Uncles ex Machina: Familial Epiphany in Euripides' Electra

At the close of Euripides’ Electra, the Dioscuri suddenly appear ‘on high’ to their distraught niece and nephew, who have just killed their mother, the divine twins’ mortal sister. This is in fact the second longest extant deus ex machina (after the final scene in Hippolytus), and the only scene in which a tragedian attempts to resolve directly the aftermath of the matricide. In this article, I argue that Castor's and Polydeuces’ sudden apparition to Orestes and Electra constitutes a specialised point of intersection between the mortal and immortal realms in Greek tragedy: familial epiphany, an appearance by a god who has an especially intimate relationship with those on stage. Euripides’ focus on the familial divine as a category accentuates various contradictions inherent to both ancient Greek theology and dramaturgy. The Dioscuri are a living paradox, ambiguously traversing the space between dead heroes and gods, managing at the same time to occupy both. They oscillate uniquely between the mortal and immortal worlds, as different sources assign different fathers to each brother, and others speak of each one possessing divinity on alternate days. As I propose, the epiphany of these ambiguous brothers crystallises the problem of the gods’ physical presence in drama. Tragedy is the arena in which gods burst suddenly into the mortal realm, decisively and irrevocably altering human action. The physical divine thus tends to be both marginal and directorial, tasked with reining in the plot or directing its future course. The appearance of the familial divine, on the other hand, can in fact obscure the resolution and future direction of a play, undermining the authority of the tragic gods. In the specific case of Electra, I contend that the involvement of the Dioscuri, who are Electra's and Orestes’ maternal uncles, produces a sense of claustrophobia at the close of the play, which simultaneously denies the resolution that is expected from a deus ex machina while also revealing the pessimistic nature of what is typically considered a reassuringly ‘domestic’ and character driven drama

Data collection and refinement statistics.

†<p><i>R</i>_merge = Σ<i>_hkl</i>Σ<i>_i</i>|<i>I_i</i>(<i>hkl</i>)−<<i>I</i>(<i>hkl</i>)>|/Σ<i>_hkl</i>Σ<i>_iI_i</i>(<i>hkl</i>), where <i>I</i> is the observed intensity and <<i>I</i>> is the average intensity of multiple observations of symmetry-related reflections.</p>‡<p><i>R</i>_factor = Σ<i>_hkl</i>|<i>F_obs</i>(<i>hkl</i>) − <i>F_calc</i>(<i>hkl</i>)|/Σ<i>_hklF_obs</i>(<i>hkl</i>), where <i>F</i>obs and <i>F</i>calc are the structure factors, deduced from measured intensities and calculated from the model, respectively.</p

Changes around the five-fold channels.

<p>Lateral view of the five-fold axis in the 80S particle (A) and the native capsid (B) of HRV2. VP1 protein is shown in blue, VP3 in red and VP4 in yellow. For clarity, only two subunits of VP1 are displayed. In the native structure, the pocket factor is displayed as orange sticks and the N-terminal α-helix (residues 1001 to 1013, not present in the crystal structure of HRV2) has been modeled according to its position in the structure of HRV16 and is shown in grey. The close-ups at the right show a top view of the five-fold axes, with residues His1138 and His1173 from the five symmetry related VP1 subunits displayed as yellow sticks. (C) Close-up of the N-terminal region of VP1 from the HRV2 native structure, seen from the inside of the virion. VP1 is shown in blue, VP2 in green and VP3 in red; the first residue seen in the 80S density map (Arg1062) is indicated with an asterisk and from then on VP1 is colored in cyan. The side chains of the residues involved in acid-labile interactions stabilizing the conformation of the VP1 N-terminus are shown as sticks and explicitly labeled.</p

Opening of the two-fold channels in the 80S empty capsid.

