African swine fever virus A137R assembles into a dodecahedron cage

ABSTRACT

African swine fever (ASF) is a highly contagious viral disease that affects domestic and wild pigs. The causative agent of ASF is African swine fever virus (ASFV), a large double-stranded DNA virus with a complex virion structure. Among the various proteins encoded by ASFV, A137R is a crucial structural protein associated with its virulence. However, the structure and molecular mechanisms underlying the functions of A137R remain largely unknown. In this study, we present the structure of A137R determined by cryogenic electron microscopy single-particle reconstruction, which reveals that A137R self-oligomerizes to form a dodecahedron-shaped cage composed of 60 polymers. The dodecahedron is literally equivalent to a T = 1 icosahedron where the icosahedral vertexes are located in the center of each dodecahedral facet. Within each facet, five A137R protomers are arranged in a head-to-tail orientation with a long N-terminal helix forming the edge through which adjacent facets stitch together to form the dodecahedral cage. Combining structural analysis and biochemical evidence, we demonstrate that the N-terminal domain of A137R is crucial and sufficient for mediating the assembly of the dodecahedron. These findings imply the role of A137R cage as a core component in the icosahedral ASFV virion and suggest a promising molecular scaffold for nanotechnology applications.

IMPORTANCE

African swine fever (ASF) is a lethal viral disease of pigs caused by African swine fever virus (ASFV). No commercial vaccines and antiviral treatments are available for the prevention and control of the disease. A137R is a structural protein of ASFV that is associated with its virulence. The discovery of the dodecahedron-shaped cage structure of A137R in this study is of great importance in understanding ASFV pathogenicity. This finding sheds light on the molecular mechanisms underlying the functions of A137R. Furthermore, the dodecahedral cage formed by A137R shows promise as a molecular scaffold for nanoparticle vectors. Overall, this study provides valuable insights into the structure and function of A137R, contributing to our understanding of ASFV and potentially opening up new avenues for the development of vaccines or treatments for ASF.

INTRODUCTION

African swine fever (ASF), caused by African swine fever virus (ASFV), is a highly infectious disease circulating in domestic and wild pig populations, which poses a serious threat to the breeding industry, world economy, and food safety. The ASF epidemic first broke out in Kenya in 1921 and subsequently became endemic in some African countries (1). In recent decades, it has rapidly spread from Africa to many European and Asian countries (2). ASFV, the only member of the family Asfarviridae and the only known DNA arbovirus, has a complex and unique multilayer structure consisting of five layers from the inside to the outside, including the core genomic DNA, a core shell, an inner membrane, a proteinaceous capsid, and the outermost viral envelope (3, 4). The ASFV particle possesses an overall spherical morphology and a diameter of about 250 nm, while its genome length ranges from 170 to 194 kb, encoding 150–167 proteins responsible for virion assembly, genome replication, and counteracting host immunity (5). Due to the complex structure of the ASFV virion and its large DNA genome, many aspects of the fundamental biology of ASFV have not been well understood. At present, there is still no effective anti-ASFV vaccine or antiviral drug available, thus culling is still the only means to control the spread of ASFV.

The A137R protein is expressed at the late stage of viral replication, which is localized in the viral factory and serves as a structural component of the complete virion (6). A previous study demonstrated that knockout of A137R significantly reduced the virulence of ASFV, suggesting that A137R is not an essential gene of ASFV but plays a crucial role in modulating its virulence (7). Pigs vaccinated intramuscularly with ASFV A137R-deletion mutant produce a strong virus-specific antibody response and are effectively protected from infection by the parental strains, indicating that the A137R-deletion mutant is a promising candidate for a live-attenuated vaccine (7). A more recent work reported that A137R may regulate ASFV virulence by interacting with TBK1 to promote autophagy-mediated lysosomal degradation of TBK1 and block nuclear translocation of interferon regulatory factor 3, thus reducing type I IFN production (8). Despite these advances, the structure of A137R and the underlying mechanisms of its biological functions remain unknown.

