Report

Architecture of African swine fever virus and implications for viral assembly

See allHide authors and affiliations

Science  01 Nov 2019:
Vol. 366, Issue 6465, pp. 640-644
DOI: 10.1126/science.aaz1439

Unveiling African swine fever virus

African swine fever virus (ASFV) is highly contagious and often lethal. With no vaccine or effective treatment, infections often require large-scale culling of pigs. Wang et al. apply cutting-edge cryo–electron microscopy techniques to determine the structure of this very large DNA virus. An 8.8-angstrom-resolution reconstruction shows the five layers of the virus, and the fourth capsid layer could be reconstructed at 4.8-angstrom resolution. The structure reveals epitopes in the major capsid protein that distinguish ASFV from other nucleocytoplasmic large DNA viruses and shows how the minor capsid proteins stabilize the capsid.

Science, this issue p. 640

Abstract

African swine fever virus (ASFV) is a giant and complex DNA virus that causes a highly contagious and often lethal swine disease for which no vaccine is available. Using an optimized image reconstruction strategy, we solved the ASFV capsid structure up to 4.1 angstroms, which is built from 17,280 proteins, including one major (p72) and four minor (M1249L, p17, p49, and H240R) capsid proteins organized into pentasymmetrons and trisymmetrons. The atomic structure of the p72 protein informs putative conformational epitopes, distinguishing ASFV from other nucleocytoplasmic large DNA viruses. The minor capsid proteins form a complicated network below the outer capsid shell, stabilizing the capsid by holding adjacent capsomers together. Acting as core organizers, 100-nanometer-long M1249L proteins run along each edge of the trisymmetrons that bridge two neighboring pentasymmetrons and form extensive intermolecular networks with other capsid proteins, driving the formation of the capsid framework. These structural details unveil the basis of capsid stability and assembly, opening up new avenues for African swine fever vaccine development.

African swine fever (ASF), first described in Kenya in 1921 (1), is a highly contagious viral disease of swine with mortality rates approaching 100% as measured by virulent isolates. Over the past decade, ASF has spread through many countries of the Caucasus, the Russian Federation, and Eastern Europe, posing a serious risk of further expansion (2). During the period from January to September 2019, the World Organisation for Animal Health (OIE, www.oie.int/en/animal-health-in-the-world/animal-diseases/african-swine-fever/) was notified by 26 countries of new or ongoing outbreaks: 13 in Europe, 10 in Asia, and 3 in Africa. With no vaccine or treatment available, culling pigs is the most effective way to contain the outbreaks, and more than 30 million pigs have been culled from 2018 to 2019. The ASF pandemics have caused estimated economic losses of $2 billion for swine production worldwide. The African swine fever virus (ASFV) is stable in the environment and transmits rapidly and efficiently among pigs.

ASFV is the only member of the Asfarviridae family and the only known DNA arbovirus (a term used to refer to any viruses that are transmitted by arthropods). Despite sharing structural, genomic, and replicative characteristics with other nucleocytoplasmic large DNA viruses (NCLDVs) (3), ASFV differs by possessing a multilayered structure and overall icosahedral morphology. Intracellular ASFV has a genome-containing nucleoid (the first layer) surrounded by a thick protein layer referred to as the core shell (the second layer), which is wrapped by an inner lipid envelope (the third layer) and an icosahedral protein capsid (the fourth layer); these four layers comprise more than 50 proteins (4, 5). Extracellular ASFV gains an external envelope (the fifth layer) as it buds through the plasma membrane (6). Large gaps in knowledge concerning the composition and structure of the infectious virion and the identification of viral proteins responsible for inducing protective immune responses in pigs hinder vaccine development.

ASFV targets macrophages, which are monocytes that are mainly present in the blood and bone marrow (7). Here we isolated porcine bone marrow (PBM) cells from 30- to 40-day-old specific pathogen-free pigs, propagated ASFV [HLJ-2018 strain, isolated from a pig spleen sample collected from an ASF outbreak farm in China (8)] in primary PBM cells, purified extracellular ASFV particles from the cell supernatants, and then inactivated the viral particles with formaldehyde. The purified virions were examined by cryo–electron microscopy (cryo-EM) (fig. S1). The extracellular ASFV particle is, on average, 260 to 300 nm in diameter (fig. S1), which is considerably larger than previous observations (~200 nm) (9) likely owing to the incompleteness of the viral particle or the dehydration treatment of the specimen before EM observation.

