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Color Plates

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Color Plate 1

(Chapter 1). Comparative genome organization. (A) The genomes of HEV-C and EMCV are drawn to scale (RNA base length), aligned at the 1D/2A junction, to illustrate relative features. The primary cleavage site (open triangle), 3C-dependent secondary cleavage sites (closed triangle), and maturation cleavage site (downward-turned arrow) are highlighted. (B) Full genome sequences for isolates of representative genera and species are shown to scale, colored according to coding features, and aligned relative to the junction of 1D/2A1. Missing 5′-terminal data (No seq) are estimated in each case as 100 bases. The 3′ poly(A) tail (40 to 100 bases) is not shown. Reference GenBank accession numbers for included sequences are as follows: enterovirus HEV-C (j02281); enterovirus HRV-A (x02316); cardiovirus EMCV (m81861); cardiovirus Theiler’s murine encephalomyelitis virus (m20562); aphthovirus FMDV-A (m14409, x00429); aphthovirus ERAV (dq268580); hepatovirus HAV (m14707); parechovirus HPev-1 (l02971); parechovirus LV (ef202833); ERBV (x96871); kobuvirus AiV (ab010145); bovine kobuvirus (ab084788); teschovirus PTV-1 (nc_003985); porcine sapelovirus (af406813); SSV (ay064708); avian sapelovirus (ay563023); SVV (dq641257); tremovirus avian encephalomyelitis virus (nc_003990); avihepatovirus DHAV (dq249299). (C) Key features of representative 5′ and 3′ UTRs are drawn to scale. Known RNA structure elements include terminal cloverleafs (CL), terminal stems (S), type 1 pseudoknots (ψ), poly(C) tracts (C), oligo pyrimidine tracts (Y), spacer segments (SP), IRESs (type I, II, III, or IV), ORF initiation codons (AUG), and ORF termination codons (circle with slash). A supplemental table listing the known start/stop positions for each RNA and/or ORF feature in this figure is available at http://virology.wisc.edu/acp.

Citation: Ehrenfeld E, Domingo E, Roos R. 2010. Color Plates, In The Picornaviruses. ASM Press, Washington, DC.
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Color Plate 2

Structures of picornavirus proteins. (A) The four virion proteins comprising a capsid assembly protomer are derived from a single (enterovirus HRV-B, HRV-14) P1 polyprotein precursor. For visual distinction, this illustration of an X-ray structure (PDB no. 4HRV) assigns individual proteins their historical color referents of blue (VP1), green (VP2), red (VP3), and gold (VP4). VP1, VP2, and VP3 each have similar wedge-shaped, eight-stranded β-barrel configurations. VP4 is on the inside of the virion and can be considered an NH extension of VP2. During particle assembly, 5 protomers combine into a pentamer, and then 12 pentamers coalesce around the viral RNA to form a completed virion. (B to I) Structures are depicted as cartoon models with α-helices in blue, β-strands in gold, and loops in green, except for 3D and 3CD, which are colored using the standard conventions for the major functional domains (the “palm” in blue, “fingers” in red, and “thumb” in green). The protein termini are labeled. (B) The X-ray structure of the aphthovirus FMDV-O leader protease Lb (PDB no. 1QOL) includes the last nine residues of the COOH-terminal extension of a (crystallographic) neighboring Lb molecule bound within the active site (yellow sticks). (C) NMR structure of the cardiovirus EMCV mengovirus leader protein (PDB no. 2BAI). Three Cys residues and one His residue coordinate Zn (pink sphere) in a CHCC zinc-finger motif. (D) X-ray structure of the enterovirus HRV-A, HRV-02) protease 2A (PDB no. 2HRV). The COOH-terminal domain coordinates a tightly bound Zn ion (pink sphere). The catalytic triad is highlighted (red). (E) NMR structure of the soluble NH-terminal domain of the enterovirus HEV-C, poliovirus 1 3A protein (PDB no. 1NG7). (F) Bundle of the 10 best-resolved NMR structures of the poliovirus 1 3B (PDB no. 2BBL). Tyr-3 (red) is a strongly conserved residue used to link this protein to the 5′ end of the genome. (G) X-ray structure of the poliovirus 1 3CD precursor (PDB no. 2IJD). The 3D active site displays a Gly-Asp-Asp (GDD) motif, which is universally conserved in all RNA-dependent RNA polymerases (yellow). (H) X-ray structure of the HAV protease 3C (PDB no. 2A4O), including a covalently bound inhibitor (acetyl-Val-Phe-amide; green sticks). The catalytic triad is highlighted (red). (I) X-ray structure of the aphthovirus FMDV-C S8c1 polymerase 3D shows the protein complexed with a template-primer RNA (PDB no. 1WNE). The GDD motif (yellow), a coordinated Mg ion (pink sphere), template RNA (orange), and primer strand (turquoise) are highlighted.

