Human noroviruses are genetically and antigenically distinct, but broad-range monoclonal antibodies capable of detecting multiple norovirus genogroups and genotypes have been described (21
). One such antibody, 5B18, is currently in use in a commercial norovirus ELISA detection kit (Denka Seiken, Japan) and was found to bind to numerous GII genotypes but not GI noroviruses (unpublished data). To describe the precise binding location of 5B18, we determined the X-ray crystal structure of the GII.10 P domain-Fab complex. We also determined the cryo-EM structure of the GII.10 VLPs in an attempt to understand the 5B18 Fab binding interaction in the context of the entire virus particle.
The 5B18 Fab binds to a face of the GII.10 P1 subdomain close to the S domain and not openly exposed at the VLP surface. Six amino acid residues on the P1 subdomain make main chain and side chain interactions with the Fab. Four of these residues are highly conserved among numerous GII norovirus genotypes (). Variation at these residues appears to be tolerated, as the 5B18 antibody detects both GII.4 VLPs, which had Thr433 (instead of Val433), and GII.12 VLPs, which had Thr433 and Ser534 (instead of Val433 and Thr534, respectively). Surprisingly, the 5B18 Fab contact residues are almost identical to those of another broad-range monoclonal antibody, MAb14-1 () (59
). Furthermore, the epitopes of two other broad-range monoclonal antibodies, NV3901 and NV3912, are in this general region (50
). The MAb14-1 antibody was shown to bind VLPs from many GII genotypes and several GI genotypes, including a GI.1 genotype, whereas the NV3901 and NV3912 antibodies were found to only bind GI genotypes. Interestingly, the 5B18, MAb14-1, and NV3901/NV3912 antibodies were raised in different mice immunized with different VLPs and their binding sites were all in close proximity on the P1 subdomain (50
). Although the precise structural binding details of MAb14-1, NV3901, and NV3912 antibodies have not been described, it suggests that the P1 subdomain was an important antigenic site for GI and GII noroviruses. Moreover, the P1 subdomain likely contained GI and GII cross-reactive epitopes. Superpositioning of published X-ray crystal structures of norovirus P domain (GI.1, GII.4, GII.9, GII.12, and GV.1) onto the GII.10 P domain-Fab complex structure showed that three of six amino acids involved in the 5B18 Fab binding were highly conserved for three norovirus genogroups and that the conformation of their side chains closely resembles those of GII.10 (see Table S2 and Fig. S4 in the supplemental material). Taken together, the results indicate that the 5B18 binding epitope represents an important site for antibody recognition ().
Initially, the X-ray crystal structure of the GI.1 VLP (53
) was used for fitting the GII.10 P domain-Fab complex and to describe the binding interaction in the context of the entire particle. The P domains of GI.1 and GII.10 matched well (root mean square deviation [RMSD], 1.3 Å), but the 5B18 Fab clashed with the GI.1 S domain (C). Indeed, the P domains in GI.1 VLPs rest on the S domains, and this necessarily placed most of the Fab structure into a position that overlapped the S domain (C). In an attempt to understand the 5B18 antibody interaction in the context of a GII VLP, the cryo-EM structure of the GII.10 VLP was determined to an ~10-Å resolution. Recent cryo-EM studies have shown that GI.1 and GV.1 norovirus capsid structures are strikingly different (30
), whereas another study indicated that GI.1 and GII.4 (Grimsby virus) capsids are highly similar (12
). The cryo-EM structure of the GII.10 VLPs showed several structural similarities to the GV.1 virion, including a raised P domain, P1-P1 subdomain contacts, and an extended hinge region (see Fig. S6 in the supplemental material). In addition, the GII.10 and GV.1 P domain dimers were rotated ~40° clockwise compared to the orientation of the GI.1 P domain dimer (data not shown). Fitting of the X-ray crystal structure of the GII.10 P domain-Fab complex into the GII.10 VLP structure showed that the P domain could be positioned unambiguously into the P domain density of the EM map; however, this placement resulted in significant overlap between Fab and neighboring P and S domains in the virus particle (). One potential explanation for this result is that the VLPs flexibly expose the P domain to the 5B18 antibody by rotating the P domains out of the conformation observed in the cryo-EM reconstruction and breaking the P1-P1 domain contacts seen in the VLP. This may be possible since the S domain-P1 subdomain connection in GII noroviruses is particularly long and flexible.