Like most NCLDVs (1013), the size (250 to 500 nm in diameter) and potential flexibility of the ASFV particle have restricted its structure determination to no better than 10 Å. We obtained an 8.8-Å resolution, icosahedrally imposed reconstruction of ASFV by averaging 43,811 particles (fig. S2). The three-dimensional reconstruction clearly shows the structure of all five layers, of which the capsid (the fourth layer) has a maximum diameter of 250 nm and the third layer is a 70-Å-thick lipid bilayer membrane that envelopes a 180-nm-diameter core shell (the second layer). The three layers adopt an overall icosahedral morphology that roughly follows the contour defined by the capsid (Fig. 1A). However, the outermost envelope and innermost nucleoid present weak density owing to the loss of some structural features resulting from icosahedral averaging.

Fig. 1 Architecture of the ASFV virion.

(A) The central slice (left) and cross section (right) of the icosahedral ASFV virion structure. The outer membrane, capsid, inner membrane, core shell, and nucleoid are colored in orange, magenta, deep blue, cyan, and gray, respectively. The radius and thickness of each layer are labeled. (B) Radially colored representations of the ASFV capsid and core shell. The T number, including the h and k vectors, is indicated. The color scale represents radial distance in angstroms. (C) Cryo-EM reconstruction of the ASFV capsid. The left half shows the trisymmetron and pentasymmetry organization, and the trisymmetrons, pentasymmetrons, and zippers (the boundaries of two neighboring trisymmetrons) are colored in yellow, light purple, and cyan, respectively. The right half shows the density of the minor capsid proteins, including the penton proteins after removing the outer capsid shell; each minor capsid protein is shown in a different color, as indicated at the bottom right. Boxes in green, red, and blue show the locations with the representative capsomer assembly patterns that will be discussed in Fig. 2D. (D) Diagrammatic organization of the minor capsid proteins and capsomers as viewed from inside the capsid. The pseudo-hexameric capsomers are outlined. The icosahedral threefold and twofold axes are shown as solid black triangles and ovals, respectively. Different minor capsid proteins, including the penton proteins, are shown as different shapes with different colors, as indicated at the bottom right. The pseudo-hexameric capsomers are labeled A, B, C, … in the trisymmetrons and a, b, c, … in the pentasymmetrons.

By using an optimized “block-based” reconstruction approach combined with gradient defocus correction, the resolution of the capsid reconstruction was improved to 4.8 Å (Fig. 1 and figs. S2 and S3). The capsid is constructed of 2760 pseudo-hexameric capsomers and 12 pentameric capsomers arranged in a triangulation number (T) = 277 icosahedral lattice (h = 7 and k = 12) (Fig. 1B). In this lattice, there are 12 pentasymmetrons (containing 30 pseudo-hexameric capsomers and a pentameric capsomer) and 20 trisymmetrons (containing 120 pseudo-hexameric capsomers); a similar organization has been observed in other NCLDVs (1012). Notably, capsomers within a trisymmetron all pack in essentially the same orientation, rotated by ~60° from the capsomers in neighboring trisymmetrons, creating 30 zippers (cleavage lines) on the capsid. Additionally, the core shell separately reconstructed to 9 Å shows 180 six-blade propeller-like capsomers with a central channel (30 Å in diameter) and 12 starfish-like pentons surrounded by 10 antennae, yielding a T = 19 icosahedral lattice (Fig. 1B).

