Structures and Mechanisms - American Chemical Society

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Chapter 22

The 3.6

ÅStructure of the Reovirus Core Particle 1

Karin M . Reinisch and Stephen C. Harrison

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Department of Cell Biology, Yale School of Medicine, 333 Cedar Street, New Haven,CT06520 Harvard University and Howard Hughes Medical Institute, Fairchild Building, 7 Divinity Avenue, Cambridge, MA 02138 2

When reoviruses enter a host cell, the virion sheds its outer coat proteins to reveal a "core". An icosaheral protein shell contains the viral dsRNA genome and does not release it even when the core reaches its destination, the cytoplasm. Instead, the core contains the necessary enzymatic machinery to transcribe the dsRNA genome into mRNA and to cap the mRNA at the 5' end. The genome consists of 10 unique segments of dsRNA, each of which encodes at least one gene, and a core transcribes several genes simultaneously and repeatedly. Thus the core, in addition merely to containing the viral genome, is a remarkably complex enzymatic assembly. (See references & for a review.) Here we discuss the atomic-resolution structure of the reovirus core particle in terms of its overall architecture and the organization of its enzymatic activities. 1

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The structure of the reovirus core particle at 3.6 Â resolution (Fig. 1A) shows the icosahedral protein shell, which includes the capping complexes. The particle is - 7 5 0 Â in diameter, and including the genome, it has a mass of 52 million daltons. A 20Â thin shell composed of 120 copies of a protein λΐ (142kDa ) directly surrounds the genome. On top of this shell, and stabilizing it, are 150 nodules of a protein σ 2 (47kDa). Finally, at each icosahedral 5-fold axis, there are pentameric complexes of a third protein, λ2 (144kDa). The pentamers form hollow turrets that perform three of four capping reactions. λ2 has three 4

© 2002 American Chemical Society Eaton et al.; Structures and Mechanisms ACS Symposium Series; American Chemical Society: Washington, DC, 2002.

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Eaton et al.; Structures and Mechanisms ACS Symposium Series; American Chemical Society: Washington, DC, 2002.


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Figure 1. A The Ca-trace of the reovirus core particle viewed down a 5-fold axis. One of twelve X2 turrets is at the center of the image, andfivemore are visible at the periphery. Multiple copies of σ2 are shown as small nodules decorating the XI surface. B. The XI surface. Copies of Xl-A are shown in dark grey, and copies of Xl-B are shown in lighter shades. Five copies of Xl-A surround the 5 fold axis, andfivecopies of XI-B are interdigitated between them to form a decameric unit. Twelve such units comprise the XI shell. Three copies of Xl-B surround the 3-fold axes. C. The XI shell with footprints outlining the σ2 binding sites. There are 150 σ2 binding sites; three of these are unique and unrelated by icosahedral symmetry. D. A ribbon diagram of Xl-A. The 5-fold axis is at the right of the molecule. E. A ribbon diagram of Xl-B. Xl-B is rendered in two colors to indicate its two subdomains and the location of the pivot point between them. The relative orientation of the subdomains about the pivot point differs in Xl-A and B. Residues 40-167 and 181-206 are also indicated in dark colors. The Zn is not shown. E. VP3-A from the Β TV core structure. Its fold is similar to that of XL F. σ2-Ι rendered so that the XI surface is toward the bottom of the page. σ2-ϋ and -Hi are very similar with small changes at the interface with the XI surface. Thisfigurehas been adapted with permission from Nature (404: 960-967), MacMillan Magazines, Ltd. 2000. ++

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enzymatic domains: a guanylyltransferase domain, a 7N methyltransferase, domain and a 2 Ό methyltransferase domain. While the interior of the core (the dsRNA and -12 transcription complexes) is not ordered icosahedrally and is therefore not visible in the crystal structure, low angle x-ray scattering experiments and low resolution electron density maps from crystallography suggest that the dsRNA is tightly packed into concentric layers that extend inward from the λΐ shell at 26Â intervals. Locally, the dsRNA could be packed hexagonally with a 30Â distance between adjacent RNA helices. Studies using electron cryomicroscopy further show that a transcription complex is tethered under each turret, so that the transcriptase may extrude the 5' end of the mRNA out of the core and into the capping complex, thus ensuring that the 5' end is not lost in the tightly packed interior of the core. Such a packing is consistent with a model previously proposed for other members of the Reoviridae family ' , in which each segment of RNA is coiled into large spools around a polymerase complex, thus keeping the genes from tangling during their concurrent and repeated transcription.


