High-Resolution Cryo-Electron Microscopy Structures of Murine Norovirus 1 and Rabbit Hemorrhagic Disease Virus Reveal Marked Flexibility in the Receptor Binding Domains

Our previous structural studies on intact, infectious murine norovirus 1 (MNV-1) virions demonstrated that the receptor binding protruding (P) domains are lifted off the inner shell of the virus. Here, the three-dimensional (3D) reconstructions of recombinant rabbit hemorrhagic disease virus (rRHDV) virus-like particles (VLPs) and intact MNV-1 were determined to ∼8-Å resolution. rRHDV also has a raised P domain, and therefore, this conformation is independent of infectivity and genus. The atomic structure of the MNV-1 P domain was used to interpret the MNV-1 reconstruction. Connections between the P and shell domains and between the floating P domains were modeled. This observed P-domain flexibility likely facilitates virus-host receptor interactions.Murine norovirus 1 (MNV-1) (3, 14, 15) and rabbit hemorrhagic disease virus (RHDV) are members of the genera Norovirus and Lagovirus of the family Caliciviridae that offer a comparison to recombinant human norovirus (rNV) virus-like particles (VLPs) for assessing the structures and roles of domains within the capsid proteins of this family of viruses. Calicivirus particles contain 180 copies of the 56- to 76-kDa major capsid protein (Orf2), which is comprised of the internal/buried N terminus (N), shell (S), and protruding (P) domains (9, 10). The S domain, an eight-stranded β-barrel, forms an ∼300-Å contiguous shell around the RNA genome. A flexible hinge connects the shell to a “protruding” (P) domain at the C-terminal half of the capsid protein, which can be further divided into a globular head region (P2) and a stem region (P1) that connects the shell domain to P2. The accompanying article (13) describes the determination of the structure of the P domain of MNV-1 to a resolution of 2.0 Å.We recently determined the cryo-transmission electron microscopy (TEM) structure of MNV-1 to ∼12-Å resolution (4) and found that, compared to rNV VLPs (10) and San Miguel sea lion virus (SMSV) (1, 2), the protruding domains are rotated by ∼40° in a clockwise fashion and lifted up by ∼16 Å. To better understand the unusual conformation of MNV-1 and whether it is unique to this particular member of the calicivirus family, the ∼8-Å cryo-TEM structures of infectious MNV-1 and the VLPs of RHDV were determined.MNV-1 was produced as previously described (4). Three liters of cell culture yielded 0.5 to 1.0 mg of purified virus with a particle/PFU ratio of less than 100. Baculovirus expression and purification of recombinant RHDV (rRHDV) VLPs were performed as previously described (8). Cryo-electron microscopy (EM) data were collected at the National Resource for Automated Molecular Microscopy (NRAMM) facility in San Diego, CA (4). Images were collected at a nominal magnification of ×50,000 at a pixel size of 0.1547 nm at the specimen level using Leginon software (12) and processed with Appion software (5). The contrast transfer function for each set of particles from each image was estimated and corrected using ACE2 (a variation of ACE [7]). Particle images were automatically selected (11). The final stacks of particle images contained 20,425 MNV virions and 7,856 rRHDV VLPs, and EMAN 3D (6) was used for the reconstructions. Resolutions were estimated by Fourier shell correlations (FSC) of the three-dimensional (3D) reconstructions and application of a cutoff of 0.5. An amplitude correction of the final electron density was performed using GroEL small-angle X-ray scattering (SAXS) data.3D reconstructions of MNV-1 and rRHDV were calculated to resolutions of 8 Å and 8.1 Å, respectively (Fig. ​(Fig.1).1). The P domains of rNV VLPs rest directly on top of the shell domain (10) (Fig. ​(Fig.1A).1A). In contrast, the P domains of MNV-1 are lifted and rotated above the shell of the capsid (4) (Fig. ​(Fig.1B).1B). At this higher resolution, there was a clear connection between the P1 domain and the shell domain in all three capsid subunits (Fig. ​(Fig.1B,1B, arrow A). Unlike the smooth protruding domains of rNV, MNV-1 has two clear “horns” (arrow B), not dissimilar to those observed for the sapoviruses (1, 2). There also are islands of density in the interior of the shell, directly beneath the 5-fold axes, that may represent ordered regions of RNA.Open in a separate windowFIG. 