Citation: Ehrenfeld E, Domingo E, Roos R. 2010. Color Plates, In The Picornaviruses. ASM Press, Washington, DC.
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Color Plate 3

Structure-based phylogenetic tree, calculated using the protomeric subunits of all the intact native picornavirus structures in the protein data bank as of October 2009 (9) (listed in Table 1). The virus structures are shown oriented such that the view is looking down an icosahedral two-fold axis of symmetry. Atoms are drawn as spheres colored according to their distance from the particle center, ranging from blue (at 120-Å radius) to red (at 170-Å radius). Enteroviruses are shown against a blue background, aphthoviruses are against a pink background, and cardioviruses and closely related viruses are against a green background. The method of calculating evolutionary distance is described in reference 64, and the tree was calculated and plotted using the PHYLIP package (20).

Citation: Ehrenfeld E, Domingo E, Roos R. 2010. Color Plates, In The Picornaviruses. ASM Press, Washington, DC.
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Color Plate 4

Architecture. (A) Ribbon outline showing the core β-barrel architecture based loosely on FMDV VP3. The nomenclature for the eight strands comprising the two β-sheets and hence the loops joining the strands is introduced. (B) Ribbon depiction of an SVDV biological protomeric subunit (1OOP [9]) color coded as follows: VP1, blue; VP2, green; VP3, red; VP4, yellow. The background blue kite shape is used to delineate this subunit within the pentameric subunit shown in panel C. (D) Ribbon depiction of a complete capsid (SVDV), with blue lines superimposed to highlight the icosahedral symmetry. (E) Close-ups of the five-fold symmetry axes in SVDV (enterovirus) and FMDV (1BBT [9]) (aphthovirus). The proteins are color coded as for panel B, and the myristate is shown in cyan. (F) β-Strands color coded according to the protein contributing to a sheet spanning the two-fold pentamer interface for SVDV (left) and FMDV (right) (colors are as defined for panel B).

Citation: Ehrenfeld E, Domingo E, Roos R. 2010. Color Plates, In The Picornaviruses. ASM Press, Washington, DC.
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Color Plate 5

Structural features. (Top left) Overlay of ribbon depictions of VP1 for reduced FMDV O (dark green), ERAV (magenta), and mengovirus (orange) (aphthoviruses and cardioviruses). For ease of identification the top left panel shows the same viruses highlighted in the corresponding colors on a structure based on the phylogenetic tree calculated for VP1 (using the method described for Color Plate 3 ). (Center panels) The left panel is a close-up view of the overlaid VP2s of ERAV, mengovirus, and FMDV VP1s overlaid together with the reduced FMDV O1BFS VP1 G-H loop structure (grey), while the two panels to the right show close-ups of the pocket in the VP1 β-barrel for these same viruses and, in addition for means of comparison, for the enteroviruses PV2L (cyan) and SVDV (lime green). A surface-rendered pocket factor is shown in cyan in both of these close-ups but can only fit in the enterovirus pocket (in aphtho- and cardioviruses, side chains fill the pocket). (Bottom panels) Color depth-cued surface renditions of FMDV O (left) and PV2L (right) (both colored using the radiusdependent scheme defined for Color Plate 3 ). One of the canyons circling the five-fold axes of enteroviruses is highlighted. Note the relatively smooth surface of FMDV.

Citation: Ehrenfeld E, Domingo E, Roos R. 2010. Color Plates, In The Picornaviruses. ASM Press, Washington, DC.
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Color Plate 6

Consensus sites. (A) Consensus antigenic sites highlighting the atoms of the relevant secondary structure using spheres color coded according to protein (using the scheme defined for Color Plate 4B ) overlaid on a grey ribbon depiction of CVA21 (data from MAb escape mutants; sites are considered to be consensus when six or more serotypes of the viruses studied shared the same site [54]). (B) Consensus receptor-binding sites shown by highlighting the relevant residues using spheres color coded according to protein on a grey ribbon depiction of CVA21 (data are from footprints of complex structures, aligned with CVA21 and considered consensus when three or more serotypes use the same site). The atomic data include all the relevant structures listed in Table 1.

