Structural determinants of rotavirus proteolytic activation

Abstract

The infectivity of rotavirus (RV), the leading cause of childhood diarrhea, hinges on the activation of viral particles through the proteolysis of the spike protein by trypsin-like proteases in the host intestinal lumen. In order to determine the structural basis of trypsin activation, we have used cryogenic electron microscopy (cryo-EM) and advanced image processing methods to compare uncleaved and cleaved RV particles. We find that the conformation of the non-proteolyzed spike is constrained by the position of loops that surround its structure, linking the lectin domains of the spike head to its body. The proteolysis of these loops removes this structural constraint, thereby enabling the spike to undergo the necessary conformational changes required for cell membrane penetration. Thus, these loops function as regulatory elements to ensure that the spike protein is activated precisely when and where it is needed to facilitate a successful infection.

Fig 1. Analysis of the atomic structures of SA11 NTR- and TR-TLP by cryo-EM.

(A, D) Cryo-EM 3DR of the NTR- (A) and TR-TLP (D) particles viewed along a 2-fold axis. The bar represents 100 Å. The surfaces are represented radially by colour coding: VP4 or VP8*/VP5* spikes (red), VP7 (yellow), VP6 (blue) and VP2 (green). The density is contoured to 2σ above the mean. (B, E) Cross-sections of the NTR- (B) and TR-TLP (E) maps, parallel to the central section, viewed along a 2-fold axis. (C, F) Close up view of the NTR (C) and TR (F) spikes indicated by a dashed square in B and E, respectively. The lectin domain of subunit VP4-C in the NTR spike is indicated by an arrowhead in C. (G, H) Atomic structure of the asymmetric subunit of NTR-TLP (G) and TR-TLP (H). VP2 chains are represented in dark green, VP6 in blue, and VP7 in yellow. The A, B and C chains of the VP4 or VP8*/VP5* spikes and their domains are represented as: foot (light green for the A chain, green for the B chain and dark green for C), body (red (A, B), orange (C)), head or lectin domains (purple (A, B), pink (C)). The binding loops between the different domains are represented in grey except for the α3-β13 loop, represented in cyan.

Fig 2. Atomic structure of the RV spike.

(A) Atomic structure of the NTR (upper panel) and TR (lower panel) spike. Each domain is named and represented following the indicated colour pattern: foot (green), stem (orange), body (red), head (violet) and VP4-C lectin domain (pink, NTR spike). (B) Each of the VP4 subunits (A, B, C) of NTR-TLP (upper panel), and TR-TLP (lower panel) are shown highlighted and represented following the same colour pattern. In both panels, the binding loops between the different domains are represented in grey except for the α3-β13 loop, represented in cyan.

Fig 3. Analysis of the atomic structure of different domains of the NTR and TR spikes.

A close-up view from the dashed boxes in the left panels of the atomic structures that have been fitted into their density maps. Within each panel (A and B) the upper row refers to the NTR spike and the lower row to the TR spike. (A) The stalk, lectin domain (VP4-C chain) and foot domains of the spikes are represented from different angles. Different residues are indicated as spheres: K29 (red), T73 (green), N221 (blue), K258 (brown), D253 (purple), T259 (yellow). (B) Side views of the body of the spike and the trypsinized (TR-VP4) or non-trypsinized (NTR-VP4) loops. Residues R231, R241, R247 are indicated in pink spheres and adjacent residues (N232, D242, A248) in blue. The α2-β1 and α3-β13 loops are shown. The molecular swapping observed in the body domain are indicated with an asterisk (*).

Fig 4. Analysis of the structural transitions of the loops and different domains of the RV spike during cell entry.

Rearrangements of the spike proteins (VP4, VP8* and VP5*) during the transition from their inactive conformation, NTR (A), through their activated form, TR (B), and intermediate (C) to their inverted conformation. (D). The domains not observed in the atomic structures, in each case, are represented schematically (panel C: lectin domains; panel D: lectin, α-amino and foot domains). The different protein domains are coloured separately to illustrate their conformational change: α-amino domains: orange, yellow, gold; lectin domains: magenta, purple and pink; α3-β14 loops: blue; body domains: red, orange and salmon; α-coiled helix and foot domains: light green, green and dark green. Loops between the α-amino and lectin domains in panels C and D are represented by dashed lines. Arrows show different residues delimiting the different coloured domains in each panel. The panels corresponding to the body domain are shown with some transparency level and highlighting the hydrophobic loops and the β14 chain in a darker tone.

Fig 5. Structural transitions of the rotavirus spike.

Schematic representation of the proposed conformational changes of the RV spike during its morphogenesis, maturation and activation. Proteins are coloured as indicated: VP6 in blue, VP7 in yellow, VP4/VP5* foot and β-barrel transparent, VP4/VP8* lectin domain A, B and C in magenta, purple and pink, respectively, and loops in grey. (A) During the late stages of ER morphogenesis, full-length VP4 monomers fold into a pre-mature three-fold symmetric structure in the eDLP. It has been proposed that the transition from this pre-mature conformation to the immature conformation is one of the driving forces for the transition of the eDLP to TLP. (B) In the immature TLP, the three unprocessed VP4 subunits are assembled as an asymmetric trimer. Two VP4 subunits. chains A and B, form the body and head of the spike joined by two internal (gray) and two external loops (light blue). The VP4-C chain folds to form the stalk of the spike to which it contributes with the β-barrel and the lectin domain. (C) Trypsin activation splits VP4 into VP5* and VP8* products in the mature spike. Trypsin proteolysis occurs at the external α3-β14 loop in three residues, R231, R241 and R247, leading to the loss of the segment 232-247 (loss of light blue loops) in the VP4-A and -B chains, and the loss of the lectin domain in the VP4-C chain (pink). (D) The activated spike is able to bind to the host cell through the interaction of VP8* lectin domains with surface glycans (light green) that result in the attachment of the virus to the cell membrane. (E) This interaction precedes the conformational change in which the lectin domains separate from the main body of the spike, exposing the hydrophobic loops of the ß-barrel domains of chains A and B. This conformational change continues with the extension of the ß-barrel domain of subunit C and ends in the intermediate conformation in which the ß-barrel domains of the three subunits adopt a symmetrical trimeric structure with the three hydrophobic loops inserted into the target membrane. F) After this interaction a further conformational transition results in the inverted conformation, in which the loops and the foot domains of the spike are insert into the membrane which is thought to provoke membrane distortions which culminate in its rupture and in the release of the DLP into the cytosol.