Differential accessibility of a rotavirus VP6 epitope in trimers comprising type I, II, or III channels as revealed by binding of a human rotavirus VP6-specific antibody

Abstract

Previous human antibody studies have shown that the human VH1-46 antibody variable gene segment encodes much of the naturally occurring human B cell response to rotavirus and is directed to virus protein 6 (VP6). It is currently unknown why some of the VH1-46-encoded human VP6 monoclonal antibodies inhibit viral transcription while others do not. In part, there are affinity differences between antibodies that likely affect inhibitory activity, but we also hypothesize that there are differing modes of binding to VP6 that affect the ability to block the transcriptional pore on double-layered particles. Here, we used a hybrid method approach for antibody epitope mapping, including single-particle cryo-electron microscopy (cryo-EM) and enhanced amide hydrogen-deuterium exchange mass spectrometry (DXMS) to determine the location and mode of binding of a VH1-46-encoded antibody, RV6-25. The structure of the RV6-25 antibody-double-layered particle (DLP) complex indicated a very complex binding pattern that revealed subtle differences in accessibility of the VP6 epitope depending on its position in the type I, II, or III channels. These subtle variations in the presentation or accessibility of the RV VP6 capsid layer led to position-specific differences in occupancy for binding of the RV6-25 antibody. The studies also showed that the location of binding of the noninhibitory antibody RV6-25 on the apical surface of RV VP6 head domain does not obstruct the transcription pore upon antibody binding, in contrast to binding of an inhibitory antibody, RV6-26, deeper in the transcriptional pore.

FIG 1

Structure of RV6-25 Fab bound to RV-DLP. Density map of the full virion structure of RV6-25–DLP complex reconstructed to 21.5-Å resolution, based on an FSC 0.5 criterion, viewed along the icosahedral 5-fold axis. The VP2, VP6, or RV6-25 Fab density is represented by cyan, gray, or salmon, respectively. The yellow circle indicates the density that represents the VP6-binding components of the third Fab. The enlarged gray box indicates a type I channel (in mesh) showing Fabs fitted into density of the third Fab. The blue ribbons represent the heavy chains, and the red ribbons represent the light chains.

FIG 2

Binding of RV6-25 Fab to VP6 trimers at the type I, II, or III channel. (A) Surface representation of RV6-25–DLP complex particle, highlighting the RV6-25 Fabs bound to the VP6 trimers (green) around the type I channel in the context of the whole virion particle (gray). (B) Surface representation of the type I pore showing two visible Fab densities bound to each VP6 trimer around the channel. At this threshold, density for the third Fab bound to each trimer is not visible. One of the Fab densities is depicted in a transparent mesh with a fitted Fab. The trimers around the type I channel are designated P trimers. (C) Surface representation of RV6-25–DLP complex particle, highlighting the RV6-25 Fabs bound to the VP6 trimers around the type II channel in the context of the whole virion particle (gray). The green color represents two of the 5-fold VP6 trimers (P) with bound Fabs, pink represents the trimers directly adjacent to the 5-fold VP6 trimers (P′) with bound Fabs, yellow represents one of the VP6 trimers at the 2-fold axis (D), and cyan represents one of the trimers directly adjacent to the VP6 trimer at the 3-fold axis (T'). (D) Surface representation of the type II pore showing the two visible Fab densities bound to the two P VP6 trimers in the adjacent type I pore and single Fab densities each bound to P′ VP6 trimers, directly adjacent to the P trimers. One of the Fab densities is depicted in a transparent mesh with a fitted Fab. Fab densities are not visible on D and T' trimers. The trimers around the type II channel are designated P, P′, D, or T'. (E) Surface representation of RV6-25–DLP complex particle, highlighting the RV6-25 Fabs bound to the VP6 trimers around the type III channel in the context of the whole virion particle (gray). The pink color represents the P′ trimer with one bound Fab density, yellow represents the two trimers at the 2-fold axis (D), light blue represents the trimer at the 3-fold axis (T), and cyan represents two of the trimers directly adjacent to the 3-fold trimer (T'). (F) Surface representation of the type III pore showing one visible Fab density bound to the P′ VP6 trimer that is also part of the adjacent type II channel and three Fab densities bound to the T trimer. One of the Fab densities bound to the T trimer is depicted in a transparent mesh with a fitted Fab. Fab densities are not visible on D or T' trimers. The trimers around the type III channel are designated P′, D, T, or T'. (G and H) Surface representation of the Fab-DLP complex highlighting all three channels (types I, II, and III) in the context of the whole particle (gray) (G) and showing the trimer designations (H). In all images, the Fab heavy and light chains are shown in red and blue, respectively. The black line, triangle, and star represent the icosahedral 2-, 3-, or 5-fold symmetry, respectively.

FIG 3

Demonstration that VP6f is a trimer in solution. Purified VP6f was run on an analytical Superdex 200 10/300 GL column (GE Healthcare) at 0.5 ml/min, in 10 mM Tris, 150 mM NaCl, pH 7.5, and compared to Bio-Rad gel filtration standards. The figure shows an overlay of these standards and VP6f. VP6f eluted between the 158,000- and 44,000-Da standards. A least-squares regression line based on these standards (excluding the 1.35-kDa standard) predicted a molecular mass of 70,254 Da for VP6f. The expected molecular mass of a VP6f trimer is 68,316 Da. These data indicate that VP6f is a trimer under dilute buffer conditions.

FIG 4

Determination of VP6 epitope for RV6-25 by deuterium exchange mass spectroscopy. (A and B) Ribbon map showing percent deuterated (% D) of VP6 alone (A) or VP6 bound to RV6-25 Fab (B). The top row shows the residue position number, the second row shows the residue, and the rest of the rows show protein dynamic features at different on-exchange time points (10, 100, or 1,000 s). As indicated in the colored bar, cold colors suggest relatively stable regions and warm colors suggest relatively flexible regions. All prolines are shown in white, because prolines do not have amide hydrogens. Residues uncovered by surface deuteration are also shown in white. (C) Difference map showing the influence of RV6-25 Fab binding to VP6 indicated by changes in % D. Blue suggests the regions that exchange slower upon Fab binding; red suggests the regions that exchange faster upon Fab binding. (D) Side view of the predicted epitope regions of RV6-25 Fab on the head domain of the VP6 structure (PDB: 1QHD). The different shades of gray represent the three protomers that make up the VP6 trimer, and the different shades of blue represent the predicted epitope regions mapped on each protomer. (E) The side view of the VP6 trimer highlighting, in red, the loops that are most likely involved in binding to RV6-25. (F) The top view of the VP6 trimer with all the predicted epitope regions visible on the structure. (G) The top view of the VP6 trimer highlighting, in red, the loops that are most likely involved in binding to RV6-25.