BACKGROUND: Myristoylation of the matrix (MA) domain mediates the transport and binding of Gag polyproteins to the plasma membrane (PM) and is required for the assembly of most retroviruses. In betaretroviruses, which assemble immature particles in the cytoplasm, myristoylation is dispensable for assembly but is crucial for particle transport to the PM. Oligomerization of HIV-1 MA stimulates the transition of the myristoyl group from a sequestered to an exposed conformation, which is more accessible for membrane binding. However, for other retroviruses, the effect of MA oligomerization on myristoyl group exposure has not been thoroughly investigated. RESULTS: Here, we demonstrate that MA from the betaretrovirus mouse mammary tumor virus (MMTV) forms dimers in solution and that this process is stimulated by its myristoylation. The crystal structure of N-myristoylated MMTV MA, determined at 1.57 Å resolution, revealed that the myristoyl groups are buried in a hydrophobic pocket at the dimer interface and contribute to dimer formation. Interestingly, the myristoyl groups in the dimer are mutually swapped to achieve energetically stable binding, as documented by molecular dynamics modeling. Mutations within the myristoyl binding site resulted in reduced MA dimerization and extracellular particle release. CONCLUSIONS: Based on our experimental, structural, and computational data, we propose a model for dimerization of MMTV MA in which myristoyl groups stimulate the interaction between MA molecules. Moreover, dimer-forming MA molecules adopt a sequestered conformation with their myristoyl groups entirely buried within the interaction interface. Although this differs from the current model proposed for lentiviruses, in which oligomerization of MA triggers exposure of myristoyl group, it appears convenient for intracellular assembly, which involves no apparent membrane interaction and allows the myristoyl group to be sequestered during oligomerization.

Department of Biochemistry Faculty of Science Charles University Prague Hlavova 8 128 40 Prague Czech Republic

Institute of Organic Chemistry and Biochemistry Academy of Sciences of the Czech Republic v v i Flemingovo nám 2 166 10 Prague Czech Republic

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Ross SR. Mouse mammary tumor virus molecular biology and oncogenesis. Viruses. 2010;2:2000–2012. doi: 10.3390/v2092000. PubMed DOI PMC

Swanstrom R, Wills JW: Synthesis, assembly, and processing of viral proteins. In: Coffin JM, Hughes SH, Varmus. HE, editors. Retroviruses. Cold Spring Harbor: Cold Spring Harbor Laboratory Press; 1997. p. 263–334. PubMed

Maurer-Stroh S, Eisenhaber F. Myristoylation of viral and bacterial proteins. Trends Microbiol. 2004;12:178–185. doi: 10.1016/j.tim.2004.02.006. PubMed DOI

Dick RA, Vogt VM. Membrane interaction of retroviral Gag proteins. Front Microbiol. 2014;5:1–11. doi: 10.3389/fmicb.2014.00187. PubMed DOI PMC

Zhou W, Parent LJ, Wills JW, Resh MD. Identification of a membrane-binding domain within the amino-terminal region of human immunodeficiency virus type 1 Gag protein which interacts with acidic phospholipids. J Virol. 1994;68:2556–2569. PubMed PMC

Bryant M, Ratner L. Myristoylation-dependent replication and assembly of human immunodeficiency virus 1. Proc Natl Acad Sci USA. 1990;87:523–527. doi: 10.1073/pnas.87.2.523. PubMed DOI PMC

Göttlinger HG, Sodroski JG, Haseltine WA. Role of capsid precursor processing and myristoylation in morphogenesis and infectivity of human immunodeficiency virus type 1. Proc Natl Acad Sci USA. 1989;86:5781–5785. doi: 10.1073/pnas.86.15.5781. PubMed DOI PMC

Freed EO, Orenstein JM, Buckler-White AJ, Martin MA. Single amino acid changes in the human immunodeficiency virus type 1 matrix protein block virus particle production. J Virol. 1994;68:5311–5320. PubMed PMC