1.Stereo diagrams (left) and thin sections (right), with radius coloring, of rNV (A), MNV-1 (B), and an rRHDV VLP (C). For rNV, the atomic coordinates (10) were used. In MNV, arrow A indicates the thin connector between the P1 and S domains. Arrow B denotes the horns found at the tips of the P2 domains. Arrow C denotes the large gap between the P1 and S domains in the rRHDV VLP. Arrow D denotes the false connectivity in rRHDV VLPs between the P1 domain and the S domain near the 5-fold axes.As with MNV-1, there is a marked gap between the P and S domains in the rRHDV VLP (Fig. ​(Fig.1C,1C, arrow C). This gap is not as pronounced as in MNV-1 because the P domains are not rotated as in MNV-1. In this electron density map, the A/B dimers appear to be touching the shell domain near the 5-fold axes. This contact difference between the A/B dimers and the C/C dimers could be the reason why the tops of the C/C dimers appear to be markedly disordered compared to the A/B dimers in rRHDV and the C/C dimers in MNV-1.Shown in Fig. ​Fig.22 is the fitting of the atomic structures of the MNV-1 P domain (13) and the rNV S domains into the MNV-1 3D reconstruction electron density. The horns (arrow A, loops A′-B′ and E′-F′) observed at the tips of the P domain match exceedingly well with the electron density. As discussed in the accompanying publication (13), the A′-B′ and E′-F′ loops displayed two discrete conformations, a closed structure, where the two loops were tightly associated, and an open structure, where the loops were splayed apart. The horns of the closed conformation fit better into the reconstruction, as the E′-F′ loop in the open form jutted out of the density at the base of the horns. The unmodified density in the lower panel of Fig. ​Fig.22 shows fine features in the shell domain and a very clear connection between the shell and P1 domains. The connections between the P1 and S domains were of sufficient quality to build a basic backbone model by uncoiling the linker region (arrow B). The P domain in the unfiltered 3D reconstruction was far less ordered than the S domain (Fig. ​(Fig.2).2). This was likely due to movement of the entire P domain with respect to the shell.Open in a separate windowFIG. 2.Fitting of the MNV-1 P domain and the rNV shell domain into the MNV-1 electron density. A, B, and C subunits are represented by blue, green, and red, respectively. The electron density is shown in transparent gray. The top panel is the 8.0-Å-resolution 3D reconstruction modified using a low-pass filter. The bottom panel is the reconstruction without modification. The horns on the tops of the P domains are denoted by arrow A. Arrow B denotes the connection between the S and P domains.Using the structure of rNV VLP P domains for modeling, the rRHDV P domains are lifted off the surface of the shell, but not rotated as with MNV-1. This places the bottom edge of the A subunit P1 domain near the S domain at the 5-fold axes. The P-domain dimers of rNV and rRHDV have a more “arch-like” shape than MNV-1. Unlike in MNV-1, the electron densities of the C/C dimers in rRHDV are far more diffuse than those of the A/B dimers (Fig. ​(Fig.3B)3B) and the connector between the S and P1 domains is not clear. During fitting, the connector region was not as extended as with MNV-1. This may afford greater flexibility, leading to more diffuse electron density.Open in a separate windowFIG. 3.Fitting of the rNV atomic structure into the rRHDV VLP electron density. The upper stereo image shows the 8.1-Å-resolution 3D reconstruction after modification by a low-pass filter. Below is the same reconstruction prior to density modification.When the atomic models for the MNV-1 P domains (13) were placed into the cryo-TEM electron density (Fig. ​(Fig.4),4), the C termini extended deep into the cores of adjacent P domains. Possible connections not accounted for by the P-domain structures were also observed in the electron density between the P domains. A bulge between the P1 and P2 domains in the 3D reconstruction indicated a possible interaction between the C termini and the adjacent P domains. These same interactions were observed in the crystal lattice. This highly mobile C terminus may be a flexible tether between the P domains in the intact virion.Open in a separate windowFIG. 4.Possible carboxyl-terminus interactions between the P domains of MNV-1. (A) Stereo image of MNV-1 calculated to 12-Å resolution with (red) and without (yellow) the last 10 residues of the P domain. (B) The calculated MNV-1 density with the carboxyl terminus removed (yellow) overlaid onto the 3D reconstruction of MNV-1 (blue). Note the strands of difference density that roughly correspond to the C terminus in panel A. (C) The C-terminus interactions observed in the structure of the MNV-1 P domains. Shown in blue and green are ribbon diagrams of an A/B P-domain dimer. In mauve is a surface rendering of the C terminus from a crystallographically related dimer. (D) Surface rendering of the final MNV-1 model with possible interactions between the P domains in MNV-1. The carboxyl termini of the A subunits (blue) interact with the counterclockwise-related B subunits around the 5-fold axes (white arrows). Around the 3-fold (quasi-6-fold) axes, the C subunits interact with the A subunits and the B subunits interact with the C subunits (orange arrows).It is absolutely clear that the hinge region between the S and P domains affords a remarkable degree of flexibility in the P domains that is not genus specific or related to differences between rVLPs and authentic virions. The simplest explanation for the role of this transition is that it gives the P domains flexibility that may be used to optimize interactions with cell receptors during attachment and entry. In this way, the P domains can increase their avidity for the cell surface by being more facile in adapting to the presentation of cellular recognition motifs.     