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λΐ, the λΐ shell, and non-equivalence The λΐ shell around the dsRNA genome is relatively smooth. Five monomers of λΐ (set A) radiate from the five fold axis, and a second set of five (set B) interdigitates with the first. Twelve such decamers together form the complete protein shell (Fig. IB). Thus far, this decameric arrangement of capsid proteins is unique to the Reoviridae ' (although it has been proposed for other dsRNA viruses with 120 protein subunits in the capsid ' ' ). The arrangement is the same for the innermost capsid proteins in the bluetongue virus core (BTV; an orbivirus) and in cytoplasmic polyhedrosis virus (CPV; a cypovirus) . Furthermore, the fold of λΐ, while without any recognizable sequence similarity, resembles that of the VP3 proteins in B T V and CPV . Figure ID and Ε illustrate the folds of λΐ and of BTV VP3 , respectively, λΐ, like the equivalent proteins in the other viruses, is an elongated flat plate. Despite its large size, the plate consists of one domain rather than several concatenated domains and is thus well suited to its role as armor for the viral genome. In all three proteins, there is an alpha helical bundle nearest the 5-fold axis, a region of criss-crossing alpha helices at the middle of the molecule, and a beta-sheeted region furthest from the 5-fold axis, λΐ assumes two conformations (Figl B,D&E), where monomers in set A are related to those in set Β by a reorientation of two subdomains (I: residues 253470 and 923-1260 and II: residues 482-922) about a pivot point. The number 9 10

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and location of the pivot points is not conserved across the Reoviridae. Sets A and Β further differ in the conformations of loops around their periphery. Most notably, residues 560-568 and 774-794 in λΙ-Α, which define the aperture at the 5-fold axis through which the mRNA is extruded into the λ2 turrets, have a different conformation in λΙ-Β or are partially disordered. Since the aperture at the 5-fold axis measures only 5.5Â in diameter, the residues bordering it probably undergo a conformational change in the course of mRNA extrusion. What is most striking in the comparison of λΙ-Α and λΙ-Β, however, is that the contacts they make with neighboring molecules differ entirely in all but one small interface across a local 2-fold dyad (see Fig IB ). The portions of λΙ-Β contacted by A differfromthose in A contacted by B. Thus, the λΐ shell, and the inner protein shells of the other studied Reoviridae, differ from all other viral capsids for which structures are known in that chemically equivalent species make almost entirely non-equivalent contacts. In such capsids, specifically those that have more than 60 protein subunits, even those subunits that are not related by icosahedral symmetry have mainly similar contacts with neighboring molecules. The first 240 residues of λΙ-Α and the first 180 residues of λΙ-Β are partially disordered inside the core. For λΙ-Β, residues 181-206 form a classic zinc finger with two histidines and two cysteines to coordinate the metal. The zinc finger is tucked between copies of λΙ-Β on the interior of the core near the 3fold axis. It cannot bind RNA in this orientation since the RNA would collide with the λΐ shell, but both this zinc finger and the corresponding one in λ Ι - Α could bind to RNA in the course of viral assembly. Further, the λΙ-Α zinc finger dangles into the core, where it could bind dsRNA even in the assembled core. A set of λΐ residues 40-167 also nest under each λΙ-Β. They do not belong to this particular λΙ-Β since residue 167 is - 9 0 Â removedfromresidue 181 but belong to some neighboring λΐ, A or B. Thus, residues 40-167 form a network of arms on the interior of the λΐ shell that may contribute to capsid stability.