Ono A, Freed EO. Binding of human immunodeficiency virus type 1 Gag to membrane: role of the matrix amino terminus. J Virol. 1999;73:4136–4144. PubMed PMC

Rhee SS, Hunter E. Myristylation is required for intracellular transport but not for assembly of D-type retrovirus capsids. J Virol. 1987;61:1045–1053. PubMed PMC

Zábranský A, Hadravová R, Stokrová J, Sakalian M, Pichová I. Premature processing of mouse mammary tumor virus Gag polyprotein impairs intracellular capsid assembly. Virology. 2009;384:33–37. doi: 10.1016/j.virol.2008.10.038. PubMed DOI

Hamard-Peron E, Muriaux D. Retroviral matrix and lipids, the intimate interaction. Retrovirology. 2011;8:15. doi: 10.1186/1742-4690-8-15. PubMed DOI PMC

Chukkapalli V, Ono A. Molecular determinants that regulate plasma membrane association of HIV-1 Gag. J Mol Biol. 2011;410:512–524. doi: 10.1016/j.jmb.2011.04.015. PubMed DOI PMC

Vlach J, Lipov J, Rumlová M, Veverka V, Lang J, Srb P, Knejzlík Z, Pichová I, Hunter E, Hrabal R, Ruml T. D-retrovirus morphogenetic switch driven by the targeting signal accessibility to Tctex-1 of dynein. Proc Natl Acad Sci USA. 2008;105:10565–10570. doi: 10.1073/pnas.0801765105. PubMed DOI PMC

Zhang G, Sharon D, Jovel J, Liu L, Wine E, Tahbaz N, Indik S, Mason A. Pericentriolar targeting of the mouse mammary tumor virus GAG protein. PLoS One. 2015;10:e0131515. doi: 10.1371/journal.pone.0131515. PubMed DOI PMC

Rhee SS, Hunter E: Amino acid substitutions within the matrix protein of type D retroviruses affect assembly, transport and membrane association of a capsid. EMBO J 1991;10:535–546. PubMed PMC

Stansell E, Tytler E, Walter MR, Hunter E: An early stage of Mason-Pfizer monkey virus budding is regulated by the hydrophobicity of the Gag matrix domain core. J Virol 2004;78:5023–5031. PubMed PMC

Stansell E, Apkarian R, Haubova S, Diehl WE, Tytler EM, Hunter E: Basic residues in the Mason-Pfizer monkey virus gag matrix domain regulate intracellular trafficking and capsid-membrane interactions. J Virol 2007;81:8977–88. PubMed PMC

Murakami T, Freed EO. Genetic evidence for an interaction between human immunodeficiency virus type 1 matrix and alpha-helix 2 of the gp41 cytoplasmic tail. J Virol. 2000;74:3548–3554. doi: 10.1128/JVI.74.8.3548-3554.2000. PubMed DOI PMC

Massiah MA, Starich MR, Paschall C, Summers MF, Christensen AM, Sundquist WI. Three-dimensional structure of the human immunodeficiency virus type 1 matrix protein. J Mol Biol. 1994;244:198–223. doi: 10.1006/jmbi.1994.1719. PubMed DOI

Rao Z, Belyaev AS, Fry E, Roy P, Jones IM, Stuart DI. Crystal structure of SIV matrix antigen and implications for virus assembly. Nature. 1995;378:743–747. doi: 10.1038/378743a0. PubMed DOI

Matthews S, Mikhailov M, Burny A, Roy P. The solution structure of the bovine leukaemia virus matrix protein and similarity with lentiviral matrix proteins. EMBO J. 1996;15:3267–3274. PubMed PMC

Christensen AM, Massiah MA, Turner BG, Sundquist WI, Summers MF. Three-dimensional structure of the HTLV-II matrix protein and comparative analysis of matrix proteins from the different classes of pathogenic human retroviruses. J Mol Biol. 1996;264:1117–1131. doi: 10.1006/jmbi.1996.0700. PubMed DOI

