Rabbit hemorrhagic disease, first described in China in 1984, causes hemorrhagic necrosis of the liver. Its etiological agent, rabbit hemorrhagic disease virus (RHDV), belongs to the Lagovirus genus in the family Caliciviridae. The detailed molecular structure of any lagovirus capsid has yet to be determined. Here, we report a cryo-electron microscopic (cryoEM) reconstruction of wild-type RHDV at 6.5 Å resolution and the crystal structures of the shell (S) and protruding (P) domains of its major capsid protein, VP60, each at 2.0 Å resolution. From these data we built a complete atomic model of the RHDV capsid. VP60 has a conserved S domain and a specific P2 sub-domain that differs from those found in other caliciviruses. As seen in the shell portion of the RHDV cryoEM map, which was resolved to ∼5.5 Å, the N-terminal arm domain of VP60 folds back onto its cognate S domain. Sequence alignments of VP60 from six groups of RHDV isolates revealed seven regions of high variation that could be mapped onto the surface of the P2 sub-domain and suggested three putative pockets might be responsible for binding to histo-blood group antigens. A flexible loop in one of these regions was shown to interact with rabbit tissue cells and contains an important epitope for anti-RHDV antibody production. Our study provides a reliable, pseudo-atomic model of a Lagovirus and suggests a new candidate for an efficient vaccine that can be used to protect rabbits from RHDV infection.

Rabbit hemorrhagic disease (RHD), first described in China in 1984, causes hemorrhagic necrosis of the liver within three days after infection and with a mortality rate that exceeds 90%. RHD has spread to large parts of the world and threatens the rabbit industry and related ecology. Its etiological agent, rabbit hemorrhagic disease virus (RHDV), belongs to the Lagovirus genus in the family Caliciviridae. Currently, the absence of a high-resolution model of any lagovirus impedes our understanding of its molecular interactions with hosts and successful design of an efficient anti-RHDV vaccine. Here, we use hybrid structural approaches to construct a pseudo-atomic model of RHDV that reveals significant differences in the P2 sub-domain of the major capsid protein compared to that seen in other caliciviruses. We identified seven regions of high sequence variation in this sub-domain that dictate the binding specificities of histo-blood group antigens. In one of these regions, we identified an antigenic peptide that interacts with rabbit tissue cells and elicits a significant immune response in rabbits and, hence, protects them from RHDV infection. Our pseudo-atomic model provides a structural framework for developing new anti-RHDV vaccines and will also help guide use of the RHDV capsid as a vehicle to display human tumor antigens as part of anti-tumor therapy.

Funding: This work was supported by grants to FS from Chinese Ministry of Science and Technology (2011CB910303), Chinese Academy of Sciences (KGCX1-YW-13) and National Natural Science Foundation of China (31021062), grants to HP from National Natural Science Foundation of China (30770439) and from State Key Laboratory of Veterinary Biotechnology (SKLVBF2011), Harbin Veterinary Research Institute, a grant to JL from National Natural Science Foundation of China (30800830) and a grant to KS from the National Institutes of Health (P41-RR005969). KZ (FS group) was supported by a Gatan China Scholarship (2011–2012). All the EM data were collected at the Center for Biological Imaging, Core Facility for Protein Sciences, Chinese Academy of Sciences. Computer time for MDFF was provided on the Blue Waters Early Science System, which is supported by the National Science Foundation (award number OCI 07-25070) and the State of Illinois. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Here, we report a pseudo-atomic model of the RHDV capsid derived through a combination of X-ray crystallography, cryoEM reconstruction, and molecular dynamics flexible-fitting (MDFF) [21] . We find that RHDV VP60 has a P2 sub-domain that differs from other caliciviruses. Furthermore, our new model reveals that certain aspects of the P2 and NTA domain structures that were previously reported [16] need reinterpretation. We also examined the putative HBGA binding sites in RHDV by mapping isolate–related sequence variations onto the P domain structure. Finally, we show that a peptide derived from a putative HBGA binding site can interact with hosts and stimulate the production of virus antibody. The new, high-resolution model of a Lagovirus presented here provides a solid framework for developing an efficacious antigen presenting system. The model yields also new insights regarding the molecular mechanisms of RHDV-host interactions.