The cryo-EM maps for the pseudo-hexameric capsomer of the capsid were improved further by local averaging of equivalent copies present in the trisymmetron to achieve a resolution of 4.1 Å (Fig. 2A and fig. S3). The backbone of the polypeptide and many side chains were clearly defined (Fig. 2A), allowing us to build an atomic model of the ASFV major capsid protein, p72 (table S1). Each pseudo-hexameric capsomer is composed of three p72 molecules, and five copies of penton protein (a protein that is not p72) constitute a pentameric capsomer. Many of the remaining uninterpreted densities decorating the inner capsid surface represent minor capsid proteins (Fig. 1C), presumably facilitating capsid assembly and maintaining the stability of the capsid shell. To determine potential identities for the minor capsid proteins, we analyzed the protein composition of PBM cell–produced extracellular ASFV particles by mass spectrometry and identified 63 viral proteins and 25 host cellular proteins (tables S2 and S3), including 13 additional viral proteins when compared with a recent proteomic analysis of Vero cell culture–adapted ASFV strain BA71 (5). The minor capsid proteins should be among these. The combination of proteomic analysis, protein abundance level in the ASFV particle, and similarity of the cryo-EM map with predicted structural features of target proteins—including protein sequence, protein secondary structure, and protein topology—led to the identification of the penton protein (H240R) and three minor capsid proteins (p17, p49, and M1249L) (Fig. 1C). Each icosahedral asymmetric unit of the outer capsid shell contains 46 pseudo-hexameric capsomers, with six of these in the pentasymmetron (a, b, c, d, e, and f) and 40 in the trisymmetron (A, B, C, D, E, F, G, H, I, J, K, L, M, N, O, P, Q, R, S, T, U, V, W, X, Y, Z, A′, B′, C′, D′, E′, F′, G′, H′, I′, J′, K′, L′, M′, and N′) (Fig. 1D). The penton and minor capsid proteins (p17, p49, and M1249L) form a complicated network immediately below the outer capsid shell, stabilizing the whole capsid (Fig. 1, C and D).

Fig. 2 Structure and organization of p72.

(A) Density maps and atomic model of the p72 trimer. Each subunit is depicted in a different color. Side-chain features are illustrated on the right. Residues with side chains are labeled. Single-letter abbreviations for the amino acid residues are as follows: E, Glu; F, Phe; G, Gly; I, Ile; K, Lys; L, Leu; N, Asn; Q, Gln; R, Arg; S, Ser; V, Val; W, Trp; and Y, Tyr. (B) Ribbon diagram of the p72 monomer. The domains base, JR1 (jelly roll 1), JR2 (jelly roll 2), and ER1 to ER4 are presented in different colors. (C) Surface presentation of the p72 trimer. ER1 to ER4 domains are depicted in the same colors as in (B); other parts are colored in gray. (D) Detailed depictions of three distinct assembly patterns according to their locations: in the trisymmetron (left), zipper (middle), and pentasymmetron (right). The identities of p72 capsomers are labeled according to the location, as in Fig. 1D. Three types of interaction mode—head to back, head to head, and back to back—are indicated by green, red, and blue triangles, respectively. Interaction areas between two capsomers are measured and marked. Notably, the capsomer interactions in the same interaction mode vary under different microenvironments. These are labeled as 1, 1′; 2, 2′; or 3, 3′, 3′′. The cartoon models depict the changed microenvironments. The diagrams in the dashed boxes show the detailed structural organization in the different microenvironments.

Given that both intracellular and extracellular ASFV forms are infectious (6), a combination of antibodies that block these two types of infection by targeting both outer membrane proteins and capsid proteins are required to confer efficient protection against ASFV. p72 acts as one of the key protective antigens, and its monoclonal antibodies were shown to neutralize virulent ASFV isolates (14, 15). On the viral capsid, p72 forms a homotrimer, with each monomer adopting a double jelly-roll structure that makes up pseudo-hexameric capsomers (also called p72 capsomers). A similar double jelly-roll fold is found in many other viral capsid proteins (Fig. 2B and figs. S4 and S5), including adenovirus (16), the phage PRD1 (17), and vaccinia virus (18). A single jelly-roll structure consisting of eight antiparallel β strands (from B to I) exists in many more viral capsid proteins, indicative of common ancestry (19). As is the case for other double jelly roll capsid proteins, four insertions within the D1E1, D2E2, F1G1, and H1I1 loops form exposed regions (ERs). The ERs, together with the N-terminal base domain, determine the specific differences between the virus types (Fig. 2, B and C, and fig. S6). Sterically, the crown of the p72 capsomer is formed by ER1 and its neighboring ER2 and orients toward the outside of the capsid, which may contribute to a conformational epitope (Fig. 2, B and C). β strands from ER3 and ER4 within the same subunit constitute a four-stranded β sheet that shapes the head of the p72 capsomer and might represent another conformational epitope (Fig. 2, B and C). ER3 surrounded by ER1, ER2, and ER4 may link these two conformational epitopes. These four ERs probably define the neutralizing epitopes and could be used to guide ASF vaccine design (Fig. 2, B and C).