σ2 clamp and more non-equivalence Decorating the exterior of the λ 1 shell, there are 150 nodules made by σ 2 , a globular and predominantly helical protein. Each σ 2 makes extensive contacts only with the λΐ shell. There are three distinct positions for σ 2 within an icosahedral asymmetric unit.



372 These three unique binding sites represent the second example of nonequivalence in the reovirus core structure. While site iii is partially similar to site ii, sites i and ii are entirely different both in terms of the secondary structure and the pattern of charged/hydrophobic/polar residues that the λΐ surface presents to σ2. σ2-ί lies over the middle of a λΙ-Α molecule, and σ2-ϋ bridges from the middle of a λΙ-Β across to the carboxy-terminal part of a λ Ι - Β from another decamer. σ2-ϋί lies on the λΐ shell directly on an icosahedral 2-fold axis in one of two equally-likely, two-fold related orientations. Consequently, σ2-ϋί has not been built into the 3.6Â electron density maps, and instead a σ2-ϋ model has been docked onto that site. It is clear, however, that the various versions of σ 2 differ only at the interface with the λΐ surface. The differences between σ2-ί and ii are subtle, and the most drastic change is an unravelled helix (residues 39-46) in σ2-ϋ with respect to σ2-ί. Chemically identical protein subunits using the same surface to make completely different intersubunit contacts have not been widely observed in crystal structures. The list of proteins involved in such non-equivalent contacts includes capsid proteins in the Reoviridae family ' , the scaffolding protein in the X174 prohead , and HIV reverse transcriptase , wherein two monomers have nonequivalent dimer interfaces. All these examples are viral proteins and constitute a testament to the strong evolutionary pressure imposed by the compactness of the viral genome. 9 10

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Two of the three sites explain why σ2 is essential in order for λΐ to form icosahedral particles, for recombinant λΐ will form icosahedral particles in mouse L fibroblasts or insect cells (Max Nibert, unpublished ) only if coexpressed with σ2: in two of its binding sites (ii,iii in FiglC) σ 2 behaves as a clamp that holds together decamers of λΐ. While σ2 does not have a clamp analog in the bluetongue virus core, some of the other turretted Reoviridae such as C P V ' and aquareovirus have proteins of a similar size positioned like σ2i and - i i . There the subunit in binding position ii probably also functions to stabilize the innermost protein shell. 15

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XI Capping Complex & the Active Sites The transcription complex in the core interior passes the newly synthesized mRNA directly into the pentameric X2 turrets (Fig 2A&B). The turrets catalyse three capping reactions: the addition of a guanosyl moiety to the 5' end of the mRNA and then the transfer of a methyl group from S-adenosyl-L-methionine (SAM) to both the N7 position of the newly added guanosine and the 2Ό of the


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first template encoded nucleotide, a guanosine in each of the ten reovirus gene segments (reactions 2-4 below). 1 9

(1) 5'-pppG-mRNA ^ 5'-ppG-mRNA + pi (2) S'-ppG-mRNA + GTP GpppG-mRNA (3) SAM + GpppG-mRNA GpppG-mRNA +SAH (4) SAM + G-pppG-mRNA GpppG '-mRNA +SAH me7


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where SAH is S-adenosyl-L-homocysteine. The turrets are about 120Â in diameter and 80Â in height and have a hollow interior (volume=2xl0 Â ). All the enzymatic active sites face the interior cavity. The top of the turret is partially closed by five flaps that, according to cryo-em reconstructions, have variable conformations . In the intact virus, the flaps grip the viral cell attachment protein σΐ, whereas in the core their conformation controls the size of the aperture at the top of the turret. 5