Here, we report the cryogenic electron microscopy (cryo-EM) structure of A137R, which provides new insights into its structural organization and functional roles in ASFV infection and pathogenesis. A137R self-oligomerizes to form a dodecahedron cage composed of 60 protomers, in which the N-terminal domain (NTD) of A137R is essential and sufficient for mediating this assembly. These findings provide important clues to understand the role of A137R as a structural component of ASFV and suggest a promising molecular scaffold for nanobiology applications.

RESULTS

A137R protein forms oligomers in solution

Recombinant A137R protein was expressed in Escherichia coli with a C-terminal 6×His tag (Fig. 1A). The Ni affinity-purified A137R protein was eluted through a HiLoad 16/600 Superdex 200 pg column with an elution volume of around 50 mL, indicating that it forms oligomers in solution. SDS-PAGE showed that the molecular weight of the A137R protein monomer was 15 kDa (Fig. 1B). Sedimentation velocity analytical ultracentrifugation analysis further confirmed that the A137R protein exists as an oligomer (~1,050 kDa) in solution (Fig. 1C).

Fig 1.

A137R forms a dodecahedron cage. (A) Schematic representation of the A137R construct. The NTD and C-terminal domain (CTD) are shown in magenta and light blue, respectively. (B) Analytical gel filtration of A137R protein. The 280 nm absorbance curve from Superdex 200 column and the SDS-PAGE migration profile of the pooled sample are shown. (C) Ultracentrifugation sedimentation profile of A137R protein. The calculated molecular weight of the indicated protein species is shown. (D) Cryo-EM map showing the density of the A137R dodecahedron. (E) The surface-rendered representations showing the electrostatic potential. The red color is attributed to an abundance of negative charges in close proximity to the surface, and the blue color signifies positive potential. (F) The hydrophobic/hydrophilic properties of A137R dodecahedron. Green represents hydrophilic regions and yellow signifies hydrophobic patches.

Cryo-EM structure determination and architecture of the A137R oligomer

The A137R oligomer displays a spherical shape under electron microscopy, with a diameter of ~18 nm (Fig. 1C and 2A and B). Ab initio 3D reconstruction revealed that the A137R oligomer was assembled in icosahedral symmetry with a Triangulation (T) of 1 (Fig. 2C). As the icosahedral vertexes are located at the center of a pentagon surrounded by five protomers, the overall morphology of the A137R oligomer is more like a hollow dodecahedral cage. By applying icosahedral symmetry, we were able to reconstruct the structure of the A137R cage at a global resolution of 3.4 Å (Fig. 1C and 2D and E; Fig. 2; Table 1). This map allowed us to derive an atomic model of A137R de novo in which the N-terminal 99 residues were successfully built whereas residues 100–137 were not resolved in the density map, suggesting a flexible C-terminal tail of A137R.

Fig 2.

Cryo-EM single-particle analysis of the A137R dodecahedron cage. (A) Representative one from 3,629 cryo-EM micrographs. (B) 2D class average images of A137R. (C) A brief workflow of cryo-EM image processing and reconstruction. (D) The Fourier shell correlation (FSC) curve for the density map at 3.4 Å resolution. (E) Local resolution distribution of the density map.

TABLE 1.

Cryo-EM data collection, refinement, and validation statistics

	A137R
Data collection and processing
Voltage (kV)	300
Electron exposure (e–/Å2)	60
Defocus range (μm)	−1.0 to −2.5
Pixel size (Å)	1.04
Symmetry imposed	I1
Final particle images (no.)	130,876
Map resolution (Å)	3.4
FSC threshold	0.143
Refinement
Initial model used (PDB code)	−
Map sharpening B factor (Å2)	−166
Model composition
Non-hydrogen atoms	50,400
Protein residues	5,940
Ligands	−
RMS deviations
Bond lengths (Å)	0.003
Bond angles (°)	0.5
Validation
MolProbity score	1.60
Clashscore	12.33
Poor rotamers (%)	0.29
Ramachandran plot
Favored (%)	98.02
Allowed (%)	1.98
Disallowed (%)	0.00

The A137R cage consists of 60 protomers, in which 5 protomers are arranged in a head-to-tail orientation to make the edge of a pentagon within one facet of the dodecahedron. Twelve of such pentagons stitch together to construct the hollow dodecahedral cage. The cage displays a regular electrostatic surface potential distribution, where the triangular vertices (the interface of three adjacent pentagons) are predominantly positively charged while the dodecahedral edges are mainly negatively charged (Fig. 1E). Overall, the cage surface is primarily hydrophilic (Fig. 1F).