Conte MR, Klikova M, Hunter E, Ruml T, Matthews S. The three-dimensional solution structure of the matrix protein from the type D retrovirus, the Mason-Pfizer monkey virus, and implications for the morphology of retroviral assembly. EMBO J. 1997;16:5819–5826. doi: 10.1093/emboj/16.19.5819. PubMed DOI PMC

McDonnell JM, Fushman D, Cahill SM, Zhou W, Wolven A, Wilson CB, Nelle TD, Resh MD, Wills J, Cowburn D. Solution structure and dynamics of the bioactive retroviral M domain from Rous sarcoma virus. J Mol Biol. 1998;279:921–928. doi: 10.1006/jmbi.1998.1788. PubMed DOI

Hatanaka H, Iourin O, Rao Z, Fry E, Kingsman A, Stuart DI. Structure of equine infectious anemia virus matrix protein. J Virol. 2002;76:1876–1883. doi: 10.1128/JVI.76.4.1876-1883.2002. PubMed DOI PMC

Riffel N, Harlos K, Iourin O, Rao Z, Kingsman A, Stuart D, Fry E. Atomic resolution structure of Moloney murine leukemia virus matrix protein and its relationship to other retroviral matrix proteins. Structure. 2002;10:1627–1636. doi: 10.1016/S0969-2126(02)00896-1. PubMed DOI

Saad JS, Ablan SD, Ghanam RH, Kim A, Andrews K, Nagashima K, Soheilian F, Freed EO, Summers MF. Structure of the myristylated human immunodeficiency virus type 2 matrix protein and the role of phosphatidylinositol-(4,5)-bisphosphate in membrane targeting. J Mol Biol. 2008;382:434–447. doi: 10.1016/j.jmb.2008.07.027. PubMed DOI PMC

Serrière J, Robert X, Perez M, Gouet P, Guillon C. Biophysical characterization and crystal structure of the Feline Immunodeficiency Virus p15 matrix protein. Retrovirology. 2013;10:64. doi: 10.1186/1742-4690-10-64. PubMed DOI PMC

Tang C, Loeliger E, Luncsford P, Kinde I, Beckett D, Summers MF. Entropic switch regulates myristate exposure in the HIV-1 matrix protein. Proc Natl Acad Sci USA. 2004;101:517–522. doi: 10.1073/pnas.0305665101. PubMed DOI PMC

Brown L, Cox C, Baptiste J, Summers H, Button R, Bahlow K, Spurrier V, Kyser J, Luttge B, Kuo L, Freed E, Summers M. NMR Structure of the myristylated feline immunodeficiency virus matrix protein. Viruses. 2015;7:2210–2229. doi: 10.3390/v7052210. PubMed DOI PMC

Prchal J, Srb P, Hunter E, Ruml T, Hrabal R. The structure of myristoylated Mason-Pfizer monkey virus matrix protein and the role of phosphatidylinositol-(4,5)-bisphosphate in its membrane binding. J Mol Biol. 2012;423:427–438. doi: 10.1016/j.jmb.2012.07.021. PubMed DOI PMC

Hill CP, Worthylake DK, Bancroft DP, Christensen AM, Sundquist WI. Crystal structures of the trimeric human immunodeficiency virus type 1 matrix protein: implications for membrane association and assembly. Proc Natl Acad Sci USA. 1996;93:3099–3104. doi: 10.1073/pnas.93.7.3099. PubMed DOI PMC

Vlach J, Srb P, Prchal J, Grocký M, Lang J, Ruml T, Hrabal R. Nonmyristoylated matrix protein from the Mason-Pfizer monkey virus forms oligomers. J Mol Biol. 2009;390:967–980. doi: 10.1016/j.jmb.2009.05.063. PubMed DOI