It is worth noting that noroviruses infect hosts by recognizing histo-blood group antigens (HBGAs) that are important host susceptibility factors [18] , and RHDV also agglutinates human erythrocytes and attaches to epithelial cells in the upper respiratory and digestive tracts of rabbits by binding to HBGAs [19] . HBGAs have recently been shown to act as attachment factors that facilitate infection and RHDV isolates from six different genetic groups bind specifically to different HBGAs [20] .

RHDV belongs to the genus Lagovirus of the family Caliciviridae, which also includes the genera Norovirus, Nebovirus, Sapovirus and Vesivirus [8] , [9] . Previous structural studies of caliciviruses include three-dimensional (3D) cryo-electron microscopic (cryoEM) reconstructions of virus-like particles (VLPs) of Murine Norovirus (MNV, Norovirus) and Feline calicivirus (FCV, Vesivirus) at 8- and 16-Å resolution, respectively [10] , [11] , and determination of the crystal structures of the Norwalk virus (NV, Norovirus) capsid at 3.4 Å [12] , native FCV virions at 3.6 Å [13] , and native virions of San Miguel sea lion virus (SMSV, Vesivirus) at 3.2 Å [14] . CryoEM reconstructions of the RHDV VLP at 8 Å [15] and the native RHDV virion at 11 Å [7] have been computed and a C α homology model of RHDV was built based on the VLP cryo-reconstruction by using the crystal structure models of SMSV and FCV [16] . However, a more complete atomic model of RHDV is still lacking. Furthermore, the P domain of VP60, which is responsible for antigenicity and binding to host tissue [17] , varies considerably across different Caliciviridae species, and hence this stimulated us to crystallize and obtain a high resolution crystal structure of this domain to provide a model that is more reliable than could be gleaned from any homology modeling approach.

The etiological agent of RHD is rabbit hemorrhagic disease virus (RHDV), which has a single-stranded, positive-sense, polyadenylated RNA genome of ∼7.5 kb [5] . Mature RHDV virions are spherical, non-enveloped particles with a T = 3, icosahedral capsid whose outer diameter varies between 32 and 44 nm and whose structure is defined by characteristic, cup-shaped depressions [6] . The only capsid protein present in RHDV, VP60, is composed of three domains, which include the N-terminal arm (NTA), the shell (S), and the protrusion (P), the latter of which is further divided into P1 and P2 sub-domains [7] .

Rabbit hemorrhagic disease (RHD) is extremely contagious in adult rabbits and is often associated with liver necrosis, hemorrhaging, and high mortality [1] . It was first described in China in 1984 [2] , and within a few years had spread worldwide [3] . RHD outbreaks still occur on almost every continent and cause significant mortality rates, being endemic in Europe, Asia, Africa, and Australia [4] . This disease has a significant impact on the rabbit industry and ecology [4] .

