Structural Insights into the Assembly of the African Swine Fever Virus Inner Capsid

Abstract

African swine fever virus (ASFV), the cause of a highly contagious hemorrhagic and fatal disease of domestic pigs, has a complex multilayer structure. The inner capsid of ASFV located underneath the inner membrane enwraps the genome-containing nucleoid and is likely the assembly of proteolytic products from the virally encoded polyproteins pp220 and pp62. Here, we report the crystal structure of ASFV p150_△NC, a major middle fragment of the pp220 proteolytic product p150. The structure of ASFV p150_△NC contains mainly helices and has a triangular plate-like shape. The triangular plate is approximately 38 Å in thickness, and the edge of the triangular plate is approximately 90 Å long. The structure of ASFV p150_△NC is not homologous to any of the known viral capsid proteins. Further analysis of the cryo-electron microscopy maps of the ASFV and the homologous faustovirus inner capsids revealed that p150 or the p150-like protein of faustovirus assembles to form screwed propeller-shaped hexametric and pentametric capsomeres of the icosahedral inner capsids. Complexes of the C terminus of p150 and other proteolytic products of pp220 likely mediate interactions between the capsomeres. Together, these findings provide new insights into the assembling of ASFV inner capsid and provide a reference for understanding the assembly of the inner capsids of nucleocytoplasmic large DNA viruses (NCLDV). IMPORTANCE African swine fever virus has caused catastrophic destruction to the pork industry worldwide since it was first discovered in Kenya in 1921. The architecture of ASFV is complicated, with two protein shells and two membrane envelopes. Currently, mechanisms involved in the assembly of the ASFV inner core shell are less understood. The structural studies of the ASFV inner capsid protein p150 performed in this research enable the building of a partial model of the icosahedral ASFV inner capsid, which provides a structural basis for understanding the structure and assembly of this complex virion. Furthermore, the structure of ASFV p150_△NC represents a new type of fold for viral capsid assembly, which could be a common fold for the inner capsid assembly of nucleocytoplasmic large DNA viruses (NCLDV) and would facilitate the development of vaccine and antivirus drugs against these complex viruses.

Keywords: African swine fever virus; cryogenic electron microscopy; inner capsid assembly; new viral capsid scaffold; nucleocytoplasmic large DNA viruses; virion structure.

FIG 1

Schematic diagrams showing the complex multilayer architectures of ASFV and faustovirus.

FIG 2

Overall structure of p150_△NC. (A) Schematic diagrams (41) showing the organization and proteolytic process of the polyprotein pp220 by the viral-encoded protease pS273R. The precursor, pp220, is colored purple. The proteolytic products p5, p34, p14, p37, p150, and p150_△NC are colored pink, gray, blue, yellow, cyan, and green, respectively. The cleavage sites are shown above the diagram and indicated by red arrows. (B) Overall structure of p150_△NC. (B, Right) Front view. (B, Left) Top view. The structure is shown as a cartoon with the N terminus, C terminus, and some main helices labeled. The helices in the central helix group are colored red, the helices in the edge helix group are colored cyan, and the β strands in the edge β strand group are colored green. (C) Diagrams (42) showing the topology of p150_△NC. The main helices and strands of p150_△NC are shown in the topology. The helices in the central helix group are colored red, the helices in the edge helix group are cyan, and the β strands in the edge β strand group are green. (D) Surface electrostatic potential of p150_△NC. The surface is colored according to the surface electrostatic potential, with red for negative potential and blue for positive potential.

FIG 3

Sequence and structure comparisons of ASFV p150_△NC and faustovirus p150_△NC. (A) Schematic diagrams (41) showing the proteolytic products of the ASFV polyprotein pp220 and the homologous faustovirus polyprotein Fp150_△NC. (B) Superimpositions of the ASFV p150_△NC structure and the predicted Fp150_△NC structure. The crystal structure of ASFV p150_△NC is colored hot pink. The predicted structure of Fp150_△NC is colored deep sky blue. The insertion domain of Fp150_△NC is marked with dashed lines. (C) Comparisons between the 2D class averages of Fp150_△NC and the projections generated from the predicted Fp150_△NC structure. (D) Predicted structure of Fp150_△NC fitted in the cryo-EM map of Fp150_△NC, which was set at a contouring level of 3.5 σ. The central helix group, edge helix group, and edge β strand groups of one monomer are colored red, cyan, and green, respectively. The insertion domain is colored hot pink.

