Structural studies of small RNA phages

1. Structural studies of small RNA phages Lars Liljas, Uppsala University Structure and Function of Large Molecular Assemblies Erice, June 12, 2006

2. Phages: all kinds of shapes T4 P2 MS2 X174

3. Phage MS2 infects E. coli The virus particles attach to bacterial f-pili

4. RNA of leviviruses codes for 3-4 proteins MS2 1 3569 130 1308 1335 1724 1761 3395 Coat (0) Replicase (0) Maturation Lysis (+1) 1678 1902 Qb • Coat protein binding site 4220 1 62 1305 1325 1743 2353 4119 Maturation-lysis Replicase Coat Read-through (A1) 2331 The genetic maps of Levivirus phage MS2 and Allolevivirus phage Qb

5. MS2: function of components • RNA: single-stranded positive sense • A-protein: attachment and entry • Coat protein: protein coat, translational repression and RNA recognition • Replicase subunit: forms active RNA-dependent RNA polymerase with host proteins • Lysis protein: lyses bacterial cell wall to release new virions

6. MS2: components • How is assembly controlled? • What determines the specificity in the binding of coat protein to the translational operator/assembly initiation site in RNA? • What is the position and conformation of the A protein? • How is the replicase working?

7. The replicase • The RNA polymerase is formed by • Replicase subunit (coded by viral RNA) • Elongation factors EF-Tu and EF-Ts • Ribosomal protein S1 (- strand synthesis)

8. The replicase RNA polymerase EF-Tu - EF-Ts complex Poliovirus, Thompson & Peersen, 2004 Kawashima et al., 1996

9. Levivirus capsid structures

10. Department of Cell and Molecular Biology, Uppsala University Karin Valegård Kerstin Fridborg Roshan Golmohammadi Sjoerd van den Worm Elin Grahn Kaspars Tars Charlotte Helgstrand Magnus Johansson Pavel Plevka Small RNA phages Department of Molecular Biology, Latvian University, Riga Maija Bundule School of Biology, University of Leeds Nicola Stonehouse Peter Stockley Wilf Horn

11. MS2 particle The virus particle is formed by • 90 coat protein dimers (180 subunits, T = 3) • 1 copy of A-protein • RNA molecule

12. MS2 particle The coat protein dimer has a central sheet facing the interior. Helices and a hairpin form the outer surface B/A C C

13. Role of A-protein Attachment to pili is due to the A-protein A fragment of the A-protein enters bacterium with viral RNA The A-protein, 393 aa, has no sequence similarity to other proteins Attempts to isolate (soluble) protein has failed

14. Role of A-protein Where is the A-protein? How does the binding trigger release of the RNA? Particle has holes at 5-fold and 3-fold axes Part of the A-protein might be exposed in one of these holes

15. Position of A-protein Fivefold and threefold axes surrounded by flexible loops Phage PRR1, Johansson et al., in prep. (Poster 26)

16. Role of coat protein Forms the protective coat Translational repressor Recognition of packaging signal in RNA MS2 capsid (one A/B dimer) viewed from outside

17. Coat protein binding to RNA A hairpin in the RNA is used both as a translational operator for the replicase and a packaging signal

18. Coat protein binding to RNA In MS2, four positions in single-stranded regions are recognized with specificity

19. RNA binding Crystals of recombinant capsids can be soaked with RNA hairpins, which enter the particles and bind to the coat protein

20. RNA binding • One RNA hairpin binds to a dimer • Binding to AB dimer is asymmetric MS2 capsid (one A/B dimer) viewed from inside

21. RNA recognition in MS2 Single-stranded regions of the RNA binds to three conserved sites in the protein

22. RNA recognition in MS2 What is determining the specificity? How are differences in specificity between different phages achieved?

23. RNA recognition in MS2: A-4 A pocket with specificity for A

24. RNA recognition in MS2: A-10 The same pocket in the other subunit with specificity for A or G

25. RNA recognition in MS2: A-10 A-10 G-10 C-10

26. RNA recognition in MS2: U-5 A binding site with specificity for U or C

27. RNA recognition in MS2: U-5 Of 9 natural and variant bases tested, 8 bind similar to wildtype U although the affinity differs by several orders of magnitude C-5: 10 A-5: 0.01 U-5: 1

28. RNA recognition in MS2: A-7 The base of A-7 has no direct contact with the protein This base has effects on the conformation that are important for binding

29. RNA binding: specificity The RNA-binding surface of the coat protein is very conserved, but Qb coat protein binds to a hairpin with different structure MS2 Qb

30. RNA binding specificity MS2 Mature Qb particles have disulfides (-->) blocking the entry of RNA segments. Mutations of two residues in MS2 leads to binding also of Qb hairpin Qb

31. RNA binding: specificity Conformation of loop very similar and the protein-RNA contacts are conserved but the basepairs are shifted MS2 hairpin Qb hairpin

32. RNA binding: specificity Phage PRR1 has still another variant of hairpin structure MS2 Qb PRR1

33. Assembly of MS2 particles Dimers assemble into correctly formed T=3 particles

34. Subunit packing in MS2 Two similar dimer conformations allow formation of quasi-equivalent contacts B/A C C

35. Role of RNA in subunit packing In leviviruses, RNA stimulates assembly: • RNA binding to dimer - induces “assembly mode” • Strong binding to one site (“initiation site”) - controls encapsidation of correct RNA

36. Control of quasiequivalence in other viruses In many small viruses, a flexible part of the coat protein is used to control the packing of subunits – used as order-disorder switches

37. Control of quasi-equivalence Leviviruses have no arms and order/disorder switches - assembly is controlled by the contact surfaces

38. Conclusions The observed specificity of coat protein binding to the viral RNA is explained by the structure of the complex The coat protein is able to form T=3 capsids without using an arm and order/disorder switches