaks to the two sub-domains of CA.39 No significant density was observed corresponding to p10 or to the fraction of MA that was also included in the construct. Consistent with the peak assignment, RSV particles assembled from Gag protein with 150 amino acid residues N-terminal to CA appeared the same as particles assembled from Gag with only 25 amino acid residues N-terminal to CA (M.C.J., unpublished data).

We have used cryo-EM to describe the morphology and architecture of immature RSV particles shed from transfected cells. We compared the radial and lateral arrangement of the Gag protein with that adopted in vitro, and with that seen in immature HIV-1 particles. These comparisons reveal conserved and variable features of the arrangement of Gag in immature retrovirus particles.

Section snippets

cryo-EM

In vitro assembled RSV Gag particles were prepared and purified as described,39 and were imaged by cryo-EM (Figure 1(a)). They appeared similar to those described by Yu et al.,39 being approximately spherical with a layered, striated structure. Because the in vitro produced particles did not have a lipid bilayer there was no distinct feature for assigning particle diameter. We therefore measured the mean radius of the particles to the innermost CA sub-domain, which was 30 (±1) nm (n=33),

The packing arrangement near the inner CA sub-domain of RSV is the same as that in HIV-1

The reflections observed in Fourier transforms of the particle centres, and in the tangential Fourier transforms, are consistent with Gag assembling to form a p3 or p6 lattice where the dimensions of the lattice are proportional to radius, at least in the vicinity of CA and the CA-NC linker region. In vitro, the RSV Gag lattice unit cell has a size of 7.8 (±0.5) nm at the inner CA sub-domain and approximately 8.9 (±0.6) nm at the outer CA sub-domain. In in vitro assembled HIV-1 Gag particles, the

Production and purification of RSV virus particles

DF1 cells were grown to 70% confluency and transfected with CMV-RSV Gag D37S using Roche FuGene reagent. Cell medium was harvested 48 h post-infection, and clarified by centrifugation at 1000g for 5 min. Immature virus-like particles were collected by centrifugation through 15% (w/v) sucrose in STE buffer (10 mM Tris (pH 7.5), 100 mM NaCl, 1 mM EDTA) at 45,000 rpm in a 50.2 Ti rotor for 30 min at 4 °C. The pellet was resuspended in 0.5 ml of STE, overlaid onto a 4 ml 10%–60% (w/w) sucrose gradient and

Acknowledgements

We thank Amanda Dalton for critical reading of the manuscript. The work was funded by a Wellcome Trust programme grant (to S.D.F.), and US NIH grant CA20081 (to V.M.V.). The BNL STEM is a US NIH supported resource centre, NIH 5-P41-EB2181 with additional support provided by the US Department of Energy. J.A.G.B. was supported by a Wellcome Trust Structural Biology Studentship, and is currently supported by a fellowship from the Alexander von Humboldt Foundation. S.D.F. is a Wellcome Trust

References (48)

  • M. van Heel et al.

    A new generation of the IMAGIC image processing system

    J. Struct. Biol.

    (1996)
  • R. Swanstrom et al.

    Synthesis, assembly, and processing of viral proteins

  • E. Morita et al.

    Retrovirus budding

    Annu. Rev. Cell Dev. Biol.

    (2004)
  • J.A.G. Briggs et al.

    Cryo-electron microscopy of mouse mammary tumor virus

    J. Virol.

    (2004)
  • J.A.G. Briggs et al.

    Structural organization of authentic, mature HIV-1 virions and cores

    EMBO J.

    (2003)
  • T. Wilk et al.

    Organization of immature human immunodeficiency virus type 1

    J. Virol.

    (2001)
  • M. Yeager et al.

    Supramolecular organization of immature and mature murine leukemia virus revealed by electron cryo-microscopy: Implications for retroviral assembly mechanisms

    Proc. Natl Acad. Sci. USA

    (1998)
  • J.A.G. Briggs et al.

    The stoichiometry of Gag protein in HIV-1

    Nature Struct. Mol. Biol.

    (2004)
  • M.C. Johnson et al.

    PR domain of Rous sarcoma virus Gag causes an assembly/budding defect in insect cells

    J. Virol.

    (2001)
  • C.P. Hill et al.

    Crystal structures of the trimeric human immunodeficiency virus type 1 matrix protein: implications for membrane association and assembly

    Proc. Natl Acad. Sci. USA

    (1996)
  • Z.H. Rao et al.

    Crystal-structure of SIV matrix antigen and implications for virus assembly

    Nature

    (1995)
  • C. Tang et al.

    Entropic switch regulates myristate exposure in the HIV-1 matrix protein

    Proc. Natl Acad. Sci. USA

    (2004)
  • R.K. Gitti et al.

    Structure of the amino-terminal core domain of the HIV-1 capsid protein

    Science

    (1996)
  • C. Tang et al.

    Structure of the N-terminal 283-residue fragment of the immature HIV-1 Gag polyprotein

    Nature Struct. Biol.

    (2002)
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    Present address: J. A. G. Briggs, Department of Chemistry and Biochemistry, Ludwig-Maximilians-Universität, Butenandtstr. 11, 81377 Munich, Germany.

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