<p>Contacts between pentamers along the two-fold and three-fold axes in the 80S empty particle (A) as compared to those present in the native HRV2 capsid (B). The capsid proteins VP1 (blue), VP2 (green) and VP3 (red) are depicted as ribbons. The position of the symmetry axes is indicated in (B). (C) Close-up view of the channel formed at the two-fold axes in the 80S particle, seen from the interior of the capsid. The VP2 and VP3 subunits surrounding the channel are represented as cartoons and the side chains of VP2 exposed at the interface are depicted as sticks. The lines crossing the cavity correspond to distances of 10.2 Å (a) and 30.5 Å (b). (D) Charge distribution on the walls of the channel at the two-fold axes. In order to show the surface of the longitudinal walls, a side view of the channel (perpendicular to the direction of the symmetry axis) has been slabbed. The surface is represented with the electrostatic potential, colored in blue and red for positive and negative charges, respectively. The relevant residues are shown as sticks and labeled. The direction of the two-fold symmetry axis is also indicated with a line and a black symbol.</p

Formation of pores in the 80S empty particle.

<p>Surface representation of a pentamer from the 80S particle (A) and the native capsid (B), seen from the outside and along the five-fold symmetry axis. In both figures, VP1 is shown in blue, VP2 in green and VP3 in red. The residues located at the outer opening of the pores in the 80S structure (Met1104, Ala1105, Gln1106, Tyr1159 and Gln1162) and the corresponding residues in the native capsid are painted in yellow. In (A), the location of one pore is explicitly highlighted with a yellow square. (C) Lateral view of the pentamer region highlighted in (A), represented as surface and slabbed to show the pore that completely crosses the capsid from the canyon floor to the capsid interior. The side chains of residues located along the pore walls are shown as sticks in blue (VP1) and red (VP3). The N-terminus of the 80S VP1 structure is also shown as sticks and the residues located at the base of the pore (Arg1062, Asp1063 and Glu1064) are labeled. The side chain of residue Arg1062 has been modeled from the native capsid structure. For orientation purposes, the direction of the five-fold symmetry axis is indicated with a line and a black symbol at the upper-left corner.</p

The hinge movement of VP1.

<p>Side by side comparison of the VP1 structures from the native virion (A) and the 80S particle (B), showing the hinge movement displacing the αA helix and the C-terminus of VP1 away from the VP1 β-barrel. The secondary structural elements are indicated on the 80S VP1 representation. The angle formed by the position of the Cα atoms from residues Ser1130, Lys1243 and Ala1105 and its value is also shown in both cases. (C) Schematic representation of the protomer expansion in the transition from the native virion (left) to the 80S particle (right). The hinge movement of VP1 (shown in blue) induces a concerted displacement of VP2 and VP3 (in green and red, respectively). To facilitate the comparison, the outer limits of the native protomer are also displayed in the 80S figure as thin lines. (D) Surface representation of the 80S protomer, seen from the interior of the particle, following the five-fold axis. VP1 is shown in blue, VP2 in green and VP3 in red. For clarity, only the Cα are displayed. The position of the symmetry axes is indicated with black symbols (E) Same view as in (D), with each residue colored according to the displacement suffered for its Cα in the transition from the native capsid to the 80S particle. The color scale, indicated as a bar, covers distances from 0 Å (in dark blue) to 5.5 Å or more (in red). The distances were calculated from a superposition of the native and the 80S protomers, using the VP1 β-barrel as a guide. The disulfide bridge linking Cys2229 and Cys3120 is shown as purple sticks and indicated with an arrowhead.</p

New Insights into Rotavirus Entry Machinery: Stabilization of Rotavirus Spike Conformation Is Independent of Trypsin Cleavage