Results/Discussion

CryoEM reconstruction of the RHDV virion Highly purified RHDV virions (Figure 1A) obtained from the livers of infected domestic rabbits were used for crystallization trials and cryoEM studies (Figure 1B). Unfortunately, we were unable to obtain any crystals of RHDV suitable for X-ray diffraction owing to its propensity to degrade with time. From cryoEM micrographs (Figure 1B), consistent with previous observations [7], [22], two distinct classes of particles were observed: intact virions containing whole genomic RNA (high density inside) and “empty” virions containing sub-genomic RNA (low density inside). The presence of these two types of particles was confirmed by image classification (Figure S1A). The cryoEM structure of RHDV that we computed from ∼36,000 images of individual particles (Figure 1C and S1B) was estimated to reach a resolution limit of 6.5/4.8 Å (Figure S1C) based on Fourier shell correlation (FSC) cutoff thresholds of 0.5 and 0.143, respectively [23], [24]. Considerably more detail was resolved in this RHDV cryo-reconstruction compared to that in our previous one at 11 Å [7]. In addition, the resolution achieved in the RHDV inner shell (radii between ∼130 and 150 Å) reached 5.5 Å (FSC 0.5 ; Figure S1C) compared to 7.0 Å (FSC 0.5 ) for structural features at larger radii (between ∼150 and 220 Å). Central cross sections of the reconstructed 3D map taken perpendicular to the icosahedral 3-, 5-, and 2-fold axes show well-resolved densities in the inner shell compared to fuzzier densities at larger radii (Figure S1B), consistent with the protruding capsomers exhibiting high flexibility [7], [15]. All secondary structural elements in the VP60 S domain were clearly resolved and, in some regions, densities corresponding to residue side chains were evident (Figure 1D). Compared to reconstructions of the RHDV VLP at 8 Å [15] and the native virion at 11 Å [7], the present result represents the most detailed view of the RHDV capsid structure and this, along with results from X-ray crystallography, enabled us to build a reliable, pseudo-atomic model. PPT PowerPoint slide

PowerPoint slide PNG larger image

larger image TIFF original image Download: Figure 1. Electron microscopy and 3D image reconstruction of RHDV. (A) Micrograph of purified, negatively stained RHDV (bar = 100 nm). (B) CryoEM micrograph of purified RHDV (bar = 40 nm). The black arrows and stars point to RHDV particles with differing amounts of internal density (also see Figure S1). (C) Reconstructed cryoEM map of the RHDV virion, color-coded by radius. Icosahedral 2-, 3- and 5-fold axes are indicated by black symbols and AB and CC capsomers are identified. On the right, the closest half of the density map has been removed to reveal internal features in the RHDV density map. The contour threshold of the cryoEM map here was set to 3.3σ above the mean. (D) Same as (C) but with the capsomer density removed to show just the RHDV inner shell. Insert: A region of the density map is shown as a transparent grey isosurface into which is fitted the backbone structures of the S domains from three copies of the RHDV major capsid protein, VP60 (A in red, B in yellow and C in blue). Magenta arrows point to a few representative density features that represent residue side chains. https://doi.org/10.1371/journal.ppat.1003132.g001 As shown previously [7], the RHDV capsid has an overall spherical shape, with a maximum outer diameter of 44 nm and an inner chamber with a diameter of 28 nm (Figure 1C). The asymmetric unit of the RHDV capsid consists of three, quasi-equivalent VP60 subunits (A, B and C) arranged with T = 3 icosahedral symmetry. The 180 VP60 subunits that comprise the capsid are organized as 90 dimers, each of which appears as an arch-like capsomer. Thirty C/C capsomers are located at the icosahedral two-fold symmetry axes and the remaining 60 A/B capsomers are located at pseudo (“local”) two-fold axes. Three A/B and three C/C dimers are positioned in alternate fashion around each icosahedral three-fold axis to form pseudo-six-fold arrangements, and five A/B dimers encircle each five-fold axis. Together, these capsomers produce a contiguous shell and 32 cup-shaped, surface depressions, the latter of which are a characteristic feature of the structure of all caliciviruses [25].