Citation: Ehrenfeld E, Domingo E, Roos R. 2010. Color Plates, In The Picornaviruses. ASM Press, Washington, DC.
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Color Plate 7

Intermediate structures. (Top) Comparison of protomers of poliovirus mature and empty (assembly intermediate) structures. The protomeric subunits are viewed from the inside, and the common structures on the surface are color coded by protein. The VP1 and VP2 N termini and VP4 (mature particle) are shown as tubes in standard protein colors (as defined for Color Plate 4B ); for the empty particle, the VP0 N terminus is shown in yellow. (Bottom) Comparison of protomers of mature ERAV and the lowpH structure (likely disassembly intermediate). The protomeric subunits are viewed from the inside, and the common structures on the surface are color coded by protein. The VP1 and VP2 N termini and VP4 are shown as tubes in the standard protein colors.

Citation: Ehrenfeld E, Domingo E, Roos R. 2010. Color Plates, In The Picornaviruses. ASM Press, Washington, DC.
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Color Plate 8

Structure of PV. (A) Depth-cued view of the intact particle with a ribbon model of a protomer, showing the prominent surface features, including star-shaped mesas at the five-fold axes, three-bladed propellers at the threefold axes, deep canyons surrounding the five-fold mesas, and saddle-shaped depressions crossing the two-fold axes. A five-fold axis, two-fold axis, and three-fold axis are indicated in red for reference. To the right of the depth-cued virion, the ribbon diagram shows the structure of the four coat protein molecules from a single protomer, with VP1 in blue, VP2 in yellow, VP3 in red, and VP4 in green. An icosahedral framework with five-fold, two-fold, and three-fold axes is provided for reference. VP1, VP2, and VP3 share a common core structure (an eight-stranded β-barrel). Each of the proteins has unique loops, carboxy-terminal extensions on the outer surface, and a long amino-terminal extension on the inner surface. (B) The amino-terminal extensions of VP1, VP2, and VP3 together with VP4 form an elaborate network on the inner surface of the capsid shell that stabilizes the virus particle. The network is viewed from the inside here. (C) The β-tube formed as five copies of the amino terminus of VP3 intertwine around the five-fold axes forms a plug which blocks an otherwise-open channel connecting the outer surface (top) and inner surface (bottom) of the virus. The β-tube is flanked by a three-stranded β-sheet formed by the hairpin from the amino terminus of VP4 and a strand from the extreme amino terminus of VP1. The myristoyl substituent at the amino terminus of VP4 (purple) mediates the interaction between the VP3 β-tube and the flanking β-sheet. This structure stabilizes the interprotomer interactions linking protomers in a pentameric subassembly. (D) A seven-stranded β-sheet formed by four strands from the β-barrel of VP3 from one pentamer, a β-hairpin from the amino terminus of VP2 from a two-fold related pentamer, and a β-strand from the amino-terminal extension of VP1 from the original pentamer. This interaction stabilizes interactions between two-fold related pentamers in the intact virion. (E) The network includes a number of prominent intraprotomer interactions, including those contributed by the amino-terminal extension of VP3 as it wraps underneath the surface of VP1 and by the amino-terminal extension of VP1 as it wraps underneath the surface of VP3. The portions of VP4 and VP3 that extend beyond the protomer at top right and a β-hairpin from the amino-terminal extension of VP2 that extends beyond the protomer at the lower left contribute to interprotomer interactions are shown.

Citation: Ehrenfeld E, Domingo E, Roos R. 2010. Color Plates, In The Picornaviruses. ASM Press, Washington, DC.
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Color Plate 9

The inner surface of the 73 empty capsids provides some clues about the mechanism of VP0 cleavage. (A) The stretch of peptide containing the scissile bond of VP0 is located at the tips of a trefoil-shaped depression in the inner surface of the empty capsid structure. Three protomers of the capsid protein are shown, surrounding a three-fold axis viewed from the inside of the capsid looking out. The depression is filled by the amino terminus of VP1 in the mature virion. (B) The trefoil-shaped depression with the ordered RNA from Cowpea chlorotic mottle virus docked into the depression. RNA binding to this site could participate in autocatalyic cleavage of VP0. (C) Stereo representation of the atomic model, showing the peptide containing the scissile bond (green/yellow boundary) with a neighboring histidine and network of water. Mutations of this histidine are impaired for VP0 cleavage.

