Architecture of the herpesvirus genomepackaging complex and implications for DNA translocation

Abstract

Genome packaging is a fundamental process in a viral life cycle and a prime target of antiviral drugs. Herpesviruses use an ATP-driven packaging motor/terminase complex to translocate and cleave concatemeric dsDNA into procapsids but its molecular architecture and mechanism are unknown. We report atomic structures of a herpesvirus hexameric terminase complex in both the apo and ADP•BeF3-bound states. Each subunit of the hexameric ring comprises three components—the ATPase/terminase pUL15 and two regulator/fixer proteins, pUL28 and pUL33—unlike bacteriophage terminases. Distal to the nuclease domains, six ATPase domains form a central channel with conserved basicpatches conducive to DNA binding and trans-acting arginine fingers are essential to ATP hydrolysis and sequential DNA translocation. Rearrangement of the nuclease domains mediated by regulatory domains converts DNA translocation mode to cleavage mode. Our structures favor a sequential revolution model for DNA translocation and suggest mechanisms for concerted domain rearrangements leading to DNA cleavage.

Keywords

dsDNA virus / genome packaging / viral maturation / terminase complex / drug target

References

[1] Adelman K, Salmon B, Baines JD (2001) Herpes simplex virus DNA packaging sequences adopt novel structures that are specifically recognized by a component of the cleavage and packaging machinery. Proc Natl Acad Sci USA 98:3086–3091

[2] Afonine PV, Grosse-Kunstleve RW, Echols N, Headd JJ, Moriarty NW, Mustyakimov M, Terwilliger TC, Urzhumtsev A,Zwart PH, Adams PD (2012) Towards automated crystallographic structure refinement with phenix. refine. Acta Crystallogr Sect D Biol Crystallogr 68:352–367

[3] Berger JM (2008) SnapShot: nucleic acid helicases and translocases. Cell 134(888–888):e881

[4] Bogner E (2002) Human cytomegalovirus terminase as a target for antiviral chemotherapy. Rev Med Virol 12:115–127

[5] Bogner E, Radsak K, Stinski MF (1998) The gene product of human cytomegalovirus open reading frame UL56 binds the pac motif and has specific nuclease activity. J Virol 72:2259–2264

[6] Chen W, Xiao H, Wang X, Song S, Han Z, Li X, Yang F, Wang L, Song J, Liu H, Cheng L (2020) Structural changes of a bacteriophage upon DNA packaging and maturation. Protein Cell.https://doi.org/10.1007/s13238-020-00715-9

[7] Dai X, Zhou ZH (2018) Structure of the herpes simplex virus 1 capsid with associated tegument protein complexes. Science 360:eaao7298

[8] Emsley P, Cowtan K (2004) Coot: model-building tools for molecular graphics. Acta Crystallogr D Biol Crystallogr 60:2126–2132

[9] Grant T, Rohou A, Grigorieff N (2018) cisTEM, user friendly software for single-particle image processing. Elife 7:e35383

[10] Guo PX, Zhang CL, Chen CP, Garver K, Trottier M (1998) Inter-RNA interaction of phage phi 29 pRNA to form a hexameric complex for viral DNA transportation. Mol Cell 2:149–155

[11] Guo PX, Schwartz C, Haak J, Zhao ZY (2013) Discovery of a new motion mechanism of biomotors similar to the earth revolving around the sun without rotation. Virology 446:133–143

[12] Heming JD, Conway JF, Homa FL (2017) Herpesvirus Capsid Assembly and DNA Packaging. Adv Anat Embryol Cell Biol 223:119–142

[13] Hilbert BJ, Hayes JA, Stone NP, Duffy CM, Sankaran B,Kelch BA (2015) Structure and mechanism of the ATPase that powers viral genome packaging. Proc Natl Acad Sci USA 112:E3792–E3799

[14] Hilbert BJ, Hayes JA, Stone NP, Xu RG, Kelch BA (2017) The large terminase DNA packaging motor grips DNA with its ATPase domain for cleavage by the flexible nuclease domain. Nucleic Acids Res 45:3591–3605

[15] Hugel T, Michaelis J, Hetherington CL, Jardine PJ, Grimes S, Walter JM, Faik W, Anderson DL, Bustamante C (2007) Experimental test of connector rotation during DNA packaging into bacteriophage phi 29 capsids. PLoS Biol 5:558–567

[16] Hwang JS, Bogner E (2002) ATPase activity of the terminase subunit pUL56 of human cytomegalovirus. J Biol Chem 277:6943–6948

[17] Mastronarde DN (2005) Automated electron microscope tomography using robust prediction of specimen movements. J Struct Biol 152:36–51

[18] Melendez DP, Razonable RR (2015) Letermovir and inhibitors of the terminase complex: a promising new class of investigational antiviral drugs against human cytomegalovirus. Infect Drug Resist 8:269–277

[19] Mettenleiter TC, Klupp BG, Granzow H (2009) Herpesvirus assembly: an update. Virus Res 143:222–234

[20] Miller JM, Enemark EJ (2016) Fundamental Characteristics of AAA+ Protein Family Structure and Function. Archaea 2016:9294307

[21] Nan Wang WC, Zhu L, Feng R, Wang J, Zhu D, Zhang X, Liu H, Rao Z, Wang X (2020) Structures of the portal vertex reveal essential protein-protein interactions for Herpesvirus assembly and maturation. Protein Cell

[22] Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC, Ferrin TE (2004) UCSF Chimera—a visualization system for exploratory research and analysis. J Comput Chem 25:1605–1612