Fledderman EL, Fujii K, Ghanam RH, Waki K, Prevelige PE, Freed EO, Saad JS. Myristate exposure in the human immunodeficiency virus type 1 matrix protein is modulated by pH. Biochemistry. 2010;49:9551–9562. doi: 10.1021/bi101245j. PubMed DOI PMC

Martin DDO, Beauchamp E, Berthiaume LG. Post-translational myristoylation: fat matters in cellular life and death. Biochimie. 2011;93:18–31. doi: 10.1016/j.biochi.2010.10.018. PubMed DOI

Doležal M, Zábranský A, Hrabal R, Ruml T, Pichová I, Rumlová M. One-step separation of myristoylated and nonmyristoylated retroviral matrix proteins. Protein Expr Purif. 2013;92:94–99. doi: 10.1016/j.pep.2013.09.003. PubMed DOI

Krissinel E, Henrick K. Inference of macromolecular assemblies from crystalline state. J Mol Biol. 2007;372:774–797. doi: 10.1016/j.jmb.2007.05.022. PubMed DOI

Zábranský A, Hoboth P, Hadravová R, Stokrová J, Sakalian M, Pichová I. The noncanonical Gag domains p8 and n are critical for assembly and release of mouse mammary tumor virus. J Virol. 2010;84:11555–11559. doi: 10.1128/JVI.00652-10. PubMed DOI PMC

Zábranský A, Sakalian M, Pichová I. Localization of self-interacting domains within betaretrovirus Gag polyproteins. Virology. 2005;332:659–666. doi: 10.1016/j.virol.2004.12.007. PubMed DOI

Sfakianos JN, Hunter E. M-PMV capsid transport is mediated by Env/Gag interactions at the pericentriolar recycling endosome. Traffic. 2003;4:671–680. doi: 10.1034/j.1600-0854.2003.00126.x. PubMed DOI

Clark J, Grznarova P, Stansell E, Diehl W, Lipov J, Spearman P, Ruml T, Hunter E. A Mason-Pfizer Monkey virus Gag-GFP fusion vector allows visualization of capsid transport in live cells and demonstrates a role for microtubules. PLoS One. 2013;8:e83863. doi: 10.1371/journal.pone.0083863. PubMed DOI PMC

Pereira LE, Clark J, Grznarova P, Wen X, LaCasse R, Ruml T, Spearman P, Hunter E. Direct evidence for intracellular anterograde co-transport of M-PMV Gag and Env on microtubules. Virology. 2014;449:109–119. doi: 10.1016/j.virol.2013.11.006. PubMed DOI PMC

Sakalian M, Hunter E. Separate assembly and transport domains within the Gag precursor of Mason-Pfizer monkey virus. J Virol. 1999;73:8073–8082. PubMed PMC

Knejzlík Z, Smékalová Z, Ruml T, Sakalian M. Multimerization of the p12 domain is necessary for Mason-Pfizer monkey virus Gag assembly in vitro. Virology. 2007;365:260–270. doi: 10.1016/j.virol.2007.03.053. PubMed DOI PMC

Sakalian M, Dittmer SS, Gandy AD, Rapp ND, Zábranský A, Hunter E. The Mason-Pfizer monkey virus internal scaffold domain enables in vitro assembly of human immunodeficiency virus type 1 Gag. J Virol. 2002;76:10811–10820. doi: 10.1128/JVI.76.21.10811-10820.2002. PubMed DOI PMC

Sommerfelt MA, Rhee SS, Hunter E. Importance of p12 protein in Mason-Pfizer monkey virus assembly and infectivity. J Virol. 1992;66:7005–7011. PubMed PMC

Srb P, Vlach J, Prchal J, Grocký M, Ruml T, Lang J, Hrabal R. Oligomerization of a retroviral matrix protein is facilitated by backbone flexibility on nanosecond time scale. J Phys Chem B. 2011;115:2634–2644. doi: 10.1021/jp110420m. PubMed DOI PMC