Citation: Ehrenfeld E, Domingo E, Roos R. 2010. Color Plates, In The Picornaviruses. ASM Press, Washington, DC.
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Color Plate 10

Structure of the virus-receptor complex. (A) Cryo-EM structure of the complex of PV with the fully glycosylated ectodomain of its receptor, PVR. The structure has been colored to show the density corresponding to the virus (blue) and the density for the receptor (grey). (B) Ribbon diagram showing a model for the receptor and the virus, built into the cryo-EM density. (C) Close-up view of the interaction of the amino-terminal domain of the receptor with the virus. The complementarity of the fit is excellent, with the receptor containing several surface features of the virus that are known to be rearranged in cell entry intermediates, including the GH loop of VP1, the EF loops of VP2 and VP3, and the carboxyl termini of VP1, VP2, and VP3.

Citation: Ehrenfeld E, Domingo E, Roos R. 2010. Color Plates, In The Picornaviruses. ASM Press, Washington, DC.
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Color Plate 11

Structure of the 135S particle. (Top left) Cryo-EM reconstruction of the 135S particle at 10 Å revealed prominent ridges of density connecting the tips of the five-fold mesa (blue dot) with the tips of the propeller-like feature surrounding the three-fold axes (red dot). This ridge, which is missing in the structure of the mature virion (top right), can be modeled as an α-helix. (Bottom left and middle) The cryo-EM density (grey) and models for the capsid proteins that were fit and refined into the density map (colors). In this view, five-fold-related protomers have been butterflied to show the surfaces that normally interact in the interprotomer interface. The first well-ordered residue in the β-barrel of VP1 is shown as an orange sphere, and the course of the amino-terminal extension of VP1 as it exits through the interface at the base of the canyon is shown as a gold tube. The helix fit into the ridge of density (tentatively assigned as residues 41 to 53 of VP1) is shown in purple, and the site of a prominent difference in the density for the intact 135S particle and the density for 135S particles in which residues 1 to 31 of VP1 are proteolytically removed are shown in green. In the left panel these features come from the neighboring subunit. (Bottom right) Cartoon representation of the model, showing the RNA inside the particle (dark blue), the VP3 plug (red), and the exiting VP1 peptide (cyan), with the helix of residues 41 to 53 (magenta). The arrows indicate that the amino-terminal amphipathic helix of VP1 residues 1 to 30 is flexibly linked to the capsid and is therefore not visible in the cryo-EM reconstruction.

Citation: Ehrenfeld E, Domingo E, Roos R. 2010. Color Plates, In The Picornaviruses. ASM Press, Washington, DC.
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Color Plate 12

Model for template selection during poliovirus translation and viral RNA replication. The figure depicts poliovirus positive-strand RNA (black line) under conditions that favor cap-independent translation (top panel) or those that favor negative-strand RNA synthesis (bottom panel). Under conditions that favor translation early during the infectious cycle (top panel), the RNA is bound by ribosomes (denoted by 80S on the gray spheres) continuously initiating and elongating nascent viral polypeptide chains (depicted by orange curly lines). Initiation of translation is facilitated by the binding of cellular protein PCBP2 to the poliovirus IRES in the 5′ noncoding region of positive-strand RNA (secondary structures with thickened black lines) and the possible bridging of RNA to the ribosome by the cellular protein, SRp20. The presence of multiple ribosomes on viral RNA may preclude the viral RNA polymerase (3D) from elongating any newly initiated negative-strand RNAs. Under conditions that favor negative-strand RNA synthesis later during the infectious cycle (bottom panel), PCBP2 has been cleaved by the accumulated 3CD (or 3C) proteinase polypeptides (as indicated by the orange arrow). This inhibits the binding of ribosomes to the IRES, thereby reducing the levels of translation initiation complex formation and releasing SRp20 from PCBP2. The cleaved form of PCBP2 can still participate in ternary complex formation with the 5′ stem-loop I structure and 3CD. This may facilitate the interaction of the 5′ ribonucleoprotein complex with the 3′ poly(A) tract via a bridging interaction with poly(A)-binding protein. This latter interaction would lead to initiation of negative-strand RNA synthesis on templates that are now cleared of translating ribosomes. The gray arrows indicate the direction of ribosomes traversing the RNA during translation, while the red arrows indicate the direction of the 3D RNA polymerase during negative-strand RNA synthesis. The blue lines represent nascent negative-strand RNAs with a VPg (small solid red circle) at their 5′ ends. The UUUUUU represents the 5′ oligo(U) tract that is templated by the 3′ poly(A) tract on genomic positive-strand RNAs. The separation of the two processes is shown for illustration purposes only, since it is likely that the viral replication cycle is a dynamic process with both activities occurring simultaneously (but not on the same template RNA). (From reference 66, with permission of the publisher.)