[23] Ren R, Ghassabi Kondalaji S, Bowman GD (2019) The Chd1 chromatin remodeler forms long-lived complexes with nucleosomes in the presence of ADP.BeF3 (-) and transition state analogs. J Biol Chem 294:18181–18191

[24] Reynolds AE, Fan Y, Baines JD (2000) Characterization of the U(L) 33 gene product of herpes simplex virus 1. Virology 266:310–318

[25] Scheres SH (2012) RELION: implementation of a Bayesian approach to cryo-EM structure determination. J Struct Biol 180:519–530

[26] Scholz B, Rechter S, Drach JC, Townsend LB, Bogner E (2003) Identification of the ATP-binding site in the terminase subunit pUL56 of human cytomegalovirus. Nucleic Acids Res 31:1426–1433

[27] Selvarajan Sigamani S, Zhao H, Kamau YN, Baines JD, Tang L (2013) The structure of the herpes simplex virus DNA-packaging terminase pUL15 nuclease domain suggests an evolutionary lineage among eukaryotic and prokaryotic viruses. J Virol 87:7140–7148

[28] Sun SY, Kondabagil K, Gentz PM, Rossmann MG, Rao VB (2007) The structure of the ATPase that powers DNA packaging into bacteriophage t4 procapsids. Mol Cell 25:943–949

[29] Sun S, Kondabagil K,Draper B, Alam TI, Bowman VD, Zhang Z, Hegde S, Fokine A, Rossmann MG, Rao VB (2008) The structure of the phage T4 DNA packaging motor suggests a mechanism dependent on electrostatic forces. Cell 135:1251–1262

[30] Sun S, Rao VB, Rossmann MG (2010) Genome packaging in viruses. Curr Opin Struct Biol 20:114–120

[31] Wang X, Peng W, Ren J, Hu Z, Xu J, Lou Z, Li X, Yin W, Shen X, Porta C (2012) A sensor-adaptor mechanism for enterovirus uncoating from structures of EV71. Nat Struct Mol Biol 19:424–429

[32] Wang X, Ren J, Gao Q, Hu Z, Sun Y, Li X, Rowlands DJ, Yin W, Wang J, Stuart DI(2015) Hepatitis A virus and the origins of picornaviruses. Nature 517:85–88

[33] Wang J, Yuan S,Zhu D, Tang H, Wang N, Chen W, Gao Q, Li Y, Liu H, Zhang X (2018) Structure of the herpes simplex virus type 2 C-capsid with capsid-vertex-specific component. Nat Commun 9:3668

[34] Wang N, Zhao D,Wang J, Zhang Y, Wang M, Gao Y, Li F, Bu Z, Rao Z,Wang X(2019) Architecture of African swine fever virus and implications for viral assembly. Science 366:640–644. https://doi.org/10.1007/s13238-020-00711-z

[35] White CA, Stow ND, Patel AH, Hughes M, Preston VG (2003) Herpes simplex virus type 1 portal protein UL6 interacts with the putative terminase subunits UL15 and UL28. J Virol 77:6351–6358

[36] Xu RG, Jenkins HT, Antson AA, Greive SJ (2017a) Structure of the large terminase from a hyperthermophilic virus reveals a unique mechanism for oligomerization and ATP hydrolysis. Nucleic acids research 45(22):13029–13042

[37] Xu RG, Jenkins HT, Chechik M, Blagova EV, Lopatina A, Klimuk E, Minakhin L, Severinov K, Greive SJ, Antson AA (2017b) Viral genome packaging terminase cleaves DNA using the canonical RuvC-like two-metal catalysis mechanism. Nucleic Acids Res 45:3580–3590

[38] Yang K, Poon AP, Roizman B, Baines JD (2008) Temperaturesensitive mutations in the putative herpes simplex virus type 1 terminase subunits pUL15 and pUL33 preclude viral DNA cleavage/packaging and interaction with pUL28 at the nonpermissive temperature. J Virol 82:487–494

[39] Yuan SA, Wang JL, Zhu DJ, Wang N, Gao Q, Chen WY, Tang H, Wang JZ, Zhang XZ, Liu HR (2018) Cryo-EM structure of a herpesvirus capsid at 3.1 angstrom. Science 360:48–60

[40] Zhang K (2016) Gctf: Real-time CTF determination and correction. J Struct Biol 193:1–12

[41] Zhang F, Lemieux S, Wu XL, St-Arnaud D, McMurray CT, Major F, Anderson D (1998) Function of hexameric RNA in packaging of bacteriophage phi 29 DNA in vitro. Mol Cell 2:141–147

[42] Zhao H, Christensen TE, Kamau YN, Tang L (2013a) Structures of the phage Sf6 large terminase provide new insights into DNA translocation and cleavage. Proc Natl Acad Sci U S A 110:8075–8080

[43] Zhao ZY, Khisamutdinov E, Schwartz C, Guo PX (2013b) Mechanism of One-Way Traffic of Hexameric Phi29 DNA Packaging Motor with Four Electropositive Relaying Layers Facilitating Antiparallel Revolution. ACS Nano 7:4082–4092

[44] Zhu D, Wang X, Fang Q, Van Etten JL, Rossmann MG, Rao Z, Zhang X (2018a) Pushing the resolution limit by correcting the Ewald sphere effect in single-particle Cryo-EM reconstructions. Nat Commun 9:1552

[45] Zhu L, Sun Y, Fan J, Zhu B, Cao L, Gao Q, Zhang Y, Liu H, Rao Z, Wang X (2018b) Structures of Coxsackievirus A10 unveil the molecular mechanisms of receptor binding and viral uncoating. Nat Commun 9:4985

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