Variation of RHDV capsomer outer surface and the putative HBGA binding sites The first step of viral entry in NV and RHDV infections involves recognition of HBGAs [18], [19]. Crystal structures of NV variants V387 and V207, bound with HBGAs, revealed that the binding sites in NV are located at the outer surface of the arch-like P dimers with both P domains contributing to the formation of the binding interfaces [18], [28]. Because the structure of the RHDV P domain bound with HBGAs is currently not available, we selected the crystal structure of NV variant V207 complexed with the non-secretor HBGA Lewis y (Ley) tetrasaccharide as a model (PDB code 3PUN) [18] to compare with our atomic model of RHDV VP60 (Figure 4A). The crystal structure of the NV V207 P dimer was superimposed onto the C/C capsomer of RHDV by aligning to one of the subunits. The relative positions of the two subunits within the dimer differ slightly between the NV V207 and RHDV models. The binding site of the Ley tetrasaccharide in the P dimer of NV V207 corresponds to loop L6 or L2 in the P domain of RHDV VP60 (Figure 4A). A surface representation of the NV P dimer shows that Ley tetrasaccharides bind to the outer portion of the dimeric interface between P domains (Figure 4B). However, this interface is completely different in RHDV (Figure 4C), and therefore, the RHDV capsomer likely utilizes distinct binding sites for HBGAs. PPT PowerPoint slide

PowerPoint slide PNG larger image

larger image TIFF original image Download: Figure 4. Structural comparison of histo-blood group antigens (HBGAs) binding sites in RHDV and NV VP60s. (A) RHDV C/C capsomer (blue and green) is superimposed with the crystal structure of the norovirus strain (VA207, GII.9) [18] (PDB code: 3PUN) P dimer (gold and magenta). A close-up, top-down view of the HBGAs binding sites in VA207 is shown at the right and compared with the RHDV P2 sub-domains. Ley represents the HBGAs Lewis y. (B, C) Surface representations of the dimeric P domains in NV VA207 (B) and RHDV (C) are shown in equivalent top views and colored as in (A, right panel). The portions of RHDV that correspond to the Ley binding sites in NV VA207 are indicated with orange dashed circles in (C). https://doi.org/10.1371/journal.ppat.1003132.g004 Though genetic diversity among RHDV isolates is far lower than that among isolates of other caliciviruses, it has been suggested that all current RHDV isolates could be assigned to one of six genetic groups and the binding specificities of HBGAs for those genetic groups have been the subject of intensive investigation recently [20]. We performed a multi-sequence alignment of VP60 among these six groups and found that seven regions of high variation (V1 to V7) distinguish these groups (Figure 5A). These regions all occur on the P2 sub-domain (Figure 5B and S7). Most significantly, these regions correspond to loops L1 to L7 in the P2 sub-domain (Figure 2 and 5). Thus, in addition to the antigenic variation contributed by these loop regions, at least some and perhaps all of these loops may give rise to different HBGA binding specificities. A relationship between variation regions and receptor binding specificity was also gleaned from the cryoEM structure of FCV bound with its receptor, fJAM-1 (feline junctional adhesion molecule 1) [11]. In addition, we found three cavities on the outer surface of the RHDV capsomer (labeled C1, C2 and C3 in Figure 5B), one or more of which might contribute to HBGA binding. Whether these are true binding sites awaits investigation by mutagenesis experiments. PPT PowerPoint slide

PowerPoint slide PNG larger image

larger image TIFF original image Download: Figure 5. Sequence alignment of VP60s of representative RHDV isolates and location of variation regions on the capsomer surface. (A) Multiple sequence alignments of VP60s among six genetic groups of RHDV isolates according to Nystrom, K. et.al. [20]. The alignment is just shown for the P2 sub-domain region for residues from 301 to 480. The GeneBank accession numbers for those isolates shown are JF438967 (G1), Z24757 (AST/89), Z49271 (RHDV-AST89), FR823355 (G2), AF231353 (NZ), FR823354 (G3), AJ535092 (95-05FR), AJ535094 (G4), Y15424 (Frankfurt), AJ006019 (Rainham), AM085133 (G5), Y15427 (Wriezen), AY926883 (Ireland12), AY928269 (Ireland19), AJ969628 (G6), DQ069280 (whn/China/01/2005), AF453761 (China/Harbin/TP), DQ205345 (JX/CHA/97), AJ303106 (00-ReuFR), AY269825 (NJ/China/1985), DQ530363 (China-Yingling(YL)), and DQ069281 (whn/China/02/2005). The seven variation regions (V1–V7) that distinguish these isolates are highlighted in yellow. Sequence alignments were performed and plotted using Chimera [39]. (B) Locations of variation regions on the RHDV capsomer surface. The degrees of sequence conservation based on the multiple sequence alignments in (A) are mapped onto the surface of the RHDV capsomer (shown in top view on the left). The most highly conserved regions (between 80 and 100% conservation) are shown in colors varying from white to cyan and then green, whereas less conserved regions (from 80 down to 43%) are represented in shades from white to violet. The seven variation regions in each of the two P2 sub-domains are indicated by dashed black ellipses and are labeled in gold in one monomer and blue in the other. The right panel shows a view of the variation sites from a different angle and three putative binding sites for HBGAs are highlighted with dashed red circles and labeled C1, C2 and C3. https://doi.org/10.1371/journal.ppat.1003132.g005