Citation: Ehrenfeld E, Domingo E, Roos R. 2010. Color Plates, In The Picornaviruses. ASM Press, Washington, DC.
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Color Plate 13

Schematic of the surface of a PV-induced vesicle, with known membrane-associated proteins of the RNA replication complex shown to scale. A cutaway section of the outer bilayer of a PV-induced membranous vesicle is shown with lipids and proteins shown to scale, and the appropriate curvature for this 400-nm vesicle is indicated. The 8- to 10-nm width of the lipid bilayer is shown, and proteins are represented as abstract shapes that indicate their relative sizes and published oligomeric forms. For PV protein 2C, an electron microscopic image of an oligomeric form has been published (1); this image is shown to scale. Although evidence has been presented that 2B proteins are oligomeric, no structural image is yet available. For poliovirus protein 3A, the NMR structure of a dimeric form of the soluble N-terminal domain has been published (73); this structure is shown at a 5× increase in scale below the bilayer. Arrows indicate the amino termini of the 3A proteins that are natively unfolded; the carboxyl termini are connected to the membrane-associated hydrophobic region.

Citation: Ehrenfeld E, Domingo E, Roos R. 2010. Color Plates, In The Picornaviruses. ASM Press, Washington, DC.
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Color Plate 14

Schematic of the surface of a PV-induced vesicle, with known polymerase-containing complexes shown to scale. A cutaway section of the outer bilayer of a membranous vesicle, 400 nm in diameter, induced during PV infection is indicated. One-sixth of the circumference of the bisected vesicle is shown, with the appropriate curvature. The 8- to 10-nm width of the lipid bilayer is indicated, with approximately 150 lipids in each leaflet of the portion of the membrane shown. Bar, 5 nm (may be used to estimate the relative sizes of the PV proteins and complexes known or suggested to be involved in RNA replication). For those proteins for which electron microscopic images of oligomeric forms are available, those images are provided and shown to scale, with permission from the original references. Proteins, their relative sizes, and any oligomeric forms that have been observed by electron microscopy or NMR, are shown as abstract shapes. For those proteins whose monomeric structures have been solved, the structures are shown at 5× magnification. In the 3CD structure, the green sequences indicate those that are most changed from those of the 3C and 3D structures.

Citation: Ehrenfeld E, Domingo E, Roos R. 2010. Color Plates, In The Picornaviruses. ASM Press, Washington, DC.
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Color Plate 15

Map of RNA-protein binding of the FMDV IRES. The schematic shows the factors required for IRES activity, depicted on the secondary structure derived from RNA probing of the entire FMDV IRES. The positions of domains 1 to 5 (or H to L) are shown at the bottom. For simplicity, only proteins whose binding site and functional involvement in IRES activity have been analyzed in detail are represented. Blue asterisks surrounding the GNRA motif mark changes in RNA accessibility to dimethyl sulfate in vivo relative to naked RNA in vitro. An arrow points to the RNase P cleavage site in vitro. Py denotes the position of a conserved polypyrimidine tract. Initiator codons, with the corresponding toeprints (depicted by stars), are highlighted in red in the sequence.

Citation: Ehrenfeld E, Domingo E, Roos R. 2010. Color Plates, In The Picornaviruses. ASM Press, Washington, DC.
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Color Plate 16

Atomic structures of picornavirus proteinases. Structures were rendered using PyMol (DeLano Scientific). (A) Structure of PV 3CD (2IJD) is shown in the inset (upper right) with the proteinase domain enlarged. The 3C proteinase domain is structurally related to the large subclass of serine proteinases. The position of the catalytic triad (H, E, and C, the latter mutated to alanine for purposes of structural determination) is shown. The RNA-binding site on the opposite face of the 3C domain, close to the 3C/3D glutamine/glycine (Q-G) cleavage site, is shown. (B) The structure of rhinovirus 2A (2HRV) reveals a bilobal structure similar to the small subclass of serine proteinases. The position of the active site is shown (H, D, and C) together with the position of the zinc atom, which performs a structural, and not catalytic, role. (C) The structure of L (1QOL) is related to thiol (papain-like) proteinases, and the position of the catalytic diad (C and H, the former mutated to alanine for purposes of structural determination) and the protruding C-terminal extension on the opposite face are shown.