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X2 consists of 7 concatenated domains (Fig 2C). The most N-terminal domain (residues 1 to 385, Fig2E) is the guanylyltransferase positioned directly on the λΐ shell. The guanylyltransferase is cup shaped, and five such domains form a ring about the icosahedral 5-fold axis. The interior of the cup contains the active site and lysines 190 and 171, which are necessary for guanylyl transfer . The reovirus guanylyltransferase domain consitutes a novel fold and bears no structural resemblance to the PBCV-1 guanylyltransferase, for which a structure has been determined recently . 21

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Residues 386-433 and 690-802 form a small, probably structural domain. The next two domains, residues 434-691 and 804-1022, are methyltransferase domains, and their folds are two different variations on the "universal" methyltransferase fold (Fig. 2D), a beta sheet with defined strand order and directionality sandwiched between alpha helices . Soaking experiments with SAH confirm that for both domains the SAM binding site coincides with that in other methyltransferases. For the more N-terminal methyltransferase domain, SAH binding is accompanied by a conformational change, in which residues 519-524 and 579-587 rearrange in order to participate in binding. While each methyltransferase domain binds SAM independently, experiments indicate that 7N methyltransfer occurs only in X2 pentamers and not in monomers. (Equivalent experiments for 2 Ό methyltransfer have not been reported.) 23,24

The carboxyterminus forms the flap, a series of three concatenated Ig-like domains. The first two domains resemble the V and C regions of an antibody light chain; the most carboxyterminal such domain is a truncated V domain. The



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Figure 2.



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Figure2. A. The pentameric Λ2 turret as viewed looking down the 5-fold icosahedral axis onto the core. One of the X2 monomers is rendered in black. B. A view of the X2 turret from the side, 90°from the view in FiglA, so that the XI surface is toward the bottom of the page. The same monomer is rendered in black. C. The "black" À2 monomer in the same orientation as in (B). The monomer is shaded by domain. The guanylyltransferase domain is grey at the lower right. The two domains near the top left and rendered dark and darker grey are methyltransferase domains I and II respectively. The light grey domain at the top left corresponds to theflap.SAH binding sites are indicated but difficult to see. D. Diagrams of the SAM-binding domains. The universal methyltransferase domain is shown at the top. Methyltransferase I (light grey) and II (dark grey) are arrayed below it so that conserved structural elements are aligned vertically. The SAM binding site is indicated. E. Ribbon diagram of the guanylyltransferase domain looking into the "cup ", so that the XI surface is behind it Lysines 171 and 190 mark the location of the active site. This figure has been adapted with permission from Nature (404:960-967), MacMillan Magazines, Ltd. 2000. 3


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conformationalflexibilityof the flap probably derives from a hinge region near residue 1023.

Organization of the Active Sites. The various active sites are ordered vertically in the core. The transcription complex is tethered inside the λΐ shell and below the XI turret ; the guanylyltransferase active sites are at the base of the turret; and one set of methyltransferase active sites is half way up, and another set is at the top of the turret. There is no biochemical evidence to establish which methyltransferase domain is the 7N and which is the 2 Ό methylase. One possibility is that the vertical ordering of the active sites reflects the temporal ordering of the reactions, so that since 7N methyltransfer always precedes 2 Ό methyltransfer, the methyltransferase near the middle of the turret methylates 7N and the methyltransferase near the top of the turret methylates the 2 Ό . Probably such a vertical ordering alone would not impose an order on the methylation reactions, and it seems likely that the reovirus 2 Ό methyltransferase, like VP39 from vaccinia , only methylates a cap structure that has been methylated previously at the 5' guanosine moiety.