FIG 4

Fitted faustovirus Fp150_△NC in the cryo-EM map of the faustovirus inner core shell. (A) Partial inner core shell structure of faustovirus obtained by fitting the hexametric and pentametric capsomeres of Fp150_△NC into the density map of the faustovirus inner protein shell (EMD-8145) (10). Voxels of the map are colored according to their radical distances to the particle center. The color scale indicates the color scheme for voxels at different distances to the particle center. One triangular facet is indicated by the red triangle in dashed lines. Positions of the icosahedral symmetry axes are indicated with numbers. The subunits in different asymmetry units are in different colors. The h vectors of the hexametric capsomeres are shown on a stripped triangular facet at the right. (B) The hexametric and pentametric capsomeres constituted by Fp150_△NC are fitted into the density map (EMD-8145) of the faustovirus inner protein shell, which was set at a contouring level of 1.9 σ (10). The central helix group, edge helix group, and edge β strand groups of one monomer are colored red, cyan, and green, respectively. The insertion domains are colored hot pink. Other monomers are colored purple.

FIG 5

The fitted p150_△NC in the cryo-EM map of the ASFV inner core shell. (A) Partial inner core shell structure of ASFV obtained by fitting the hexamer and pentamer of p150_△NC into the density map of the ASFV inner core shell extracted from the cryo-EM density map EMD-0815 (4). Voxels of the map are colored according to their radical distances to the particle center. The color scale indicates the color scheme for voxels at different distances to the particle center. One triangular facet is indicated by the red triangle in dashed lines. Positions of the icosahedral symmetry axes are indicated with numbers. The h and k vectors of the hexametric capsomeres are shown on a stripped triangular facet at the right. (B) The hexametric and pentametric capsomers formed by p150_△NC are fitted into the density map of the ASFV inner core shell. (B, Right) One hexametric capsomere constituted by six p150_△NC molecules is fitted into the density map (EMD-0815) of the ASFV core shell, which was set at a contouring level of 3.9 σ (4). The fitting resulted in a CC value of 0.55, with only the symmetry restrained on each monomer. (B, Left) One pentametric capsomere constituted by six p150_△NC molecules is fitted into the density map of the ASFV core shell. The fitting resulted in a CC value of 0.71, with only the symmetry restraint on each monomer. The central helix group, edge helix group, and edge β groups of one monomer are colored red, cyan, and green, respectively. Other monomers are colored purple.

FIG 6

Surface electrostatic potential of the ASFV and faustovirus inner capsid capsomeres. Surface-rendered representations of the ASFV and faustovirus capsomeres. The surfaces are colored according to the electrostatic potential, with red for negative potential and blue for positive potential.

FIG 7

Comparisons of the ASFV and faustovirus inner capsid shells. (A) Images showing the central slices of the icosahedral faustovirus (left) and ASFV (right) inner capsid shells. The thickness of the inner capsid shells is indicated. (B) Comparison of the organization of the capsomeres in the faustovirus and ASFV inner capsid shells.

FIG 8

Structural comparisons of capsid proteins from the four viral capsid lineages. Representative structures from the four viral capsid lineages are compared, including the major capsid protein P3 of the bacteriophage PRD1 (A), which belongs to the adeno-like viral capsid lineage; the capsid proteins VP1 to VP4 of the human poliovirus 1 Mahoney (B), which belongs to the picorna-like viral capsid lineage; the major capsid protein gp5 of the bacteriophage HK97 (C), which belongs to the HK97-like viral capsid lineage; the inner core protein VP3 of the bluetongue virus (BTV1) (D), which belongs to the BTV-like viral capsid lineage (24, 43–45); and the ASFV p150 (E).