兔出血症病毒与细小病毒抗原相关性试验

用间接ELISA、ELISA交叉阻断法和交叉血凝抑制试验对兔出血症病毒(RHDV)与6种细小病毒进行抗原相关性试验。用间接ELISA证实,RFIDV与它们有轻度交叉关系,其抗原相关值分别为:小鼠细小病毒(MVM)5.59％;鹅细小病毒(GPV)3.54％;猪细小病毒(PPV)1.76％;水貂肠炎病毒0.7％。细小病毒间的抗原相关值:MEV与PPV为31.6％,MEV与MVM为35.36％;而CPV与MEM、PPV、MVM的相关值均为零,即无相关性。在ELISA交叉阻断法中证实:犬细小病毒(CPV)、猫泛白细胞减少症病毒(FPV)和MEV均不能阻断RHDV与其抗体结合,仅GPV有轻度阻断作用,其最大阻断率为40％。在血凝交叉抑制试验中,未发现RHDV与细小病毒及其相应抗体间存在交叉抑制现象。以上结果表明RHDV与细小病毒在血清学方面有轻度相关性。     

兔出血症病毒结构多肽分析

粗提病毒经Ｓｅｐｈａｒｏｓｅ４Ｂ层析后,获得纯化的兔出血症病毒（ＲＨＤＶ）。提纯病毒经过ＳＤＳ－ＰＡＧＥ经考马斯亮蓝染色显示Ａ、Ｂ、Ｃ、Ｄ、Ｅ、Ｆ和Ｇ７条多肽,凝胶扫描显示Ａ为ＲＨＤＶ主要结构多肽。用多抗和单抗作免疫转印分析,证实Ａ、Ｂ、Ｃ、Ｄ、Ｅ、和Ｇ为结构多肽,此６条结构多肽间的抗原关系十分密切。     

兔出血症细小病毒血清学的研究

应用免疫双扩散和酶联免疫吸附分析(ELISA)对七个不同地区兔出血症病原分离物进行了血清学比较研究。本试验用免疫双扩散和ELISA,证明七个兔出血症病毒分离物之间存在着基本相同的血清学关系,其中A_2R—3和H.E.二株抗原之间存在较大差异.证实我国流行的兔出血症病毒属于一个血清型,但不同地区的分离株可能出现亚型。     

兔出血症病毒在体外诱导细胞凋亡

The apoptosis of RK13 cells induced by RHDV was investigated with DAPI staining,DNA ladder,Caspase 3 activity and flow cytometry,etc.The results showed that nuclear staining of infected cells with DAPI showed gradually morphological changes of the nuclei.As shown in the paper,a canonic oligonucleosome-sized DNA ladder was observed in cells harvested at 24h,48h and 72h post-infection,confirming that DNA fragmentation was induced by RHDV infection.The results of flow cytometry showed that about 63 % of cells ...     

兔出血症病毒复制子的构建及其在RK-13细胞中的复制

为研究兔出血症病毒(RHDV)的复制机制、病毒与宿主之间的相互作用以及致病机制等,创建一个安全、有效的技术平台,在前期构建的RHDV侵染性克隆基础上,将病毒的衣壳蛋白编码区删除,保留了RHDV复制必需的所有蛋白酶基因和两端的非编码区,构建了RHDV复制子。试验结果证明,将该复制子RNA导入RK13细胞中后,能够进行高水平的复制和表达。     

兔出血症病毒体外复制的研究

在几种动物细胞上,采用同步感染的方法研究了兔出血症病毒(RHDV)的复制特性。病毒感染细胞后(PI)48—72小时可观察到明显的细胞病变,血凝效价可增高5—10倍。病毒对细胞传代代数不同,敏感性也不同。在兔婴肾(RK)和兔婴肺(RL)细胞上以4—8代最为敏感。采用免疫荧光染色法,经病毒感染48—72小时的细胞中可观察到特异性荧光。细胞增殖的病毒经PEG-DS浓缩,Sepharose 4B柱层析提纯后,在电镜下可观察到完整的病毒粒子,将此病毒回接健康实验兔可致100％死亡。免疫双扩散和免疫电泳试验表明,细胞增殖的病毒抗原与来自病兔肝的RHDV抗血清之间产生明显的沉淀带。SDS-PAGE分析病毒获得四条多肽,其分子量大小与病兔肝组织提取的病毒蛋白多肽分子量相比略有差异。     

Detection of Viral Proteins after Infection of Cultured Hepatocytes with Rabbit Hemorrhagic Disease Virus

The calicivirus rabbit hemorrhagic disease virus (RHDV), which replicates predominantly in the livers of infected rabbits, cannot be propagated in tissue culture. To enable the performance of in vitro studies, rabbit hepatocytes were isolated by liver perfusion and gradient centrifugation. After inoculation with purified RHDV, more than 50% of the cells proved to be infected. Protein analyses led to the detection of 13 RHDV-specific polypeptides within the infected cells. These proteins were assigned to defined regions of the viral genome, resulting in a refined model of RHDV genome organization.     

