Immature retroviral particles are assembled by self-association of the structural polyprotein precursor Gag. During maturation the Gag polyprotein is proteolytically cleaved, yielding mature structural proteins, matrix (MA), capsid (CA), and nucleocapsid (NC), that reassemble into a mature viral particle. Proteolytic cleavage causes the N terminus of CA to fold back to form a β-hairpin, anchored by an internal salt bridge between the N-terminal proline and the inner aspartate. Using an in vitro assembly system of capsid-nucleocapsid protein (CANC), we studied the formation of virus-like particles (VLP) of a gammaretrovirus, the xenotropic murine leukemia virus (MLV)-related virus (XMRV). We show here that, unlike other retroviruses, XMRV CA and CANC do not assemble tubular particles characteristic of mature assembly. The prevention of β-hairpin formation by the deletion of either the N-terminal proline or 10 initial amino acids enabled the assembly of ΔProCANC or Δ10CANC into immature-like spherical particles. Detailed three-dimensional (3D) structural analysis of these particles revealed that below a disordered N-terminal CA layer, the C terminus of CA assembles a typical immature lattice, which is linked by rod-like densities with the RNP.

Institute of Organic Chemistry and Biochemistry Academy of Sciences of the Czech Republic v v i IOCB and Gilead Research Center Prague Czech Republic

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Accola MA, Hoglund S, Gottlinger HG. 1998. A putative alpha-helical structure which overlaps the capsid-p2 boundary in the human immunodeficiency virus type 1 Gag precursor is crucial for viral particle assembly. J. Virol. 72: 2072–2078 PubMed PMC

Accola MA, Strack B, Gottlinger HG. 2000. Efficient particle production by minimal gag constructs which retain the carboxy-terminal domain of human immunodeficiency virus type 1 capsid-p2 and a late assembly domain. J. Virol. 74: 5395–5402 PubMed PMC

Bohmova K, et al. 2010. Effect of dimerizing domains and basic residues on in vitro and in vivo assembly of Mason-Pfizer monkey virus and human immunodeficiency virus. J. Virol. 84: 1977–1988 PubMed PMC

Borsetti A, Ohagen A, Gottlinger HG. 1998. The C-terminal half of the human immunodeficiency virus type 1 Gag precursor is sufficient for efficient particle assembly. J. Virol. 72: 9313–9317 PubMed PMC

Briggs JA, Johnson MC, Simon MN, Fuller SD, Vogt VM. 2006. Cryo-electron microscopy reveals conserved and divergent features of gag packing in immature particles of Rous sarcoma virus and human immunodeficiency virus. J. Mol. Biol. 355: 157–168 PubMed

Briggs JA, et al. 2009. Structure and assembly of immature HIV. Proc. Natl. Acad. Sci. U. S. A. 106: 11090–11095 PubMed PMC

Briggs JA, et al. 2004. The stoichiometry of Gag protein in HIV-1. Nat. Struct. Mol. Biol. 11: 672–675 PubMed

Campbell S, Vogt VM. 1995. Self-assembly in vitro of purified Ca-Nc proteins from Rous sarcoma virus and human immunodeficiency virus type 1. J. Virol. 69: 6487–6497 PubMed PMC

Cheslock SR, et al. 2003. Charged assembly helix motif in murine leukemia virus capsid: an important region for virus assembly and particle size determination. J. Virol. 77: 7058–7066 PubMed PMC

Cornilescu CC, Bouamr F, Yao X, Carter C, Tjandra N. 2001. Structural analysis of the N-terminal domain of the human T-cell leukemia virus capsid protein. J. Mol. Biol. 306: 783–797 PubMed

Datta SA, et al. 2011. Solution properties of murine leukemia virus Gag protein: differences from HIV-1 Gag. J. Virol. 85: 12733–12741 PubMed PMC

de Marco A, et al. 2010. Conserved and variable features of Gag structure and arrangement in immature retrovirus particles. J. Virol. 84: 11729–11736 PubMed PMC

de Marco A, et al. 2010. Structural analysis of HIV-1 maturation using cryo-electron tomography. PLoS Pathog. 6: e1001215. PubMed PMC

