Crystal Structure of the Vaccinia Virus DNA Polymerase Holoenzyme Subunit D4 in Complex with the A20 N-Terminal Domain

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Vaccinia virus is the prototype of the orthopoxvirus genus which includes other pathogens infecting humans and variola virus which was eradicated in the late 70's. Vaccinia virus DNA synthesis relies on three proteins: these are E9, the DNA polymerase bound to its heterodimeric cofactor D4/A20. To date, the molecular mechanism involved in poxvirus DNA replication remains poorly understood. Here, we present the high-resolution crystal structure of a complex formed by D4 and the first 50 residues of A20 (A201–50) that are necessary and sufficient for binding. The structure of D4/A201–50 reveals the contact surface engaged in the D4/A20 interaction in great detail. Interestingly, we could show that known small molecule inhibitors of vaccinia virus DNA synthesis selected for their ability to interfere with the D4/A20 interface could be docked onto the D4 surface where they mimic several aspects of the interacting A20 molecule. Finally, we present a model of D4/A20 in complex with DNA that allows us to discuss the role of mutations affecting the D4/A20 cofactor. Altogether, our structure gives new insights into the assembly of the vaccinia virus DNA polymerase cofactor and will be useful for the design of new antiviral compounds targeting the D4/A20 interaction.

Vyšlo v časopise: Crystal Structure of the Vaccinia Virus DNA Polymerase Holoenzyme Subunit D4 in Complex with the A20 N-Terminal Domain. PLoS Pathog 10(3): e32767. doi:10.1371/journal.ppat.1003978
Kategorie: Research Article
prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.ppat.1003978

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Zdroje

1. Boyle K, Traktman P (2009) Poxviruses. In Cameron, C.E., Gotte, M., Raney, K.D., editors. Viral Genome Replication. New York: Springer. pp. 225–246.

2. BoyleKA, ArpsL, TraktmanP (2007) Biochemical and genetic analysis of the vaccinia virus d5 protein: Multimerization-dependent ATPase activity is required to support viral DNA replication. J Virol 81 : 844–859.

3. De SilvaFS, LewisW, BerglundP, KooninEV, MossB (2007) Poxvirus DNA primase. Proc Natl Acad Sci U S A 104 : 18724–18729.

4. UptonC, StuartDT, McFaddenG (1993) Identification of a poxvirus gene encoding a uracil DNA glycosylase. Proc Natl Acad Sci U S A 90 : 4518–4522.

5. BoyleKA, StanitsaES, GresethMD, LindgrenJK, TraktmanP (2011) Evaluation of the role of the vaccinia virus uracil DNA glycosylase and A20 proteins as intrinsic components of the DNA polymerase holoenzyme. J Biol Chem 286 : 24702–24713.

6. SeleC, GabelF, GutscheI, IvanovI, BurmeisterWP, et al. (2013) Low-resolution structure of vaccinia virus DNA replication machinery. J Virol 87 : 1679–1689.

7. IshiiK, MossB (2002) Mapping interaction sites of the A20R protein component of the vaccinia virus DNA replication complex. Virology 303 : 232–239.

8. McCraithS, HoltzmanT, MossB, FieldsS (2000) Genome-wide analysis of vaccinia virus protein-protein interactions. Proc Natl Acad Sci U S A 97 : 4879–4884.

9. McDonaldWF, TraktmanP (1994) Vaccinia virus DNA polymerase. In vitro analysis of parameters affecting processivity. J Biol Chem 269 : 31190–31197.

10. StanitsaES, ArpsL, TraktmanP (2006) Vaccinia virus uracil DNA glycosylase interacts with the A20 protein to form a heterodimeric processivity factor for the viral DNA polymerase. J Biol Chem 281 : 3439–3451.

11. WardTM, WilliamsMV, Traina-DorgeV, GrayWL (2009) The simian varicella virus uracil DNA glycosylase and dUTPase genes are expressed in vivo, but are non-essential for replication in cell culture. Virus Res 142 : 78–84.

