Figure 1.
Atomic model of the VP4 spike.
Ribbon representation (top) of the atomic structure of the VP4 spike (PDB entry 3IYU) color-coded to represent the spike foot (green), stalk (red), body (red) and head (purple). As indicated (VP4-A, -B and -C), each of the subunits is highlighted in color, while the remaining molecules are shown in grey. The amino- and carboxy-terminal regions of the three molecules, located contacting one another beneath the VP7 layer [6], contribute equally to the trimeric foot of the structure. The asymmetric central stalk is formed mainly by the VP5* β-barrel domain of VP4-C, which lies almost parallel to the particle surface. The distal part of the spike, constructed by equal contributions of the A and B molecules, contains the body (built by the VP5* β-barrel domains) and the globular heads (formed by VP8* lectin domains). The unusual structure of VP4-C appears to arise from asymmetries on the VP7 trimers that surround the base of the spike, which allows for distinct interactions with the base of the VP5*-C β-barrel. The globular VP8* lectin domain of the VP4-C molecule is not accounted for in the resolved structure, and is presumed to be released during proteolysis. Diagram of the organization of the VP4 chain (bottom), color-coded as in top. The VP4 proteolytic products (VP5* and VP8*) and domains are labeled. Residues delimiting domains and trypsin cleavage sites are indicated.
Figure 2.
Analysis of SA11 NTR- and TR-TLP by SDS-PAGE and cryo-electron microscopy.
(A, B) Coomassie blue-stained SDS-PAGE gels of purified SA11 TLP grown in the absence (A) or presence (B) of trypsin. Positions of rotavirus structural proteins (VP) are indicated. Unprocessed spike protein VP4 and its proteolytic products VP8* and VP5* are highlighted in grey. (C, D) Cryo-electron micrographs of NTR (C) and TR (D) particles. Arrowheads indicate examples of clearly defined spikes projecting from the virion surface. The bar represents 50 nm.
Figure 3.
Single particle three-dimensional structures of SA11 NTR- and TR-TLP.
(A, D) Surface-shaded representation of the outer surfaces of NTR (A) and TR (D) particles, viewed along an icosahedral 2-fold axis. The surfaces are radially color-coded to represent VP4 or VP8*/VP5* spikes (red), VP7 (yellow), and VP6 (blue). The density is contoured at 1σ above the mean. The bar represents 100 Å. (B, E) Transverse sections, 2.8 Å thick, taken from the maps of NTR- (B) and TR-TLP (E), parallel but displaced 34 Å from the central section, viewed along a 2-fold axis (darker, denser). Arrows indicate spikes in the surface-shaded representations in A and D and their corresponding densities in B and E. For the NTR map, the relative density of the spike contained ∼50% of the shell density; for the TR map, relative density was ∼55%. (C, F) Close up view of the NTR (C) and TR (F) spike represented as in A and D.
Figure 4.
Infectivity assay of in vitro trypsin-treated TLP.
(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 in vitro 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.
Figure 5.
3DR of SA11 TLP from cryo-electron tomography.
(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.
Figure 6.
Tomogram averaging and classification of NTR and TR spikes.
(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).
Figure 7.
Model of the conformational states of the rotavirus spike in NTR- and TR-TLP.
NTR spikes have a flexible VP4-C lectin domain at the base of the stalk (top, left). NTR Class 1 represents the average of more distal positions (black asterisks) and class 2 is generated by the average of more central positions (white asterisk) of the flexible lectin domain. The proteolytic processing of spike components releases the VP4-C lectin domain (if an additional cleavage at Lys29 is produced) or increases its flexibility in TR class 1 spikes (bottom, left). The fraction of NTR (top, right) and TR (bottom, left) subvolumes in which no spike density is detected could correspond to a mixture of positions without spikes (or with damaged spikes) and with highly flexible spikes.