Ehrlich LS, Agresta BE, Carter CA. 1992. Assembly of recombinant human immunodeficiency virus type 1 capsid protein in vitro. J. Virol. 66: 4874–4883 PubMed PMC

Erlwein O, et al. 2011. Investigation into the presence of and serological response to XMRV in CFS patients. PLoS One 6: e17592. PubMed PMC

Fischer N, et al. 2008. Prevalence of human gammaretrovirus XMRV in sporadic prostate cancer. J. Clin. Virol. 43: 277–283 PubMed

Furuta RA, et al. 2011. No association of xenotropic murine leukemia virus-related virus with prostate cancer or chronic fatigue syndrome in Japan. Retrovirology 8: 20. PubMed PMC

Ganser BK, Li S, Klishko VY, Finch JT, Sundquist WI. 1999. Assembly and analysis of conical models for the HIV-1 core. Science 283: 80–83 PubMed

Ganser-Pornillos BK, Yeager M, Sundquist WI. 2008. The structural biology of HIV assembly. Curr. Opin. Struct. Biol. 18: 203–217 PubMed PMC

Gitti RK, et al. 1996. Structure of the amino-terminal core domain of the HIV-1 capsid protein. Science 273: 231–235 PubMed

Gross I, Hohenberg H, Huckhagel C, Krausslich HG. 1998. N-terminal extension of human immunodeficiency virus capsid protein converts the in vitro assembly phenotype from tubular to spherical particles. J. Virol. 72: 4798–4810 PubMed PMC

Gross I, Hohenberg H, Krausslich HG. 1997. In vitro assembly properties of purified bacterially expressed capsid proteins of human immunodeficiency virus. Eur. J. Biochem. 249: 592–600 PubMed

Gross I, et al. 2000. A conformational switch controlling HIV-1 morphogenesis. EMBO J. 19: 103–113 PubMed PMC

Hilditch L, et al. 2011. Ordered assembly of murine leukemia virus capsid protein on lipid nanotubes directs specific binding by the restriction factor, Fv1. Proc. Natl. Acad. Sci. U. S. A. 108: 5771–5776 PubMed PMC

Hohn O, et al. 2009. Lack of evidence for xenotropic murine leukemia virus-related virus(XMRV) in German prostate cancer patients. Retrovirology 6: 92. PubMed PMC

Hong P, Li J, Li Y. 2010. Failure to detect xenotropic murine leukaemia virus-related virus in Chinese patients with chronic fatigue syndrome. Virol. J. 7: 224. PubMed PMC

Johnson MC, Scobie HM, Vogt VM. 2001. PR domain of rous sarcoma virus Gag causes an assembly/budding defect in insect cells. J. Virol. 75: 4407–4412 PubMed PMC

Keller PW, Johnson MC, Vogt VM. 2008. Mutations in the spacer peptide and adjoining sequences in Rous sarcoma virus Gag lead to tubular budding. J. Virol. 82: 6788–6797 PubMed PMC

Khorasanizadeh S, Campos-Olivas R, Summers MF. 1999. Solution structure of the capsid protein from the human T-cell leukemia virus type-I. J. Mol. Biol. 291: 491–505 PubMed

Kingston RL, et al. 2000. Structure and self-association of the Rous sarcoma virus capsid protein. Structure 8: 617–628 PubMed

Klikova M, Rhee SS, Hunter E, Ruml T. 1995. Efficient in-vivo and in-vitro assembly of retroviral capsids from Gag precursor proteins expressed in bacteria. J. Virol. 69: 1093–1098 PubMed PMC