Besides surrounding the penton to fill the pentasymmetron, p72 capsomers also pack together to form the trisymmetron and zipper (Fig. 1, C and D). p72 capsomers adopt different arrangements to allow three distinct assembly patterns: “head to back,” “head to head,” and “back to back” (Fig. 2D). Within the trisymmetron, all p72 capsomers are arranged head to back to create two types of trimers of p72 capsomers (labeled 1 and 1′ in Fig. 2D; 1 has less interaction at the quasi-equivalent threefold axis, whereas 1′ has tight associations at the quasi-equivalent threefold axis). In the zipper and pentasymmetron, p72 capsomers possess all three contact modes, of which head-to-head associations exist in two forms (labeled 2 and 2′ in Fig. 2D) and back-to-back interactions occur in three different microenvironments (labeled 3, 3′, and 3′′ in Fig. 2D). The head-to-head interactions in the zipper are straight and tight with a four-stranded β sheet contributed from two adjacent ER1s, whereas the corresponding interactions in the pentasymmetron are considerably decreased owing to the relative rotation of the p72 capsomer caused by higher curvature in the pentasymmetron (Fig. 2D). By contrast, back-to-back contacts in the zipper are the weakest when compared with their counterparts in the pentasymmetron, where a four-helix bundle is formed in the microenvironment of 3′′ (Fig. 2D). Overall, the back-to-back contacts in the zipper and all three modes of contacts in the pentasymmetron seem insufficient to facilitate the assembly of higher-order structures, which suggests that minor capsid proteins are needed to build the networks that trigger the assembly of the whole capsid.

Correlated with fewer contacts among capsomers, interactions in the pentasymmetron are strengthened by all three minor capsid proteins and the penton (Fig. 3). The penton at the fivefold vertices of the outer capsid exhibits a single jelly roll and a globular cap (Fig. 3A), suggesting that the penton protein is different from, yet homologous to, p72, consistent with structural observations in some known large double-stranded DNA viruses (e.g., PBCV-1, mavirus, PRD1, and others). Flexible fitting of the mavirus penton protein structure into our map was straightforward, with the jelly-roll fold well aligned (Fig. 3A). A protein BLAST search with the mavirus penton sequence against all ASFV open reading frames identified a homolog with 37.7% sequence similarity, H240R, an uncharacterized but essential virion protein (5) (fig. S7 and table S2). H240R is 240 residues in length, enriched with β strands, and predicted to possess a single jelly-roll fold and a ~70–amino acid N-terminal extension (fig. S8), which further supports the identity of the penton in ASFV. Underneath the pentons, weak lantern-like densities (~9 Å) are observed connecting the penton and inner membrane and associating five neighboring capsomers, presumably playing roles in the assembly of the vertices (Fig. 3, A and C). Previous studies reported that the putative capsid protein p49 (B438L) is required for the formation of the capsid vertices (20) and is located in close proximity to the capsid vertices (5). Additionally, p49, which behaves as an integral membrane protein, is not involved in particle transportation from the virus assembly sites to the plasma membrane (20), suggesting that it might be positioned at the inner shell of the capsid. Based on these observations, we propose that the lantern-like densities are five copies of p49 (Fig. 3, A and C).

Fig. 3 Extensive intermolecular networks from minor capsid proteins underpin capsid stability.