RHDV variation region V1 (loop L1) contributes to host interaction and is a major neutralization site Variation region V1 is a contiguous stretch of mostly hydrophilic residues on loop L1 (a.a. 304–314) (Figure 5A) and is highly flexible in crystals as evidenced by high crystallographic B-factors (Figure S8A and B). Given that L1 is the most exposed loop on the surface of the RHDV capsomer and that it lies juxtaposed to three putative HBGA binding pockets (Figure 5B), this loop is hypothesized to be a primary determinant of RHDV host interaction such that it represents an effective epitope in RHDV. Also, the sequence of this loop constitutes the most diverse region in VP60 in RHDV isolates (Figure S8C) and suggests that this sequence plays a critical role in defining RHDV antigenicity. To test our hypothesis, we designed two peptides, NJ85 (a.a. 300–318) and NJ85Δ (missing 4 residues N 308 ATN 311 of the loop L1 on the most exposed position of the capsomer), derived from the VP60 protein of the RHDV NJ85 isolate strain. Each peptide was synthesized with an N-terminally-labeled fluorescent isothiocyanate (FITC) and then used as a reagent to analyze receptor-binding activity in rabbit hepatocytes, primary splenocytes, and kidney (RK13) cells from healthy male, New Zealand white rabbits. Both peptides bound to the surfaces of the hepatocytes and primary splenocytes, but neither one bound to RK13 cells (Figure 6A). This suggests that the hepatocyte and splenocyte cells express receptors capable of binding both peptides, but that rabbit kidney cells do not, which concurs with previous studies on the specific tissue distributions of RHDV [29], [30]. This binding assay also suggested that at least one of the RHDV-host interaction sites resides at the top surface of the capsomer (i.e., in the loop). However, the four residues (308–311) at the top part of the capsomer, which vary the most across isolates (Figure S8C), unexpectedly did not affect interactions between RHDV and its host. PPT PowerPoint slide