Knox K, et al. 2011. No evidence of murine-like gammaretroviruses in CFS patients previously identified as XMRV-infected. Science 333: 94–97 PubMed

Krausslich HG, Facke M, Heuser AM, Konvalinka J, Zentgraf H. 1995. The spacer peptide between human immunodeficiency virus capsid and nucleocapsid proteins is essential for ordered assembly and viral infectivity. J. Virol. 69: 3407–3419 PubMed PMC

Kremer JR, Mastronarde DN, McIntosh JR. 1996. Computer visualization of three-dimensional image data using IMOD. J. Struct. Biol. 116: 71–76 PubMed

Kuznetsov YG, Ulbrich P, Haubova S, Ruml T, McPherson A. 2007. Atomic force microscopy investigation of Mason-Pfizer monkey virus and human immunodeficiency virus type 1 reassembled particles. Virology 360: 434–446 PubMed

Li S, Hill CP, Sundquist WI, Finch JT. 2000. Image reconstructions of helical assemblies of the HIV-1 CA protein. Nature 407: 409–413 PubMed

Liang C, Hu J, Whitney JB, Kleiman L, Wainberg MA. 2003. A structurally disordered region at the C terminus of capsid plays essential roles in multimerization and membrane binding of the Gag protein of human immunodeficiency virus type 1. J. Virol. 77: 1772–1783 PubMed PMC

Lombardi VC, et al. 2009. Detection of an infectious retrovirus, XMRV, in blood cells of patients with chronic fatigue syndrome. Science 326: 585–589 PubMed

Ludtke SJ, Baldwin PR, Chiu W. 1999. EMAN: semiautomated software for high-resolution single-particle reconstructions. J. Struct. Biol. 128: 82–97 PubMed

Ma YM, Vogt VM. 2002. Rous sarcoma virus Gag protein-oligonucleotide interaction suggests a critical role for protein dimer formation in assembly. J. Virol. 76: 5452–5462 PubMed PMC

Ma YM, Vogt VM. 2004. Nucleic acid binding-induced Gag dimerization in the assembly of Rous sarcoma virus particles in vitro. J. Virol. 78: 52–60 PubMed PMC

Macek P, et al. 2009. NMR structure of the N-terminal domain of capsid protein from the Mason-Pfizer monkey virus. J. Mol. Biol. 392: 100–114 PubMed

Mortuza GB, et al. 2008. Structure of B-MLV capsid amino-terminal domain reveals key features of viral tropism, gag assembly and core formation. J. Mol. Biol. 376: 1493–1508 PubMed

Mortuza GB, et al. 2004. High-resolution structure of a retroviral capsid hexameric amino-terminal domain. Nature 431: 481–485 PubMed

Paprotka T, et al. 2011. Recombinant origin of the retrovirus XMRV. Science 333: 97–101 PubMed PMC

Phillips JM, Murray PS, Murray D, Vogt VM. 2008. A molecular switch required for retrovirus assembly participates in the hexagonal immature lattice. EMBO J. 27: 1411–1420 PubMed PMC

Pornillos O, Ganser-Pornillos BK, Yeager M. 2011. Atomic-level modelling of the HIV capsid. Nature 469: 424–427 PubMed PMC

Raisch KP, et al. 2003. Molecular cloning, complete sequence, and biological characterization of a xenotropic murine leukemia virus constitutively released from the human B-lymphoblastoid cell line DG-75. Virology 308: 83–91 PubMed

Robinson MJ, Erlwein O, McClure MO. 2011. Xenotropic murine leukaemia virus-related virus (XMRV) does not cause chronic fatigue. Trends Microbiol. 19: 525–529 PubMed

Rumlova-Klikova M, Hunter E, Nermut MV, Pichova I, Ruml T. 2000. Analysis of Mason-Pfizer monkey virus Gag domains required for capsid assembly in bacteria: role of the N-terminal proline residue of CA in directing particle shape. J. Virol. 74: 8452–8459 PubMed PMC

