Human enterovirus 71 uncoating captured at atomic resolution

Abstract

Human enterovirus 71 (EV71) is the major causative agent of severe hand-foot-and-mouth diseases (HFMD) in young children, and structural characterization of EV71 during its life cycle can aid in the development of therapeutics against HFMD. Here, we present the atomic structures of the full virion and an uncoating intermediate of a clinical EV71 C4 strain to illustrate the structural changes in the full virion that lead to the formation of the uncoating intermediate prepared for RNA release. Although the VP1 N-terminal regions observed to penetrate through the junction channel at the quasi-3-fold axis in the uncoating intermediate of coxsackievirus A16 were not observed in the EV71 uncoating intermediate, drastic conformational changes occur in this region, as has been observed in all capsid proteins. Additionally, the RNA genome interacts with the N-terminal extensions of VP1 and residues 32 to 36 of VP3, both of which are situated at the bottom of the junction. These observations highlight the importance of the junction for genome release. Furthermore, EV71 uncoating is associated with apparent rearrangements and expansion around the 2- and 5-fold axes without obvious changes around the 3-fold axes. Therefore, these structures enabled the identification of hot spots for capsid rearrangements, which led to the hypothesis that the protomer interface near the junction and the 2-fold axis permits the opening of large channels for the exit of polypeptides and viral RNA, which is an uncoating mechanism that is likely conserved in enteroviruses.

Importance: Human enterovirus 71 (EV71) is the major causative agent of severe hand-foot-and-mouth diseases (HFMD) in young children. EV71 contains an RNA genome protected by an icosahedral capsid shell. Uncoating is essential in EV71 life cycle, which is characterized by conformational changes in the capsid to facilitate RNA release into host cell. Here we present the atomic structures of the full virion and an uncoating intermediate of a clinical C4 strain of EV71. Structural analysis revealed drastic conformational changes associated with uncoating in all the capsid proteins near the junction at the quasi-3-fold axis and protein-RNA interactions at the bottom of the junction in the uncoating intermediate. Significant capsid rearrangements also occur at the icosahedral 2- and 5-fold axes but not at the 3-fold axis. Taking the results together, we hypothesize that the junction and nearby areas are hot spots for capsid breaches for the exit of polypeptides and viral RNA during uncoating.

FIG 1

Purification of EV71 virions and characterization of the uncoating intermediate. (A) Purification of EV71 virions. The protein composition of the virion, which was purified as described in Materials and Methods, was determined using 15% SDS-PAGE analysis. Lane 1, molecular mass marker; lane 2, purified full virions. The calculated molecular masses of VP1, VP2, VP3, and VP4 are 32.6 kDa, 27.7 kDa, 26.4 kDa, and 7.5 kDa, respectively. The purified full virions contain all four structural proteins, i.e., VP1, VP2, VP3, and VP4. (B) Cryo-EM images of samples from the crystallization drops containing crystals of the uncoating intermediate (left) and purified naturally occurring empty particles (right). The internal density corresponded to the RNA genome in the uncoating intermediate, whereas the empty particle was completely devoid of internal density. The presence of the RNA genome inside the particle suggests that these are uncoating intermediates. (C) The uncoating intermediate is more protease sensitive than the full virion. The proteolytic sensitivities of the full virion and the uncoating intermediate were assessed by trypsin digestion as described in Materials and Methods. The digested samples were analyzed by Western blot using a VP1 antibody. Lane 1, hanging drops containing crystals of the uncoating intermediate. Lane 2, hanging drops containing crystals of the uncoating intermediate digested with trypsin for 1 h at 37°C. The truncated VP1 is indicated with an arrow. Lane 3, hanging drops containing crystals of the full virion. Lane 4, hanging drops containing crystals of the full virion digested with trypsin for 1 h at 37°C. VP1 in the full virion is resistant to trypsin digestion. (D) The VP4 in the uncoating intermediate is protected from α-chymotrypsin digestion. The uncoating intermediate was treated by α-chymotrypsin digestion as described in Materials and Methods. The digested samples were analyzed by Western blotting using a VP4 antibody, with VP2 as a loading control. Lane 1, hanging drops containing crystals of the uncoating intermediate were incubated at 25°C. Lane 2, hanging drops containing crystals of the uncoating intermediate were digested with α-chymotrypsin for 1 h at 25°C. The VP4 in the uncoating intermediate was resistant to protease digestion.