PowerPoint slide PNG larger image

larger image TIFF original image Download: Figure 6. Variation region V1 of RHDV VP60. (A) Binding assay of three types of rabbit tissue cells (hepatocytes, splenocytes, and RK13 cells) for peptides derived from loop L1. (B) Western blot assay against RHDV for the sera containing anti-NJ85-KLH and anti-NJ85Δ-KLH antibodies. The black arrow indicates the band of the major capsid protein VP60 (see also Figure S9). (C) Virus neutralization assays for the sera raised against the NJ85 and NJ85Δ peptides. Rabbits were challenged by an RHDV sample that was mixed with the respective sera and monitored for the number of animals that survived as a function of time. (D) Efficiency of KLH-conjugated peptides (NJ85-KLH and NJ85Δ-KLH) in developing vaccines. Rabbits immunized with NJ85-KLH, NJ85Δ-KLH and KLH (control), were subjected to challenge against the HYD strain of RHDV. Each experimental group consisted of five rabbits and survival was monitored every day. https://doi.org/10.1371/journal.ppat.1003132.g006 To explore whether the protruding loop L1 of the capsomer can function as an antigenic site and induce an effective host immune response, we coupled the NJ85 and NJ85Δ peptides with keyhole limpet hemocyanin (KLH) (KLH-NJ85 and KLH-NJ85Δ) and used these two constructs to immunize rabbits. Western blot and ELISA analyses showed that antibody titer induced by KLH-NJ85 is about ten-fold higher than that induced by KLH-NJ85Δ (Figure 6B and S9). The RHDV hemagglutination inhibition assay revealed that the inhibition titers of the serum raised by those two peptides were 1∶64 for KLH-NJ85 and 1∶32 for KLH-NJ85Δ, respectively (Figure S10). As a result, although the highly exposed four residues (a.a.308–311) at the top of the capsomer are not required for host cell interaction, they do elicit a strong immunological response from the host. We next investigated whether antibodies raised by those two peptides could neutralize RHDV and protect rabbits. Fifteen rabbits were divided into three groups of five. The sera that were raised by KLH-NJ85 and KLH-NJ85Δ were diluted 32-fold. An aliquot of each (800 µL) was mixed with 256 hemagglutination units of RHDV and incubated at 37°C for 1 hour, and then the mixture was given to rabbits intranasally. The serum from specific pathogen free rabbits was used as a negative control. In the negative control group, one of the five rabbits died within 24 hours after inoculation, three of the five rabbits died within 72 hours, and the fifth succumbed within 96 hours. In the groups of rabbits inoculated with sera raised by KLH-NJ85 and KLH-NJ85Δ, the rabbits were continuously housed and monitored every 24 hours for 10 days and all ten rabbits survived (Figure 6C and Table S2). Furthermore, virus challenge with RHDV displayed 100% immune protection in the two groups of rabbits vaccinated separately with KLH-NJ85 and KLH-NJ85Δ (Figure 6D). In a control group that was vaccinated in parallel with KLH, two of the five rabbits died within 48 hours after challenge whereas the other three succumbed within 72 hours. Each virus challenge experiment was repeated four times and yielded consensus results (Table S3). These experiments demonstrated that loop L1 in the P2 sub-domain of VP60 forms an epitope on RHDV, and peptides derived from this loop are sufficient to stimulate rabbits to produce antibodies that immunize them against RHDV infection. It is noteworthy that previous structural studies of a norovirus/Fab complex suggested the two loops (A′-B′ and E′-F′) in the P2 sub-domain of MNV to contact antibody [10], [31]. When we superimposed the crystal structures of the RHDV and MNV P2 sub-domains, we found that the L1 and L5 loops of RHDV correspond, respectively, to the two loops in MNV (Figure S11). Consequently, our results with RHDV concur with those of at least one other calicivirus. It is unclear why peptides NJ85Δ and NJ85 provide equal protection from RHDV challenge when titers of the sera elicited by them differ. Hence, we performed an immunological assay to determine the expression levels of cytokines in the sera raised by KLH-NJ85 and KLH-NJ85Δ. Four cytokines (IL 2, IFN γ, IL 6, and IL 10) were detected by ELISA. Specific pathogen free rabbit serum was used as a negative control. Except for IL 6, expression levels of IL 2, IFN γ, and IL 10 in sera raised in response to challenges by both KLH-NJ85 and KLH-NJ85Δ were higher than the negative control (Figure S12). It was known that high levels of IL 2 proliferate activated T cells and high levels of IFN γ activate macrophages, neutrophils, and NK cells, and then promote cell-mediated immunity for antiviral effects [32]. Also, high levels of IL10 promote B-cell proliferation and antibody responses [32]. As a result, though NJ85Δ and NJ85 lead to different titers of antibodies, both are able to stimulate similar levels of cytokine expression and activate a cell-immune response that allows rabbits to resist challenges from lethal doses of RHDV.