Satterfield BC, et al. 2011. Serologic and PCR testing of persons with chronic fatigue syndrome in the United States shows no association with xenotropic or polytropic murine leukemia virus-related viruses. Retrovirology 8: 12. PubMed PMC

Schutzer SE, Rounds MA, Natelson BH, Ecker DJ, Eshoo MW. 2011. Analysis of cerebrospinal fluid from chronic fatigue syndrome patients for multiple human ubiquitous viruses and xenotropic murine leukemia-related virus. Ann. Neurol. 69: 735–738 PubMed

Sfanos KS, et al. 2008. A molecular analysis of prokaryotic and viral DNA sequences in prostate tissue from patients with prostate cancer indicates the presence of multiple and diverse microorganisms. Prostate 68: 306–320 PubMed

Shin CH, et al. 2011. Absence of XMRV retrovirus and other murine leukemia virus-related viruses in patients with chronic fatigue syndrome. J. Virol. 85: 7195–7202 PubMed PMC

Still A, Huseby D, Barklis E. 2011. Analysis of the N-terminal region of the murine leukemia virus nucleocapsid protein. Virus Res. 155: 181–188 PubMed PMC

Switzer WM, et al. 2010. Absence of evidence of xenotropic murine leukemia virus-related virus infection in persons with chronic fatigue syndrome and healthy controls in the United States. Retrovirology 7: 57. PubMed PMC

Ulbrich P, et al. 2006. Distinct roles for nucleic acid in in vitro assembly of purified Mason-Pfizer monkey virus CANC proteins. J. Virol. 80: 7089–7099 PubMed PMC

Urisman A, et al. 2006. Identification of a novel Gammaretrovirus in prostate tumors of patients homozygous for R462Q RNASEL variant. PLoS Pathog. 2: e25. PubMed PMC

van Heel M, Harauz G, Orlova EV, Schmidt R, Schatz M. 1996. A new generation of the IMAGIC image processing system. J. Struct. Biol. 116: 17–24 PubMed

Verhaegh GW, et al. 2011. Prevalence of human xenotropic murine leukemia virus-related gammaretrovirus (XMRV) in Dutch prostate cancer patients. Prostate 71: 415–420 PubMed

von Schwedler UK, et al. 1998. Proteolytic refolding of the HIV-1 capsid protein amino-terminus facilitates viral core assembly. EMBO J. 17: 1555–1568 PubMed PMC

von Schwedler UK, Stray KM, Garrus JE, Sundquist WI. 2003. Functional surfaces of the human immunodeficiency virus type 1 capsid protein. J. Virol. 77: 5439–5450 PubMed PMC

Wang MQ, Goff SP. 2003. Defects in virion production caused by mutations affecting the C-terminal portion of the Moloney murine leukemia virus capsid protein. J. Virol. 77: 3339–3344 PubMed PMC

Wildova M, et al. 2008. The effect of point mutations within the N-terminal domain of Mason-Pfizer monkey virus capsid protein on virus core assembly and infectivity. Virology 380: 157–163 PubMed PMC

Wilk T, et al. 2001. Organization of immature human immunodeficiency virus type 1. J. Virol. 75: 759–771 PubMed PMC

Wright ER, et al. 2007. Electron cryotomography of immature HIV-1 virions reveals the structure of the CA and SP1 Gag shells. EMBO J. 26: 2218–2226 PubMed PMC

Yeager M, Wilson-Kubalek EM, Weiner SG, Brown PO, Rein A. 1998. Supramolecular organization of immature and mature murine leukemia virus revealed by electron cryo-microscopy: implications for retroviral assembly mechanisms. Proc. Natl. Acad. Sci. U. S. A. 95: 7299–7304 PubMed PMC

Yu F, et al. 2001. Characterization of Rous sarcoma virus Gag particles assembled in vitro. J. Virol. 75: 2753–2764 PubMed PMC

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