FIG 2

Overall structures of EV71 full virions and the uncoating intermediate. (Left) Radius-colored surface representation of the EV71 full virion viewed along the 2-fold axis. The surface is colored from blue to red according to the distance from the particle center (blue represents the closest). (Middle) Ribbon representations of the full virion (colored red) and the uncoating intermediate (colored blue). Only half of each capsid shell is represented, as an ∼80-Å slab, to illustrate the expansion of the uncoating intermediate with respect to the full virion. The position of the 2-fold axis of the particle is indicated. (Right) Radius-colored surface representation of the uncoating intermediate viewed along the 2-fold axis. The surface is colored as in the left panel.

FIG 3

Structural changes in the protomer and individual capsid proteins during uncoating. (A) Structural comparison of the protomer in the full virion and the uncoating intermediate. A surface representation of the EV71 full virion viewed along the 2-fold axis is shown, with VP1, VP2, and VP3 colored magenta, yellow, and cyan, respectively. Cartoon representations of the protomer with VP1, VP2, and VP3 in the uncoating intermediate are colored magenta, yellow, and cyan, respectively, whereas their counterparts in the full virion are colored gray. The positions of the icosahedral symmetry elements are indicated. (B) Superposition of VP1. Residues 1 to 297 are modeled in the full virion and colored red, whereas residues 72 to 296 are modeled in the uncoating intermediate and colored blue. (C) Superposition of VP2. The proteins are colored as in panel B. Residues 9 to 254 and residues 16 to 47 and 54 to 250 are modeled in the full virion and the uncoating intermediate, respectively. (D) Superposition of VP3. The proteins are colored as in panel B. Residues 1 to 242 and residues 1 to 175 and 189 to 236 are modeled in the full virion and the uncoating intermediate, respectively. (E) Structure of VP4, colored as in panel B. Residues 12 to 69 of VP4 were modeled from well-defined electron density in the full virion. (F) Comparison of the VP1 pockets in the full virion (red, with the pocket factor shown in green) and the uncoating intermediate (blue). Cys225 near the pocket region is shown in yellow.

FIG 4

Structure-based sequence alignments of the capsid proteins VP1, VP2, and VP3 from different EV71 strains. Capsid protein sequences used for the alignment include those of the clinical EV71 C4 strain used in this study (4N53) and of two other EV71 strains (denoted 3VBS and 4GMP) whose capsid proteins have been structurally determined. The secondary structure elements for the EV71 full virion and the uncoating intermediate are shown at the top and bottom of the sequence alignment, respectively. The residue numbers are those in the EV71 full virion. Conserved residues are shown in white with a red background. Helices and strands are labeled according to standard picornavirus nomenclature and are represented by coils and arrows, respectively. The blue triangles indicate the residues that are variable between 4N53 and 3VBS. The black triangles indicate the residues that are variable between 4N53 and 4GMP. The VP1 GH loop, VP3 GH loop, and residues 48 to 53 of VP2 are boxed with blue rectangles and correspond to the disordered regions in the structure of the uncoating intermediate. This figure was produced using ESPript (50).

FIG 5

Structural changes at the protomer interface. (A) Top view of the 5-fold axis channel in the virion. The surfaces are colored from blue to red according to their distance from the particle center (blue represents the closest). Four mutations between the clinical C4 strain and 3VBS in the capsid proteins are exposed on the viral surface as indicated. The triangle is drawn around the quasi-3-fold axis (surrounded by VP1 and VP2 from one protomer and VP3 from a neighboring protomer). The variable residues between the clinical C4 strain and 3VBS that are exposed on the capsid surface are colored blue (Glu98 and Cys225 of VP1), cyan (Ser144 of VP2), and black (Ser93 of VP3). (B) Hydrogen bonding network around the 5-fold axis. The amino group in the side chain of Lys182 (colored black) interacts with Asp185 (colored red) of a neighboring VP1 through hydrogen bonds. This interaction network around the 5-fold axis channel is likely conserved among human enterovirus species A and D. (C) Structural changes at the junction during uncoating. The structure of the virion is shown on the left, whereas that of the uncoating intermediate is shown on the right. VP1, VP2, and VP3 are colored magenta, yellow, and cyan, respectively. The GH loop of VP1 is colored green, residues 48 to 53 of VP2 are colored blue, and the GH loop of VP3 is colored orange. During uncoating, conformational changes occur in the GH loop of VP1, whereas residues 48 to 53 in VP2 and the GH loop in VP3 become disordered.