Schuck P. Size-distribution analysis of macromolecules by sedimentation velocity ultracentrifugation and Lamm equation modeling. Biophys J. 2000;78:1606–1619. doi: 10.1016/S0006-3495(00)76713-0. PubMed DOI PMC

Mueller U, Darowski N, Fuchs MR, Förster R, Hellmig M, Paithankar KS, Pühringer S, Steffien M, Zocher G, Weiss MS. Facilities for macromolecular crystallography at the Helmholtz-Zentrum Berlin. J Synchrotron Radiat. 2012;19:442–449. doi: 10.1107/S0909049512006395. PubMed DOI PMC

Kabsch W. XDS. Acta Crystallogr Sect D Biol Crystallogr 2010; 66:125–132. PubMed PMC

Sheldrick GM. Experimental phasing with SHELXC/D/E: combining chain tracing with density modification. Acta Crystallogr D Biol Crystallogr. 2010;66(Pt 4):479–485. doi: 10.1107/S0907444909038360. PubMed DOI PMC

Pape T, Schneider TR. HKL2MAP: a graphical user interface for macromolecular phasing with SHELX programs. J Appl Crystallogr. 2004;37:843–844. doi: 10.1107/S0021889804018047. DOI

Schneider TR, Sheldrick GM. Substructure solution with SHELXD. Acta Crystallogr D Biol Crystallogr. 2002;58:1772–1779. doi: 10.1107/S0907444902011678. PubMed DOI

Cowtan K. The Buccaneer software for automated model building. 1. Tracing protein chains. Acta Crystallogr D Biol Crystallogr. 2006;62:1002–1011. doi: 10.1107/S0907444906022116. PubMed DOI

Emsley P, Cowtan K. Coot: model-building tools for molecular graphics. Acta Crystallogr D Biol Crystallogr. 2004;60:2126–2132. doi: 10.1107/S0907444904019158. PubMed DOI

Pannu NS, Murshudov GN, Dodson EJ, Read RJ. Incorporation of prior phase information strengthens maximum-likelihood structure refinement. Acta Crystallogr D Biol Crystallogr. 1998;54:1285–1294. doi: 10.1107/S0907444998004119. PubMed DOI

Žáková L, Kletvíková E, Veverka V, Lepšík M, Watson CJ, Turkenburg JP, Jiráček J, Brzozowski AM. Structural integrity of the B24 site in human insulin is important for hormone functionality. J Biol Chem. 2013;288:10230–10240. doi: 10.1074/jbc.M112.448050. PubMed DOI PMC

Case DA, Babin V, Berryman JT, Betz RM, Cai Q, Cerutti DS, T.E. Cheatham I, Darden TA, Duke RE, Gohlke H, Goetz AW, Gusarov S, Homeyer N, Janowski P, Kaus J, Kolossváry I, Kovalenko A, Lee TS, LeGrand S, Luchko T, Luo R, Madej B, Merz KM, Paesani F, Roe DR, Roitberg A, Sagui C, Salomon-Ferrer R, Seabra G, Simmerling CL, et al.: AMBER 14. 2014.

Hornak V, Abel R, Okur A, Strockbine B, Roitberg A, Simmerling C: Comparison of multiple amber force fields and development of improved protein backbone parameters. Proteins Struct Funct Genet 2006;712–725. PubMed PMC

Bayly CCI, Cieplak P, Cornell WD, Kollman PA. A well-behaved electrostatic potential based method using charge restraints for deriving atomic charges: the RESP model. J Phys… 1993; 97:10269–80.

Wang J, Wolf RM, Caldwell JW, Kollman PA, Case DA. Development and testing of a general Amber force field. J Comput Chem. 2004;25:1157–1174. doi: 10.1002/jcc.20035. PubMed DOI

Differences and commonalities in plasma membrane recruitment of the two morphogenetically distinct retroviruses HIV-1 and MMTV