Structure of the A137R protomer

We then analyzed the structure of one A137R protomer in detail. The A137R protomer is mainly composed of two α-helices, one 3₁₀ helix, and four β-strands (Fig. 3A through C). This topology represents the NTD of A137R with residues 1–99. The C-terminal domain (CTD) of A137R is not visible in our structure, maybe due to its flexibility. A gun-like shape is formed by each protomer, with the long Helix α1 forming the barrel and the remaining parts forming the grip. Furthermore, each protomer establishes stable interactions through multiple binding interfaces, collectively creating a hollow dodecahedral edge. The N-terminal α1 helix makes the edge of each pentagonal facet and mediates the inter-facet interactions, whereas the C-terminal grip is surrounded within the pentagon, only involved in intra-facet interactions. Besides, the electrostatic potential patterns of the inside and outside surfaces of A137R protomer are remarkably different (Fig. 3D).

Fig 3.

Structure of a protomer of A137R. (A) Sequence annotation of the A137R molecule, with its NTD and CTD domains highlighted in magenta and light blue, respectively. The secondary structure of the NTD is based on our structural analysis; CTD density is not visible in the structure. (B) The structure of a protomer of A137R. (C) Topological view of the A137R protomer. α: α-helix; β: β-strand; η: 3₁₀ helix. (D) The surface-rendered representations showing the electrostatic potential of a protomer of A137R.

Molecular basis underpinning the assembly of A137R cage

We then analyzed the molecular interactions within the A137R cage in detail. Each A137R protomer interacts with five adjacent protomers through different interfaces. These interfaces create distinct interaction patterns and contribute differently to the assembly of the dodecahedron cage structure. Among the five interaction interfaces, interfaces I and IV (Fig. 4A and C) are responsible for creating the pentagonal shape of the resulting pentamer. Residues M1, K7, L8, E11, and F19 in Helix α1 from one protomer interact with residues F34, Y44, F46, and M53 in the grip of an adjacent protomer through hydrophobic and van der Waals interactions. Interfaces II and III (Fig. 4B), occurring at the triangular vertices where three pentagons meet, are stabilized by three symmetric salt bridges between residue E2 of one protomer and residue K13 from its neighbor. Finally, interface V contributes to the interaction between adjacent pentamers, involving several amino acids from Helix α1 and Helix α2, including R21, W24, I31, L35, E70, F74, and N77. These observations reveal an elaborate and delicate interaction network that stabilizes the dodecahedral assembly of the A137R cage.

Fig 4.

Assembly basis of the A137R dodecahedron cage. One A137R protomer (magenta) in the dodecahedron interacts with five nearby molecules through five different interaction interfaces, leading to the formation of five distinct interaction sites. Interfaces I (green, A) and IV (cyan, C) contribute to the pentagonal pentamer formation, and interfaces II (yellow, B) and III (pink, B) contribute to the pentagonal triangular vertex formation, and interface V (purple) contribute to two neighboring pentamer interaction. Residues involved in the interactions are shown as sticks and labeled.

NTD of A137R is sufficient for forming a dodecahedron

Although the full-length A137R was used for structural analysis, we were unable to resolve the C-terminal tail in the density map. We hypothesized that the C-terminal region of A137R might be dispensable for the formation of the dodecahedron assembly. To test this hypothesis, we constructed a truncated version of A137R, including residues 1–99 (A137R_1–99), and successfully purified this mutant protein. Through analytical gel filtration (Fig. 5A) and analytical ultracentrifugation sedimentation (Fig. 5B) experiments, we confirmed that the A137R_1–99 formed oligomers in solution with a molecular weight of ~60 protomers, indicating that the NTD alone is sufficient to form a dodecahedron, consistent with our structural observations.

Fig 5.

N-terminal domain of A137R forms a dodecahedron in solution. (A) Analytical gel filtration of A137R-NTD protein. The 280 nm absorbance curve from Superose 6 column and the SDS-PAGE migration profile of the pooled sample are shown. (B) Ultracentrifugation sedimentation profile of A137R-NTD protein.