FIG 6

Structural changes at the 2-fold axis channel. VP1, VP2, and VP3 are colored magenta, yellow, and cyan, respectively. The 2-fold channel in the full virion is shown on the left, whereas the 2-fold channel in the uncoating intermediate is shown on the right. The 2-fold axis channel expands during uncoating.

FIG 7

Capsid rearrangements during uncoating. (A) The absolute distance shift for every Cα atom (×10) during EV71 uncoating was mapped onto the full virion structure. The surface is colored from red to blue according to the relative distance shift (blue represents the lowest shift). (B) The inner surface of the full virion is colored as in panel A, showing the internal rearrangements during uncoating. (C) The absolute distance shift for every Cα atom (×10) during HRV2 uncoating was mapped onto the full virion structure. The surface is colored from red to blue according to the relative distance shift (blue represents the lowest shift).

FIG 8

Capsid-RNA interactions in the EV71 uncoating intermediate. (A) EV71 A-particle, showing density extending from the capsid shell that interacts with the viral RNA genome. The fitted uncoating intermediate crystal structure is depicted in a ribbon representation with VP1, VP2, and VP3 colored magenta, yellow, and cyan, respectively. The A-particle cryo-EM density is depicted as a gray mesh. Residue 72 of VP1 is colored in green, and residues 32 to 36 of VP3 are colored in orange, both of which interact with the inner RNA density. (B) The fitted uncoating intermediate crystal structure is depicted in a ribbon representation with VP1, VP2, and VP3 colored magenta, yellow, and cyan, respectively. The A-particle cryo-EM density is depicted as a gray mesh. Residue 16 of VP2 is colored red and does not interact with the inner RNA density.

FIG 9

Structural comparison between the EV71 uncoating intermediate and the CVA16 A-like particle. (A) Structural comparison of the protomer in the EV71 uncoating intermediate with that in the CVA16 135S-like particle. VP1, VP2, and VP3 in EV71 are colored magenta, yellow, and cyan, respectively, and those in CVA16 are colored gray. Thr175 and Tyr189 in VP3 mark the beginning and end of the disordered regions in the determined uncoating intermediate structure. Ala47 and Thr54 in VP2 mark the beginning and end of the disordered region in the determined uncoating intermediate structure. (B) Superposition of VP1. Residues 72 to 296 of VP1 in EV71 (colored blue) and residues 62 to 210 and 219 to 297 in CVA16 (colored orange) are modeled. The ordered region in VP1 begins at Ser72 (colored blue) of the determined uncoating intermediate structure. The ordered region in VP1 of the CVA16 135S-like particle begins at Asn62 (colored orange). (C) Superposition of VP2. The proteins are colored as in panel B. Residues 16 to 47 and 54 to 250 in EV71 and residues 6 to 136 and 142 to 249 in CVA16 are modeled. Ala47 and Thr54 indicate the beginning and end of the disordered regions in EV71. Ala136 and Glu142 indicate the beginning and end of the disordered regions in CVA16. (D) Superposition of VP3. The proteins are colored as in panel B. Residues 1 to 175 and 189 to 236 in EV71 and residues 1 to 179 and 185 to 236 in CVA16 are modeled. Tyr185 and Ala179 (orange) indicate the beginning and end of the disordered regions in CVA16. (E) Side views showing different positions of the VP1 N terminus in the EV71 uncoating intermediate (left) and the CVA16 135S-like particle (right). EV71 is colored blue, whereas CVA16 is colored red. The gray surface representation shows the surface of the capsid pentamer from the side view. A stretch of polypeptide was observed to traverse the capsid in CVA16, whereas this region is disordered in EV71. (F) Structures at the junction in CVA16. VP1, VP2 and, VP3 are colored magenta, yellow, and cyan, respectively. The GH loop in VP1 is colored green, and residues 142 to 146 in VP2 are colored blue. Portions of the GH loop of VP1 and the EF loop of VP2 (residues 137 to 141), both facing the junction channel, are disordered.