The structural differences between the GI.1 and GII.10 norovirus VLPs do not appear to be a consequence of sequence diversity, since the GI.1 and GII.4 VLP structures are similar and distinct from the GV.1 virion and GII.10 VLP structures. Moreover, the VLP preparation and cryo-EM techniques appear to be essentially the same (54
). Two factors that may have affected the particle structures were the insect cell type and the pH of the VLPs. The GI.1 VLPs were expressed in Spodoptera frugiperda
(Sf9) cells, purified by CsCl ultracentrifugation, and then resuspended in water (pH not described in text) (53
), and the GII.10 VLPs were expressed in Trichopulsia ni
(H5) cells, purified by CsCl ultracentrifugation, and then resuspended in PBS (pH 7.3). We note parenthetically that the cryo-EM structures of hepatitis E virus VLPs expressed in Sf9 and H5 cells are similar, although the processing of the viral protein appeared different (38
). Our EM results showed that GII.10 VLPs were intact particles at pH 5.3, 6.3, and 7.3, while another study found that the diameter of norovirus VLPs remained virtually unchanged at pH 3 to 7 but appeared smaller at pH 8 (2
). This suggests that the insect cell line and water/PBS (neutral pH) did not affect the overall structure of the VLPs. However, another study has shown that a pH change from 7.6 to 5.0 could cause large structural changes in Nudaurelia capensis
ω virus VLPs (43
). It is possible that these varied conformations do not represent different, stable norovirus structures but are rather all part of a wide spectrum of conformations afforded by the flexible tether between the P and S domains. From previous studies (30
), it is clear that this “floating P domain” conformation is independent of whether the sample is a VLP or infectious virion. Since this extended conformation is now observed in rabbit hemorrhagic disease virus (also a calicivirus, genus Lagovirus
), it also cannot be dependent upon calicivirus genera. It is possible that the energy differences between the conformations represented by these viruses is relatively small and that subtle protein-protein interaction differences favor one conformation under particular conditions. It would be particularly interesting to examine the conformations of these viruses under a broad range of conditions that mimic the expected environments during the viral life cycle. Such changes in virion structure have been observed with numerous other viruses (3
). In the case of GV.1 norovirus, where there is an animal model (69
) and infectious clone (66
), it would also be important to determine what role this flexible tether region has in the replication of the virions and pathology of the disease.
It is important to note that the observed ELISA binding of 5B18 IgG may not occur with intact VLPs. It is possible that denatured or partially broken VLPs or the presence of contaminating GII.10 VP1 was responsible for the binding observed in the ELISA (19
). However, it is known that high pH (8.3 or above), partially breaks or denatures norovirus VLPs (2
). Despite this pH dependence, the titer remained almost identical, especially in the comparison between pHs 7.3 to 9.3 (), suggesting that only intact or structurally stable virions are being detected. Moreover, the 5B18 antibody could detect GII.10 VLPs that were bound to the plates via histo-blood group antigens, which required a dimeric interaction (22
; also unpublished data). Finally three other antibodies, MAb14-1 and NV3901/NV3912, which bound in close proximity to the 5B18 were all shown to detect VLPs (50
). These data therefore favor a model in which apparently intact norovirus capsids can indeed bind the 5B18 antibody (and other antibodies) despite significant steric clashes with the VLP structure.
Viruses often use remarkable conformational changes in their envelope or capsid structures to protect their genetic material by waiting for the proper cellular trigger to release their genome into the host cell. For example, the hemagglutinin spike in influenza undergoes a drastic pH-dependent conformational change in the endosome that initiates membrane fusion (8
). Similarly large pH-dependent changes have been observed with the enveloped flaviviruses (31
) and alphaviruses (36
). Such changes due to environmental cues can expose or hide antigenic sites (e.g., see references 41
, and 47
). Viruses can also receive cues via interactions with cellular receptors, as is the case with human rhinovirus (25
). Viruses also undergo small, dynamic structural changes, “breathing” (6
), that are probably a prelude to the far larger conformational changes that occur during uncoating. These dynamic motions can transiently expose more-conserved antigenic sites that can be leveraged in designing vaccines (29
). However, the fact that these norovirus antibodies are recognizing deeply occluded portions of the P1 domain in apparently intact virions represents a different kind of viral dynamic: for this recognition to occur, the P domains must be capable of extremely large conformational changes without any obvious environmental cue. Such recognition would probably involve just one or a few P domains of a VLP being recognized by antibody 5B18; indeed, images of VLPs after incubation with an excess of antibody 5B18 for 1 h at 37°C (the same incubation used in the ELISA) shows them to be intact, with bound IgG difficult to detect (see Fig. S7 in the supplemental material).
Other antibodies have recently been described that bind to occluded sites on virions. With West Nile virus, the fusion loop-specific antibody E53 recognizes an epitope that should be inaccessible on mature virions. However, this antibody could neutralize mature West Nile virus in a time- and temperature-dependent manner, indicating a role of virus “breathing” or conformational dynamics in antibody recognition (17
). With HIV-1, broadly neutralizing antibodies against the membrane-proximal external region of the virus do not appear to recognize the native viral spike (11
), again implicating conformational rearrangements to permit antibody recognition. These studies, along with the present study on norovirus recognition by 5B18, suggest substantial flexibility in certain virus particles as being important biologically for antibody-mediated recognition.
In summary, we have shown that a broadly reactive monoclonal antibody binds to an occluded site on the GII.10 P1 subdomain. The binding site was in close proximity to other monoclonal antibody binding sites, suggesting that the site contained an immunodominant region. We also found that the GII.10 VLP structure was more closely related to a GV.1 virion structure than to a GI.1 VLP structure and has marked flexibility in the P domains. These studies suggest that the P domain of noroviruses is capable of adopting variable conformations with respect to the S domain. Despite the vaunted diversity of noroviruses, especially on the exposed outer surface of the virion, one mechanism to achieve near pan-recognition by antibody may be to target a highly conserved domain interface that is dynamically exposed to the environment.