12. MullaneyJ, MossHW, McGeochDJ (1989) Gene UL2 of herpes simplex virus type 1 encodes a uracil-DNA glycosylase. J Gen Virol 70 (Pt 2) 449–454.

13. ReddySM, WilliamsM, CohenJI (1998) Expression of a uracil DNA glycosylase (UNG) inhibitor in mammalian cells: varicella-zoster virus can replicate in vitro in the absence of detectable UNG activity. Virology 251 : 393–401.

14. PrichardMN, DukeGM, MocarskiES (1996) Human cytomegalovirus uracil DNA glycosylase is required for the normal temporal regulation of both DNA synthesis and viral replication. J Virol 70 : 3018–3025.

15. ChenR, WangH, ManskyLM (2002) Roles of uracil-DNA glycosylase and dUTPase in virus replication. J Gen Virol 83 : 2339–2345.

16. MillnsAK, CarpenterMS, DeLangeAM (1994) The vaccinia virus-encoded uracil DNA glycosylase has an essential role in viral DNA replication. Virology 198 : 504–513.

17. HolzerGW, FalknerFG (1997) Construction of a vaccinia virus deficient in the essential DNA repair enzyme uracil DNA glycosylase by a complementing cell line. J Virol 71 : 4997–5002.

18. De SilvaFS, MossB (2003) Vaccinia virus uracil DNA glycosylase has an essential role in DNA synthesis that is independent of its glycosylase activity: catalytic site mutations reduce virulence but not virus replication in cultured cells. J Virol 77 : 159–166.

19. SchonhoftJD, KosowiczJG, StiversJT (2013) DNA translocation by human uracil DNA glycosylase: role of DNA phosphate charge. Biochemistry 52 : 2526–2535.

20. ParikhSS, MolCD, SlupphaugG, BharatiS, KrokanHE, et al. (1998) Base excision repair initiation revealed by crystal structures and binding kinetics of human uracil-DNA glycosylase with DNA. EMBO J 17 : 5214–5226.

21. ParikhSS, PutnamCD, TainerJA (2000) Lessons learned from structural results on uracil-DNA glycosylase. Mutat Res 460 : 183–199.

22. WongI, LundquistAJ, BernardsAS, MosbaughDW (2002) Presteady-state analysis of a single catalytic turnover by Escherichia coli uracil-DNA glycosylase reveals a “pinch-pull-push” mechanism. J Biol Chem 277 : 19424–19432.

23. StuartDT, UptonC, HigmanMA, NilesEG, McFaddenG (1993) A poxvirus-encoded uracil DNA glycosylase is essential for virus viability. J Virol 67 : 2503–2512.

24. DalesS, MilovanovitchV, PogoBG, WeintraubSB, HuimaT, et al. (1978) Biogenesis of vaccinia: isolation of conditional lethal mutants and electron microscopic characterization of their phenotypically expressed defects. Virology 84 : 403–428.

25. IshiiK, MossB (2001) Role of vaccinia virus A20R protein in DNA replication: construction and characterization of temperature-sensitive mutants. J Virol 75 : 1656–1663.

26. KlempererN, McDonaldW, BoyleK, UngerB, TraktmanP (2001) The A20R protein is a stoichiometric component of the processive form of vaccinia virus DNA polymerase. J Virol 75 : 12298–12307.

27. PunjabiA, BoyleK, DeMasiJ, GrubishaO, UngerB, et al. (2001) Clustered charge-to-alanine mutagenesis of the vaccinia virus A20 gene: temperature-sensitive mutants have a DNA-minus phenotype and are defective in the production of processive DNA polymerase activity. J Virol 75 : 12308–12318.

28. Druck ShudofskyAM, SilvermanJE, ChattopadhyayD, RicciardiRP (2010) Vaccinia virus D4 mutants defective in processive DNA synthesis retain binding to A20 and DNA. J Virol 84 : 12325–12335.

29. SchormannN, GrigorianA, SamalA, KrishnanR, DeLucasL, et al. (2007) Crystal structure of vaccinia virus uracil-DNA glycosylase reveals dimeric assembly. BMC Struct Biol 7 : 45.