Citation: Ehrenfeld E, Domingo E, Roos R. 2010. Color Plates, In The Picornaviruses. ASM Press, Washington, DC.
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Color Plate 17

Ribbon diagrams of the structure of FMDV 3D polymerase in complex with an RNA template primer (PDB ID 1WNE), shown in different views. (Top) Conventional orientation displaying the right-hand conformation. The polymerase is depicted in gray, with the fingers, palm, and thumb subdomains specifically labeled. The six different conserved structural motifs of the palm and fingers domains are colored as follows: A, brown; B, green; C, red; D, blue; E, orange; F, cyan; G, magenta. (Bottom) Top-down view (90° upward rotation relative to the structure shown at the top), with the same color codes. Amino acids G62 and M296, involved in resistance of picornaviruses to R, are indicated. (Based on references 62 and 62a.)

Citation: Ehrenfeld E, Domingo E, Roos R. 2010. Color Plates, In The Picornaviruses. ASM Press, Washington, DC.
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Color Plate 18

Structure and interactions of the FMDV 3D-VPg-UMP complex (PDB ID 2F8E). The FMDV polymerase is shown in grey, the primer protein VPg in yellow, and the UMP in purple. VPg lines the RNA-binding cleft of the 3D polymerase, positioning its Tyr-3 hydroxyl group as a molecular mimic of the free 3′-hydroxyl group of a nucleic acid primer at the active site for nucleotidylylation. The two insets on the right side show close-ups of the interactions established between VPg and different polymerase residues. In the active site, the hydroxyl group of the Tyr-3 side chain was found covalently attached to a UMP molecule by a phosphodiester linkage. (Upper inset) Two metal ions (dark grey spheres) participate in the uridylylation reaction. Metal 1 bridges the catalytic aspartate, Asp338 of motif C (red), and the O of the tyrosine side chain, now covalently bound to phosphate α of UMP. Metal 2 coordinates the carboxyl group of Asp245 of motif A (brown), the O1 oxygen of phosphate α, and the hydroxyl group of Ser298 within loop β9-α11, next to motif B (dark green). The conserved Tyr336 of motif C and the positively charged residues K164, R168, K172, and R179 of motif F (cyan) also participate in the uridylylation process. (Lower inset) In addition to the interactions in the polymerase active site, the lower inset shows the different residues of motifs F (R168; cyan) and E (K387 and R388; orange) that together with residues within the first helix of the thumb subdomain (amino acids from T407 to I411; grey) interact with the central part of VPg. Finally, the FMDV 3D residues Gly216 and Cys217 (grey), in the fingers subdomain, establish hydrophobic contacts with VPg at the exit of the polymerase cavity. (Based on references 59 and 60 with publisher permission.)

Citation: Ehrenfeld E, Domingo E, Roos R. 2010. Color Plates, In The Picornaviruses. ASM Press, Washington, DC.
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Color Plate 19

Structure and interactions of the FMDV polymerase, RNA template primer, and rNTP substrates. The polymerase is shown in grey in the central panel, with the conserved motifs, involved in contacts with the RNA molecule, highlighted in different colors. The template and primer strands of the RNA molecule are shown in yellow. The upper inset on the right side shows a close-up of the interactions in the polymerase active site after AMP incorporation, PPi release (sand), and the positioning of the new incoming UTP (blue) close to the active site, as seen in the structure of the FMDV 3D-RNA-ATP/ UTP complex (PDB ID 2E9Z). The residues that establish contacts are shown as sticks and the hydrogen bonds as dashed lines, in black. The UTP is located close to the nucleotide-binding pocket bound to the polymerase/template/primer complex through a metal ion and the basic residues of motif F (cyan). The ribose and base moieties of UTP establish additional contacts with Asn307 of motif B (lime green) and Ser298 of the β9-α11 loop (dark green). The ribose-binding pocket is partially occluded by the side chains of Asp245, Thr303, and Asn307, which are connected by hydrogen bonds. The lower inset on the right side shows a close-up of the interactions in the polymerase active site with RTP (PDB ID 2E9R). The incoming nucleotide analog (pink) is located adjacent to the 3′ terminus of the primer and is base-paired to the template acceptor base. The position of the RTP base is further stabilized by interactions with residues of motifs A (brown) and B (lime green) and the loop β9-α11 (dark green). The triphosphate moiety is hydrogen bonded to different residues of motifs A and F (cyan) and interacts with one metal ion (dark green sphere). (Based on reference 61.)