(A) Cryo-EM map of the penton complex (top). Each subunit of the penton is depicted in a different color, and the pentameric p49 is colored in magenta. The putative homologous structure of the mavirus penton protein is fitted into the ASFV penton cryo-EM map (bottom). (B) Structures of the p17 minor capsid proteins that glue together capsomers within each trisymmetron. Three copies of p17 molecules from one p72 capsomer are depicted in three colors (red, green, and blue), and p72 capsomers are shown as cartoons. (C) Overview of the intermolecular contacts at the inner capsid. One zipper and two neighboring pentasymmetrons are shown with the p72 capsomers labeled. The view is from the inside of the capsid, and the color scheme is the same as that in Fig. 1D. The diagrams labeled (i) to (iv) show enlarged views of the interactions indicated by the dashed boxes.

Inner membrane protein p17 (D117L) is an essential and highly abundant protein required for the assembly of the capsid and icosahedral morphogenesis (21). Our reconstructions show repeated densities underneath the p72 capsomers. The snake-shaped structure consisting of three continuous α helices matches well with the secondary structure prediction of the ectodomain of p17 (Fig. 3B and fig. S9) and supports the protein abundance level analysis, suggesting that the density is p17 (table S2). p17 closely associates with the base domain of p72, and three copies of p17 encircle each p72 capsomer in the inner capsid shell, firmly anchoring p72 capsomers on the inner membrane (Fig. 3B). Interestingly, tight interdigitations of three p17s from three adjacent p72 capsomers mediate interactions among three capsomers under the type 1 microenvironment (Figs. 2D and 3B). These, together with interactions among three capsomers under the type 1′ microenvironment, guide an ordered packing of the capsomers in the trisymmetron, which comprises most of the capsid.

The microenvironments within pentasymmetrons and zippers are, however, more complicated. Here, the skeleton protein M1249L holds 34 capsomers together, not only fixing one pentasymmetron but also linking two neighboring pentasymmetrons (Figs. 1D and 3C). The essential 1249–amino acid–long M1249L is predicted to be full of coils (fig. S10) and exhibits a fiber-like configuration with two terminal lobes (~150 residues per lobe). The fiber part is about 100 nm in length and possesses 30 extended helices (~30 residues per helix) (Figs. 1D and 3C). M1249L starts from capsomers b and c (contacting a) within one pentasymmetron, extends along the outer edge of the zipper (capsomers c-e, d-f, B-A…N-E, M-D, and L-C), and ends with the capsomers B and C, interacting with the capsomer e from a neighboring pentasymmetron (Fig. 3C). The capsomer pairs (b-c and B-C), which normally show the weakest interactions (~150 Å2) in the mode of back to back (types 3′ and 3), are now held together by the two lobes of M1249L (Figs. 2D and 3C). Additionally, M1249L extensively interacts with p17 and p72 capsomers to form the rigid zipper structure, where capsomers tightly contact each other in the head-to-head mode (type 2) to generate 17 geminate capsomers (d:B, e:C, f:D, A:E, K:F, T:G, B′:H, I′:I, and J:J) and 30 zippers construct the capsid framework. Surprisingly, p17 in the zipper adopts a distinct conformation compared with the one in the trisymmetron (Fig. 3, B and C). Double lasso structures formed by two sets of p17 pairs (Fig. 3C, red and blue) at quasi-equivalent twofold axes and tight intertwinements of p17s (Fig. 3C, red and green) and M1249L at the edge largely facilitate associations of capsomers along the fiber of M1249L, ensuring correct assembly of the zipper.