DISCUSSION

Emerging and re-emerging viruses continue to pose a threat to public health (9). ASFV is highly contagious and causes lethal diseases in both domestic pigs and wild boar victims. A deep understanding of the structure and function of viral proteins is necessary for developing effective strategies to prevent and treat ASFV infections.

The cryo-EM structure of A137R presented in this study reveals a unique dodecahedral cage structure composed of 60 protomers. This structure is stabilized by interactions between protomers through multiple binding interfaces, with the N-terminus being more outward and the C-terminus being more inward. Our biochemical assays further demonstrate that the N-terminal domain of A137R is sufficient for forming the dodecahedron, indicating that this region is crucial for A137R high-order assembly. No similar structure was found in the Dali database. The arrangement of viral proteins with dodecahedral symmetry is extremely rare. Although several viral proteins forming dodecahedral structures have been reported, for example, Adenovirus 3 (Ad3) penton (10, 11), the structure of the A137R dodecahedron differs from these proteins in several ways. First, the monomers of A137R have a gun-like shape, which is different from the globular shape of the penton base protein. The small molecular weight of A137R monomer results in the formation of hollow dodecahedral structures. Second, the A137R dodecahedron is made up of 60 protomers, whereas the Ad3 penton protein forms a dodecahedron made up of 12 pentons (12 penton bases with 12 protruding trimeric fibers). Furthermore, while the Ad3 penton proteins have been extensively studied for their structural and functional roles in virus assembly, the biological functions of the A137R dodecahedron are still unclear. One possible function of this dodecahedral cage is that it may serve as a core scaffold to facilitate the assembly of the icosahedral viral capsid as A137R is a structural component of the complete virion. Further studies are required to fully understand the role of the A137R dodecahedron in ASFV infection and interactions with host immune responses.

The unique structure of the A137R dodecahedron suggests its potential use as a nanoparticle carrier for vaccine development. The dodecahedral cage composed of 60 polymers provides a stable and organized platform for the presentation of antigenic epitopes, which could induce a strong immune response. Several studies have reported successful applications of virus-like particles (VLPs) as vaccines against various viral diseases, including hepatitis B and human papillomavirus infection (12, 13). VLPs are structural mimics of viruses but lack infectious genetic material, making them a safe and effective vaccine candidate. Similarly, the A137R dodecahedron could be an attractive alternative to VLPs due to its unique structure and functional characteristics. The hollow dodecahedral edge formed by A137R could provide an attractive platform for the presentation of proper antigen as a vaccine candidate. The unique shape and organization of the dodecahedron could enhance the immune response and potentially induce long-lasting immunity against ASFV infection. Future studies could explore the feasibility of using this protein as a vaccine carrier candidate and evaluate its safety and efficacy in animal models.

Our study provides a potential target for the development of antiviral therapies against ASFV. Targeting the assembly of the dodecahedral cage may disrupt the potential functions of A137R and ultimately reduce the virulence of ASFV. Overall, our results provide important insights into the structure and function of A137R, which may be useful for the development of effective strategies for controlling and treating ASF outbreaks. However, the exact role and function of A137R in ASFV pathogenesis still remain unknown, and further work is needed to determine the functional role of A137R.

In conclusion, the unique structure of the A137R protein forming the hollow dodecahedral edge provides new insights into virus assembly and potential applications in vaccine development. Further studies could explore the potential for utilizing these unique features for therapeutic interventions against ASFV.