<div><p>The infectivity of rotavirus, the main causative agent of childhood diarrhea, is dependent on activation of the extracellular viral particles by trypsin-like proteases in the host intestinal lumen. This step entails proteolytic cleavage of the VP4 spike protein into its mature products, VP8* and VP5*. Previous cryo-electron microscopy (cryo-EM) analysis of trypsin-activated particles showed well-resolved spikes, although no density was identified for the spikes in uncleaved particles; these data suggested that trypsin activation triggers important conformational changes that give rise to the rigid, entry-competent spike. The nature of these structural changes is not well understood, due to lack of data relative to the uncleaved spike structure. Here we used cryo-EM and cryo-electron tomography (cryo-ET) to characterize the structure of the uncleaved virion in two model rotavirus strains. Cryo-EM three-dimensional reconstruction of uncleaved virions showed spikes with a structure compatible with the atomic model of the cleaved spike, and indistinguishable from that of digested particles. Cryo-ET and subvolume average, combined with classification methods, resolved the presence of non-icosahedral structures, providing a model for the complete structure of the uncleaved spike. Despite the similar rigid structure observed for uncleaved and cleaved particles, trypsin activation is necessary for successful infection. These observations suggest that the spike precursor protein must be proteolytically processed, not to achieve a rigid conformation, but to allow the conformational changes that drive virus entry.</p></div

3DR of SA11 TLP from cryo-electron tomography.

<p>(A, C) Slice through the xy plane of the reconstructed cryo-electron tomograms of NTR- (A) and TR-TLP (C). The bar represents 100 nm. White arrowheads indicate examples of electrodense structures inside particles. Black arrowheads indicate examples of spikes on the outer particle surface of the virions. (B, D) Gallery of central slices through extracted NTR- (B) and TR-TLP (D). (E, G) Surface-rendered model of the averaged NTR- (E) and TR-TLP (G) calculated from the extracted subtomograms and viewed along an icosahedral 2-fold axis. The bar represents 100 Å. (F, H) Close up view of the NTR (C) and TR (F) spike represented as in E and G.</p

Tomogram averaging and classification of NTR and TR spikes.

<p>(A, B) Surface-rendered model of averaged tomograms, reference-free classified, for NTR (A) and TR (B) spikes. Top and middle rows show two side views of the averages related by a 90 degree rotation. The bottom row shows the top view of the averages. Arrowheads indicate an extra density at the base of the spike stalk in the class 2 NTR average, which is absent in NTR and TR class 1 averages. (C) Surface-rendered model of the class 1 TR spike fitted with the VP4 atomic model (PDB entry 3IYU). VP8* molecules A and B are in purple, VP5* molecules A and B are in red, and VP5* C is in orange. The last resolved residue of VP8* (Lys²⁹, purple) and the first resolved residue of VP5* (Glu²⁶⁴, orange) are indicated by spheres for the VP4-C molecule. (D) Surface-rendered model of the class 2 NTR spike fitted with the VP4 atomic model and represented as in C. (E) Close up view of the NTR spike fitted with a single VP8* lectin domain superimposed on the extra density detected at the base of the stalk. First (Leu⁶⁵, cyan) and last (Leu²²⁴, yellow) residues for the fitted domain are represented as spheres (arrowheads).</p

Infectivity assay of <i>in vitro</i> trypsin-treated TLP.

<p>(A, B) Coomassie blue-stained SDS-PAGE gels of purified SA11 (A) and OSU (B) TLP grown in the absence (NTR) or presence (TR) of trypsin. Samples were mock-incubated (-Trp) or incubated <i>in vitro</i> with 100 BAEE units/ml of trypsin (+Trp) (30 min, 37°C). The positions of the structural proteins (VP) are indicated. The unprocessed spike protein VP4 and its proteolytic products VP8* and VP5* are highlighted in grey. (C, D) Determination of specific infectivity of SA11 (C) and OSU (D) TLP by fluorescent focus assay in the absence of trypsin. (E, F) Determination of infectivity of SA11 (E) and OSU (F) TLP by plaque-forming assay in the presence of trypsin. Data are shown as mean ± SD. FFU, focus-forming units. PFU, plaque-forming units. * p<0.02, ** p<0.005.</p