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What ensures the efficiency of the capping reaction? While the different active sites are close spatially, they are not contiguous. Nor is there any evident groove that links active sites. The capping efficiency probably results from the container like nature of the turret. Although the XI flap domains form a lid for the turret, efficient capping occurs even in the absence of the flap domains : the polymerase pins the 3' end of the mRNA in place at the bottom of the turret, and the cylindrical turret walls restrict the 5' end in two dimensions. Thus reovirus traps the 5' substrate in a cavity densely occupied by fifteen active sites. Indeed, the major theme underlying the organization of active sites, including the location of the polymerase, seems to be that of not losing the substrate. 26

Conclusion The reovirus core structure is among the largest structures determined to date by x-ray crystallography. We have focussed here on a description of the structure in terms of the non-equivalent interactions made by both lambal and σ2. The capsids of the reovirus family do not conform to the theory of quasiequivalence proposed for icosahedral viruses by Caspar and Klug in 1962 , and stable, non-equivalent interactions appear to evolve more readily than previously 27


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imagined. We have found that the arrangement and the fold of λ 1 is universal for members of the reovirus family and that the arrangement and the structures of the other capsid proteins vary and reflect different replication and entry strategies. And finally, we have begun to understand how in this instance organizing multiple enzymatic functions into an efficient machine depends on not completely releasing the substrate until its processing is complete.

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378 14. Kohlstaedt, L. Α., Wang, J., Friedman, J. M., Rice, P. A. & Steitz, T. A. Crystal structure of 3.5A resolution HIV-1 reverse transcriptase complexed with an inhibitor. Science 256, 1783-1790 (1992). 15. Xu, P., Miller, S. & Joklik, W. K. Generation of reovirus core-like particles in cells infected with hybrid vaccinia viruses that express genome segments L1, L2, L3, and S2. Virology 197, 726-731 (1993). 16. Hill, C. L. et al. The structure of cypovirus and the functional organization of dsRNA viruses. Nature Structural Biology 6, 565-568 (1999). 17. Zhang, H. et al. Visualization of protein-RNA interactions in cytoplasmic polyhedrosis virus. Journal of Virology 73, 1624-1629 (1999). 18. Shaw, A. L., Samal, S. K., Subramanian, K. & Prasad, Β. V. V. The structure of aquareovirus shows how the different geometries of the two layers of capsid are reconciled to provide symmetrical interactions and stabilization. Structure 4, 957-967 (1996). 19. Furuichi, Y., Muthukrishnan, S., Tomasz, J. & Shatkin, A. Mechanism and formation of reovirus mRNA 5'-terminal blocked and methylated sequence GpppG pC. The Journal of Biological Chemistry 251, 5043-5053 (1976). 20. Dryden, K. A. et al. Early steps in reovirus infection are assosciated with dramatic changes in supramolecular structure and protein conformation: analysis of virions and subviral particles by cryoelectron microscopy and image reconstruction. The Journal of Cell Biology 122, 1023-1041 (1993). 21. Luongo, C. L . , Reinisch, Κ. M . , Harrison, S. C. & Nibert, M . L. Identification of the Guanylyltransferase Region and Active Site in Reovirus mRNA Capping Protein 12. Journal of Biological Chemistry 275, 28042810 (2000). 22. Håkansson, Κ., Doherty, A. J., Shuman, S. & Wigley, D. B. X-Ray crystallography reveals a large conformational change during guanyl transfer by mRNA capping enzymes. Cell 89, 545-553 (1997). 23. Schluckebier, G., O'Gara, M., Saenger, W. & Cheng, X. Universal catalytic domain structure of AdoMet-dependent methyltransferases. Journal of Molecular Biology 247, 16-20 (1995). 24. Hodel, A. E., Gershon, P. D., Shi, X. & Quiocho, F. A. The 1.85Åstructure of the vaccinia protein VP39: a bifunctional enzyme that participates in the modification of both mRNA ends., (1996). 25. Hu, G., Gershon, P. D., Hodel, A. E. & Quiocho, F. A. mRNA cap recognition: dominant role of enhanced stacking interactions between methylated bases and protein aromatic side chains. Proceedings of the National Academy of Science USA 96, 7149-7154 (1999). 26. Luongo, C. L. et al. Localization of a C-terminal region of λ2 protein in reovirus cores. Journal of Virology 71, 8035-40 (1997). 27. Caspar, D. L. D. & Klug, A. in Cold Spring Harbor Symp. Quant. Biol. 124 (1962). m7

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Structures and Mechanisms - American Chemical Society

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