ASFV assembly begins with the appearance of viral inner membrane precursors, which presumably derive from the endoplasmic reticulum, and then proceeds into icosahedral intermediates and icosahedral particles by the progressive assembly of the capsid layer (21, 22). During the early stage of viral assembly, the inner membranes are flexible and bear various morphologies, even open structures, with various mobile inner membrane proteins—for example, p17—floating on them. The capsid appears to be assembled under the guidance of the inner membrane proteins and minor capsid proteins, but no preassembled arrays or symmetrons have been observed in the infected cells. By combining previous experimental observations and our structural analysis, we can propose a detailed hypothesis for a further stage in understanding ASFV capsid assembly. First, the ability of p49 to associate with the membrane mediates the docking of the penton complex to the inner membrane, where it recruits capsomers (capsomer a) to form the penton core (Fig. 4), initiating the assembly. This is consistent with the in vivo assembly of the mimivirus capsid, which starts from the fivefold vertex and proceeds gradually to complete the capsid shell (23, 24). Second, the skeleton unit M1249L with two capsomer pairs (b-c and B-C) attaches to the penton core; meanwhile, skeleton units, penton cores, and p17 can move around on the inner membranes, increasing their chance to form higher-order assemblies (Fig. 4). Under the guidance of p17 capsomers, skeleton protein M1249L and p17 contribute to the formation of the zippers, which connect neighboring penton cores and gradually construct a polyhedral framework (Fig. 4). Accompanying the formation of the polyhedral framework, capsomers fill in the trisymmetrons to complete the capsid assembly (Fig. 4). In our model, skeleton protein M1249L serves as the backbone for the construction of the capsid framework and determines the size of the capsid. In line with this, fiber-like proteins with similar functions have also been observed in PRD-1 (17), PBCV-1 (25), and Bam35 (26), suggesting a similar assembly pathway.

Fig. 4 The proposed assembly pathway for the ASFV capsid.

(A) Formation of the penton cores on the inner membrane. The ASFV capsid assembly begins with appearance of viral inner membrane precursors that envelop the core shell and nucleoid progressively. During the early stage, the inner membranes exhibit generally open structures, with various inner membrane proteins, for example, p17, floating on them. The penton complexes associate with the inner membrane and then recruit p72 capsomers to form the penton cores, initiating the assembly. (B) Correct assembly of the zipper structures mediated by skeleton protein M1249L and p17 and p72 capsomers. (C) Construction of the polyhedral cage by 12 pentasymmetrons and 30 zippers. (D) Accompanying the formation of the polyhedral framework, p72 capsomers fill in the trisymmetrons to complete the capsid assembly.

The ASFV architecture at near-atomic resolution reported here allows us to take the first steps toward understanding what drives the assembly of the capsid and the basis for its stability. In addition, the structural details, and the p72 atomic structure in particular, can guide the rational design of an epitope-focused immunogen, which will affect the development of new strategies for vaccine intervention against ASFV infections.

Supplementary Materials

science.sciencemag.org/content/366/6465/640/suppl/DC1

Materials and Methods

Figs. S1 to S10

Tables S1 to S3

References (2742)

References and Notes

Acknowledgments: We thank Z. Liu, G. Jiang, Q. Sun, S. Cao, X. Huang, B. Zhu, and G. Ji for cryo-EM data collection and the Electron Microscopy Facility of ShanghaiTech University and the Center for Biological imaging (CBI) in the Institute of Biophysics for EM work. Work was supported by the African Swine Fever Research Emergency Program of the Chinese Academy of Sciences (KJZD-SW-L06), the Strategic Priority Research Program (XDB29010000 and XDB08020200), the National Key Research and Development Program (2017YFC0840300, 2018YFC1200600, and 2018YFA0900801), Center for Biosafety Mega-Science, CAS, and Applied Technology Research and Development Project of Heilongjiang Province (GA19B301). X.W. was supported by the Ten Thousand Talent Program and the NSFS Innovative Research Group (no. 81921005). Author contributions: N.W., D.Z., Jia.W., Y.Z., Y.G., F.L., M.W., Jin.W., and X.W. performed experiments; X.W., Z.B., and Z.R. designed the study; all authors analyzed data; and X.W., Z.B., and Z.R. wrote the manuscript. Competing interests: All authors have no competing interests. Data and materials availability: Cryo-EM density maps of ASFV; the vertex, facet, and edge blocks; the asymmetric unit; and averaged map of the p72 capsomer have been deposited in the electron microscopy data bank under accession codes EMD-0815, EMD-0813, EMD-0812, EMD-0811, EMD-0810, and EMD-0814, respectively, and the atomic coordinate of p72 has been deposited in the Protein Data Bank under accession code 6L2T. Live ASFV is restricted to BSL3+ laboratories and may be subject to import controls.

Stay Connected to Science

Navigate This Article