Structural basis for DNA replication by the African swine fever virus polymerase

Figures

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  Figure 1. ASFV DNA polymerase β (Pol β) colocalizes with the viral factory in infected cells (A) Immunofluorescence analysis of porcine alveolar macrophages (PAMs) infected with a recombinant ASFV expressing Flag-tagged Pol β. Infected cells are marked by eGFP fluorescence (green). Pol β was visualized using an anti-Flag antibody (red), while nuclei and viral factories were stained with DAPI (blue). Image was taken at 10× magnification. Scale bars = 30 μm. (B) A magnified view (60× magnification) demonstrates the colocalization of Pol β (red) with the viral factory (blue). The viral factory is outlined by a white dashed circle. Colocalization was analyzed using ImageJ. Scale bars = 5 μm.

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  Figure 2. Biochemical and structural characterization of the ASFV Pol β-ΔC (A) Schematic of the domain architecture of the ASFV Pol β-ΔC construct (residues 1–999). The N-terminal domain (NTD), exonuclease (Exo) domain, and the canonical DNA polymerase domains (palm, fingers, thumb) are indicated. (B) Size-exclusion chromatography (SEC) profile of purified Pol β-ΔC, showing a single, symmetrical elution peak consistent with a monomeric state. Inset: Coomassie blue-stained SDS-PAGE gel of the peak fractions confirming protein purity and expected molecular weight. (C) Cryo-EM density map and structural model of apo Pol β-ΔC at an overall resolution of 2.93 Å. Individual domains are colored according to the schematic in (A).

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  Figure 3. Cryo-EM structures of ASFV Pol β-ΔC in apo and DNA-bound states (A) Structural comparison of two distinct apo conformations (state 1 and state 2) of Pol β-ΔC. A conformational shift of ∼6.2° in the thumb domain (colored orange and light blue, respectively) results in a more closed conformation in state 2. (B) Cryo-EM density map and model of the Pol β-ΔC-DNA complex. The protein domains are colored as follows: NTD (yellow), Exo (purple), palm (blue), fingers (orange), and thumb (light blue). The DNA template strand is shown in red, and the primer strand is in green-yellow. Arrows indicate the DNA template entry path toward the polymerase active site. (C) The DNA-binding interface. Left: Surface view showing the primer-template DNA duplex docked to the polymerase. Right: Close-up view highlighting key residues (R342, K344, Y495, T152) that form interactions with the template strand. (D) The polymerase active site shown with a bound dCTP substrate. The incoming dCTP (green-yellow) is positioned against the template base dG-4 (red), coordinated by two Mg2+ ions (yellow spheres) and key residues from the palm and fingers domains. (E) The exonuclease (Exo) active site. The conserved…

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  Figure 4. Conformational changes in ASFV Pol β-ΔC upon DNA binding (A) Structural alignment of Pol β-ΔC in the DNA-bound replicating state (multi-colored) and the apo state (pink). Right: Close-up views show significant conformational changes in the thumb and fingers domains, which move by approximately 32° and 20° respectively to enclose the DNA substrate. (B) Structural comparison of the replicating states of ASFV Pol β-ΔC and the Mpox virus DNA polymerase F8 (PDB: 8HM0). The MPXV F8 structure is colored wheat. Notable differences are highlighted in the thumb, Exo, and fingers domains, particularly the presence of Insert 2 in the thumb domain of F8.

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  Figure 5. Oligomerization is critical for the polymerase activity of ASFV Pol β-ΔC (A) DNA primer extension assays comparing the polymerase activity of wild-type (WT) Pol β-ΔC and an oligomerization-defective triple mutant (R875A/E877A/Q878A). Left: Denaturing urea-PAGE gels showing primer extension products at various enzyme concentrations. The red arrowheads indicate the full-length 38 nt DNA extension product. Right: Quantification of enzyme relative activities. Data are presented as mean ± SD from three independent experiments. Statistical significance was determined by a Student’s t test (∗p < 0.05, ∗∗∗p < 0.001). (B) Representative cryo-EM 2D class averages of Pol β-ΔC bound in the presence of dsDNA, confirming a stable tetrameric assembly. Each panel shows a distinct 2D class. Scale bars = 170 Å. (C) Cartoon representation of the Pol β-ΔC tetramer. The four protomers (A, B, C, and D) are colored blue, orange, light purple, and pink, respectively. (D) Close-up views of the interfaces between protomers. Key residues forming the oligomerization interface, including R875, E877, and Q878, are shown as sticks.