30. SchormannN, SommersCI, PrichardMN, KeithKA, NoahJW, et al. (2011) Identification of protein-protein interaction inhibitors targeting vaccinia virus processivity factor for development of antiviral agents. Antimicrob Agents Chemother 55 : 5054–5062.

31. KrissinelE, HenrickK (2007) Inference of macromolecular assemblies from crystalline state. J Mol Biol 372 : 774–797.

32. NuthM, HuangL, SawYL, SchormannN, ChattopadhyayD, et al. (2011) Identification of inhibitors that block vaccinia virus infection by targeting the DNA synthesis processivity factor D4. J Med Chem 54 : 3260–3267.

33. PearlLH (2000) Structure and function in the uracil-DNA glycosylase superfamily. Mutat Res 460 : 165–181.

34. EllisonKS, PengW, McFaddenG (1996) Mutations in active-site residues of the uracil-DNA glycosylase encoded by vaccinia virus are incompatible with virus viability. J Virol 70 : 7965–7973.

35. MullardA (2012) Protein-protein interaction inhibitors get into the groove. Nat Rev Drug Discov 11 : 173–175.

36. FlusinO, SaccucciL, Contesto-RichefeuC, HamdiA, BardouC, et al. (2012) A small molecule screen in yeast identifies inhibitors targeting protein-protein interactions within the vaccinia virus replication complex. Antiviral Res 96 : 187–195.

37. SaccucciL, CranceJM, ColasP, BickleM, GarinD, et al. (2009) Inhibition of vaccinia virus replication by peptide aptamers. Antiviral Res 82 : 134–140.

38. SilvermanJE, CiusteaM, ShudofskyAM, BenderF, ShoemakerRH, et al. (2008) Identification of polymerase and processivity inhibitors of vaccinia DNA synthesis using a stepwise screening approach. Antiviral Res 80 : 114–123.

39. JainAN (2003) Surflex: fully automatic flexible molecular docking using a molecular similarity-based search engine. J Med Chem 46 : 499–511.

40. GerardFC, Ribeiro EdeAJr, AlbertiniAA, GutscheI, ZaccaiG, et al. (2007) Unphosphorylated rhabdoviridae phosphoproteins form elongated dimers in solution. Biochemistry 46 : 10328–10338.

41. DimasiN, FlotD, DupeuxF, MarquezJA (2007) Expression, crystallization and X-ray data collection from microcrystals of the extracellular domain of the human inhibitory receptor expressed on myeloid cells IREM-1. Acta Crystallogr Sect F Struct Biol Cryst Commun 63 : 204–208.

42. BattyeTG, KontogiannisL, JohnsonO, PowellHR, LeslieAG (2011) iMOSFLM: a new graphical interface for diffraction-image processing with MOSFLM. Acta Crystallogr D Biol Crystallogr 67 : 271–281.

43. WinnMD, BallardCC, CowtanKD, DodsonEJ, EmsleyP, et al. (2011) Overview of the CCP4 suite and current developments. Acta Crystallogr D Biol Crystallogr 67 : 235–242.

44. McCoyAJ, Grosse-KunstleveRW, AdamsPD, WinnMD, StoroniLC, et al. (2007) Phaser crystallographic software. J Appl Crystallogr 40 : 658–674.

45. EmsleyP, LohkampB, ScottWG, CowtanK (2010) Features and development of Coot. Acta Crystallogr D Biol Crystallogr 66 : 486–501.

46. MurshudovGN, SkubakP, LebedevAA, PannuNS, SteinerRA, et al. (2011) REFMAC5 for the refinement of macromolecular crystal structures. Acta Crystallogr D Biol Crystallogr 67 : 355–367.

47. McNicholasS, PottertonE, WilsonKS, NobleME (2011) Presenting your structures: the CCP4mg molecular-graphics software. Acta Crystallogr D Biol Crystallogr 67 : 386–394.

48. KabschW (1976) Solution for Best Rotation to Relate 2 Sets of Vectors. Acta Crystallographica Section A 32 : 922–923.