Citation: Ehrenfeld E, Domingo E, Roos R. 2010. Color Plates, In The Picornaviruses. ASM Press, Washington, DC.
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Color Plate 20

In vitro and structural analyses of picornavirus RdRp fidelity. An in vitro biochemical assay, developed by the Cameron lab, uses a symmetrical, RNA primer-template that permits the measure of incorporation kinetics and fidelity of purified polymerase enzyme. This system permits the dissection of polymerase activity into five steps. Studies with the wild-type and G64S enzymes showed that step 2 is a critical step in determining RdRp fidelity. The crystal structure of the poliovirus RdRp revealed that a hydrogen bond network in which residue 64 participates is altered by the G64S change. (Adapted from references 4, 6, and 92.)

Citation: Ehrenfeld E, Domingo E, Roos R. 2010. Color Plates, In The Picornaviruses. ASM Press, Washington, DC.
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Color Plate 21

IRES types of the 12 classified (names in grey ovals) and 3 likely further genera within the (A) and the 4 genera within the (B). The distribution of the type IV IRESs shows several inconsistencies with the branching order of the different picornavirus genera. A structurally similar and likely evolutionarily related type IV IRES is also found in the flavivirus genera (containing the human pathogen hepatitis C virus) and . The tree was constructed by neighbor joining using amino acid sequence distances between sequences in the 3D regions (positions 5862 to 7365, as numbered in the poliovirus Leon strain [accession number K01392]) from representative members of each species and genus. The tree is based on the region of identifiable homology in the RNA-dependent RNA polymerase (NS5[B]) between positions 8395 and 8759 (numbered as in the hepatitis C virus type 1 prototype sequence H77 [accession number AF011751]). (Panel A is an update of a previous analysis [31].)

Citation: Ehrenfeld E, Domingo E, Roos R. 2010. Color Plates, In The Picornaviruses. ASM Press, Washington, DC.
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Color Plate 22

(A) Phylogenetic analysis of the 5′ UTR of prototype sequences of HEVs, color coded by species (A to D). Sequence labels indicate the serotype and accession number. Abbreviations: CVA, coxsackie A virus; CVB, coxsackie B virus; EV, enterovirus; E, echovirus; PV, poliovirus. (B) Phylogenetic analysis of the 5′ UTR of prototypic and available complete genome sequences of HRV-A to -C and partial 5′ UTR sequences representing the divergent species C 5′ UTR group. The trees were constructed by neighbor joining using uncorrected nucleotide sequence distances from the whole 5′ UTR (A) or between positions 291 and 616 for HRV (positions numbered according to the NC_001490 reference sequence [serotype 14]). Bootstrap resampling was used to determine robustness of the groupings; values of ≥70% are shown.

Citation: Ehrenfeld E, Domingo E, Roos R. 2010. Color Plates, In The Picornaviruses. ASM Press, Washington, DC.
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Color Plate 23

(A) Phylogenetic grouping of isolates of different serotypes of HEV species C (including poliovirus serotypes 1 to 3) in a series of phylogenetic trees generated from consecutive 300-base fragments across the genome. Below the map is a genome diagram of HEVs, drawn to scale and numbered according to the poliovirus P3/Leon/37 sequence (accession number K01392). (B) Segregation scores for consecutive fragments across genomes of HEV-A to -C, where zero ( axis) represents perfect phylogenetic segregation by assigned group (serotypes) and 1 represents the calculated value with no association between phylogeny and group assignment. (C) Mean pairwise amino acid sequence distances between sequences in consecutive 300-base fragments across the genome; values were averaged over a window size of 3 for the three enterovirus species. (D) Phylogenetic compatibility matrix between trees generated from different genomic regions of HEV-B. The matrix shows phylogenetic compatibility scores between trees generated from consecutive 300-base fragments of genome alignments of each virus group, color coded according to the key (compatibility scores from 0.0 to 1.5). Phylogenetically compatible regions are shown in deep blue. (The components of this figure are modified from illustrations in reference 66.)

Citation: Ehrenfeld E, Domingo E, Roos R. 2010. Color Plates, In The Picornaviruses. ASM Press, Washington, DC.
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Color Plate 24

(A) Phylogenetic tree of a region in the 3D gene (representative of the NS region) of isolates of echovirus 30 collected worldwide between 2001 and 2007. Echovirus 30 sequences were resolved into a total of 38 phylogenetically distinct lineages, with the main clades color coded (those with >3 members are shown in black). Echovirus 30 clades were highly interspersed with those of other species B serotypes (unfilled circles, labeled by serotype designation and accession number, as for Color Plate 22 ). (B) Turnover of echovirus 30 in Europe over the last decade (240 isolates), showing the relative abundances of isolates with different 3D sequences (color coded as for the phylogenetic tree in panel A). Isolates with rarer 3D clades (two or fewer occurrences) are individually labeled. The tree was constructed by neighbor joining using maximum composite likelihood nucleotide sequence distances from the 3D gene (positions 6968 to 7151, numbered as for the poliovirus P3/Leon/37 strain [accession number K01392]). Bootstrap resampling was used to determine robustness of the groupings; values of ≥70% are shown. The components in this illustration are modified from reference 44.