MATERIALS AND METHODS

Gene cloning, protein production, and purification

The A137R protein full-length DNA sequence of Pig/HLJ/18 virus strain (GenBank entry number MK333180.1) (14) was incorporated into the pET-21a vector fused at its C-terminus with a hexa-histidine tag using NdeI and XhoI restriction sites. Transformed E. coli strain BL21 (DE3) clones were grown in Luria-Bertani (LB) medium containing 100 µg/mL ampicillin to an OD₆₀₀ of 0.6–0.8 at 37°C. Expression of the recombinant proteins was induced by the addition of 0.5 mM isopropyl-β-D-1-thiogalactopyranoside, and incubation was continued for further 16 h at 16°C. Cells were harvested by centrifugation at 7,000 × g for 15 min at 4°C and then resuspended in lysis buffer (20 mM Tris–HCl, pH 8.0, and 150 mM NaCl) and further homogenized with a low-temperature ultra-high pressure cell disrupter (JNBIO, China). The lysate was clarified by centrifugation at 20,000 × g for 60 min at 4°C. The supernatant was purified by metal affinity chromatography using a HisTrap HP 5 mL column (GE Healthcare). Proteins were eluted using the lysis buffer supplemented with 300 mM imidazole. The proteins were further purified by gel filtration chromatography using a HiLoad 16/600 Superdex 200 pg column (GE Healthcare) with a running buffer of 20 mM Tris–HCl pH 8.0 and 50 mM NaCl, and the collected protein fractions were concentrated to 10 mg/mL using a membrane concentrator with a molecular weight cutoff of 10 kDa (Millipore).

Biochemical characterization of A137R protein

The purified protein was analyzed using an analytical gel filtration assay with a calibrated HiLoad 16/600 Superdex 200 pg column (GE Healthcare). The sample was further analyzed with SDS-PAGE. The analytical ultracentrifugation assay was performed according to a previously reported method (15). The proteins were prepared in 20 mM Tris, pH 8.0, and 150 mM NaCl at a concentration of A₂₈₀ = 0.8. The assay was performed on an optimal ProteomeLab XL-I analytical ultracentrifuge (Beckman Coulter) at a speed of 48,000 rpm. The molecular mass analysis was performed with the XL-I data analysis software.

Cyro-EM sample preparation and data acquisition

The purified A137R protein solution was diluted to 1.0 mg/mL, and then about 3.0 µL of liquid was applied to glow-discharged copper grids (Quantifoil, R1.2/1.3, 300 mesh), which were blotted for 3 s at a temperature of 4°C and a humidity level of 100% using Vitrobot Mark IV (Thermo Fisher Scientific). Cryogenic specimens were loaded onto 300 kV Titan Krios transmission electron microscope (Thermo Fisher Scientific). The data were collected automatically by Serial EM software using beam-image shift imaging scheme. Images were recorded with a K2-subunit detector using super resolution mode with a calibrated pixel size of 1.04 Å. Each stack was exposed at a dose rate of 10 e⁻¹ pixel⁻¹ s⁻¹ and an accumulative dose of 60 e⁻¹ Ų and was fractioned into 30 movie frames. The final defocus ranges were −1.0 to −2.5 µm.

Image processing and 3D reconstruction

The movie frames were aligned using MotionCor2 (16) and the contrast transfer function (CTF) values of each micrograph were determined using CTFFind4 (17). All classification and reconstruction procedures were performed using Relion-3.0 (18). Fifty micrographs were selected for automatic particle picking using Laplacian-of-Gaussian blob detection and were subjected to two-dimensional (2D) classification to generate templates for autopicking against the entire data set. A total of ~380,000 particles were selected from 3,629 micrographs and were reduced to ~210,000 particles after two rounds of 2D classification. We generated the ab initio model (four classes) using Relion-3.0 and selected the best class low-passed to 60 Å as the initial reference model for 3D classification using D5 symmetry. After 3D classification, a clean data set of 130,876 particles from two classes was selected and subjected to 3D refinement, which yielded a reconstruction map at 4.3 Å. When we analyzed the density map, we found that it showed icosahedral symmetry. To further improve the map resolution, we performed another round of 3D refinement with I1 symmetry, where we obtained a better map with a resolution at 3.4 Å. The local resolution maps were evaluated by ResMap (19).

Model building

The quality of map is good enough for manually modeling ab initio. The initial coordinates were refined against the corresponding maps using PHENIX (20) with secondary structure restraints and Ramachandran restraints applied. And then, we performed manual model building to improve local fit using COOT (21). The stereochemical quality of each model was assessed using MolProbity (22). Structural figures were prepared by Pymol (https://pymol.org/) and ChimeraX (23).

DATA AVAILABILITY

The cryo-EM density map and atomic coordinates of A137R have been submitted and deposited in the Electron Microscopy Data Bank (EMDB) with entry ID EMD-38214 and the Protein Data Bank (PDB) with entry ID 8XB8.