Expression of Enzymatically Active Rabbit Hemorrhagic Disease Virus RNA-Dependent RNA Polymerase in Escherichia coli

The rabbit hemorrhagic disease virus (RHDV) (isolate AST/89) RNA-dependent RNA-polymerase (3D^pol) coding region was expressed in Escherichia coli by using a glutathione S-transferase-based vector, which allowed milligram purification of a homogeneous enzyme with an expected molecular mass of about 58 kDa. The recombinant polypeptide exhibited rifampin- and actinomycin D-resistant, poly(A)-dependent poly(U) polymerase. The enzyme also showed RNA polymerase activity in in vitro reactions with synthetic RHDV subgenomic RNA in the presence or absence of an oligo(U) primer. Template-size products were synthesized in the oligo(U)-primed reactions, whereas in the absence of added primer, RNA products up to twice the length of the template were made. The double-length RNA products were double stranded and hybridized to both positive- and negative-sense probes.     

Complete Genomic Sequences of Rabbit Hemorrhagic Disease Virus G1 Strains Isolated in the European Rabbit Original Range

The complete genomic sequences of rabbit hemorrhagic disease virus (RHDV) strains isolated in 1995 (CB137) and 2006 (CB194) from wild European rabbits from Portugal are described. The strains were isolated in the original range of the European rabbit and assigned to genogroup 1 (G1), which is known to have persisted only in the Iberian Peninsula. ORF2 of isolate CB137 might encode a shorter minor structural protein, VP10.     

兔出血症病毒衣壳蛋白基因在毕赤酵母中的表达

将RHDVVP60基因插入酵母转移载体pPICZB中转化毕赤酵母菌GS115株 ,经筛选获得染色体基因组中整合入VP60基因的重组酵母菌。以甲醇诱导培养后经SDS-PAGE和Westernblot检测表达产物 ,在60kD处出现一特异蛋白条带 ,表明RHDV的衣壳蛋白得到了成功表达。血凝试验表明 ,表达的重组蛋白具有血凝特性 ,可以凝集人“O”型红细胞 ,血凝价达 2.8,同时 ,该血凝性可被抗RHDV的高免血清所抑制。经电镜观察 ,重组酵母表达的衣壳蛋白可以在酵母菌体内自聚成大小约4.0nm ,和天然RHDV病毒子在物理形态上类似的病毒样颗粒 (VLPs)。该病毒样颗粒与兔抗RHDV高免血清作用后可被凝集成团 ,表明该VLPs与天然RHDV在抗原性上也极为相似。     

兔出血症病毒核酸的某些理化性质的研究

本文对我国无锡分离的兔出血症病毒A_2R-3毒株核酸的某些理化性质进行了研究。采用孚尔根染色、二苯胺反应和核酸酶解实验证实病毒核酸为DNA类型。吖啶橙染色、甲醛反应、核酸酶S_1消化和核酸热变性实验表明病毒核酸为单链型。核酸电泳呈单一组分。电境观察显示核酸分子链呈线状,平均长度约为2.15μ。计算分子量约为2.1—2.5×10~6d。核酸碱基组盛为A25.34、T29.37、G23.85、C21.43、(G C)克分子百分比值为45.28。结合以前的报道、我们认为:兔出血症病毒可以归类于细小病毒科。     

免出血症病毒的形态与结构的研究

免出血症病毒无锡株A_2R—3的完整病毒颗粒呈球形,廿面体等轴对称、无囊膜、其直径约为33—37nm。空心病毒粒子亦可看到,其核心直径为21—25nm。病毒衣壳包裹在颗粒的最外层,由紧密连结的子粒所组成,子粒排列规则,呈管状结构。其长度约为5—6nm,中心孔径为2—3nm。廿面体对称的等边三角形的面由6个子粒所构成,即每条边上排列3个子粒。据此推算出病毒衣壳的子粒总数为42,分负数为4,三角形面数为80,结构单位数为240。     

Oral Immunization using Tuber Extracts from Transgenic Potato Plants Expressing Rabbit Hemorrhagic Disease Virus Capsid Protein

Rabbit hemorrhagic disease, which is caused by a calicivirus, is a lethal infection of adult animals that is characterized by acute liver damage and disseminated intravascular coagulation. In this study, we report the production of the major structural protein VP60 of rabbit hemorrhagic disease virus in transgenic tubers of potato plants and its use as an oral immunogen in rabbits.