Citation: Ehrenfeld E, Domingo E, Roos R. 2010. Color Plates, In The Picornaviruses. ASM Press, Washington, DC.
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Color Plate 25

(A) Sequence divergence in the capsid and NS gene regions between the 74 serotypes of HRV-A (each of the 3,321 pairwise comparisons is individually plotted). (B) Nucleotide sequence divergence scan between two representative HRV-A serotypes (types 16 and 44) falling within the main group, representing the typical degrees of divergence in S and NS regions between picornavirus serotypes. Also shown are two selected outliers with similar degrees of sequence divergence in the capsid region but different variabilities in the NS region, indicative of past recombination.

Citation: Ehrenfeld E, Domingo E, Roos R. 2010. Color Plates, In The Picornaviruses. ASM Press, Washington, DC.
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Color Plate 26

3ABC mediates cleavage of the IFN-signaling adaptor protein MAVS (106). (A) Laser-scanning confocal microscopy images showing colocalization of ectopically expressed 3ABC-Flag and MitoTracker, a mitochondrial marker. (B) Confocal microscopy images demonstrating that HM175/18f infection of FRhK-4 (fetal rhesus kidney) cells abolishes expression of the adaptor protein MAVS. (Reproduced from [106] with permission. Copyright 2007, National Academy of Sciences, USA.)

Citation: Ehrenfeld E, Domingo E, Roos R. 2010. Color Plates, In The Picornaviruses. ASM Press, Washington, DC.
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Color Plate 27

Earliest detection of FMDV in vivo. Localization of FMDV strain O1 Manisa in the nasopharyngeal mucosa-associated lymphoid tissue (MALT) of a bovine 6 h after aerosol exposure. (a) Immunohistochemical detection of FMDV capsid antigen within superficial MALT. Magnification, ×4. The insert (magnification, ×40) shows a higher magnification of the region of interest (dashed line). (b, c, and d) Simultaneous immunofluorescent localization of FMDV capsid (red) and cytokeratin (green) with nuclear counterstain (blue) on a serial section of the tissue shown in panel a. In the merged image (d), FMDV colocalizes with cytokeratin in nasopharyngeal epithelial cells (yellow/orange), indicating that these are the primary infection sites in cattle. Magnification, ×40. (Images courtesy of Jonathan Arzt.)

Citation: Ehrenfeld E, Domingo E, Roos R. 2010. Color Plates, In The Picornaviruses. ASM Press, Washington, DC.
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Color Plate 28

FMDV induces degradation of NF-кB. (Left) Bovine epithelial cells infected with FMDV serotype A12 wild-type show that by 4 h postinfection, L (green) is present in the cytoplasm and the nucleus of an infected cell while activated NF-кB (red) is concentrated in the nucleus. (Right) Progression of the infection leads to a complete disappearance of NF-кB concurrent with an increased presence of L in the nuclei of infected cells. Nuclei were stained with 4′,6-diamidino-2-phenylindole. (Images courtesy of Fayna Diaz-San Segundo.)

Citation: Ehrenfeld E, Domingo E, Roos R. 2010. Color Plates, In The Picornaviruses. ASM Press, Washington, DC.
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Color Plate 29

Mutation of the L SAP domain affects nuclear retention and degradation of NF-кB. (a) L (green) is present in the cytoplasm and nucleus of FMDV wild-type-infected cells. Infected cells are indicated by the presence of VP1 (1D) (red; in all panels). (b) L(green) is localized only to the cytoplasm of FMDV SAP mutant-infected cells. (c) NF-кB (green) is not detected in FMDV wild-type infected cells but is detected in the nuclei of bystander cells. (d) NF-кB (green) is detected in the nuclei of FMDV SAP mutant-infected and bystander cells. Bovine epithelial cells were infected with FMDV, and images were taken at 6 h postinfection. (Images courtesy of Fayna Diaz-San Segundo.)

Citation: Ehrenfeld E, Domingo E, Roos R. 2010. Color Plates, In The Picornaviruses. ASM Press, Washington, DC.
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