Cryo-EM structure of the EBV ribonucleotide reductase BORF2 and mechanism of APOBEC3B inhibition

Viruses use a plethora of mechanisms to evade immune responses. A recent example is neutralization of the nuclear DNA cytosine deaminase APOBEC3B by the Epstein-Barr virus (EBV) ribonucleotide reductase subunit BORF2. Cryo-EM studies of APOBEC3B-BORF2 complexes reveal a large >1000-Å2 binding surface composed of multiple structural elements from each protein, which effectively blocks the APOBEC3B active site from accessing single-stranded DNA substrates. Evolutionary optimization is suggested by unique insertions in BORF2 absent from other ribonucleotide reductases and preferential binding to APOBEC3B relative to the highly related APOBEC3A and APOBEC3G enzymes. A molecular understanding of this pathogen-host interaction has potential to inform the development of drugs that block the interaction and liberate the natural antiviral activity of APOBEC3B. In addition, given a role for APOBEC3B in cancer mutagenesis, it may also be possible for information from the interaction to be used to develop DNA deaminase inhibitors.


The PDF file includes:
Figs. S1 to S13 Table S1 Legends for movies S1 and S2

Other Supplementary Material for this manuscript includes the following:
Movies S1 and S2   Select view of the BORF2 LLI insertion and the corresponding regions of the human RNR a subunit (pdb: 6aui, chain A; tan) and the E. coli RNR a subunit (pdb:6w4x, chain B; green). Cryo-EM map (left) for the LLI region of BORF2 (blue mesh). (B) Ribbon schematics showing connectivity between BORF2 insertions LLI and SHI and A3Bctd. The left schematic emphasizes core interactions and the right schematic provides additional BORF2 structure including the SHI. BORF2 residues that interact with A3Bctd are labeled. (C) A faded ribbon schematic showing the extensive interactions that occur between the SHI and LLI and also between these novel insertions and the protein core. Top: Cryo-EM structure of EBV-BORF2 in comparison to AlphaFold-predicted structures of the RNR large subunit from EBV (light blue), KSHV (yellow), and HSV-1 (pink). The actual and predicted SHI and LLI domain regions depicted in red. Bottom: Multiple sequence alignment of two regions of EBV BORF2 (GenBank V01555.2), KSHV ORF61 (GenBank QFU18774.1), and HSV-1 ICP6 (GenBank QFQ61410.1). BORF2 SHI and LLI domain regions are colored red. Sequence alignment was done using Clustal Omega multiple sequence alignment tool. (A) Structures of A3Bctd (this study), A3A-ssDNA (pdb: 5sww), and A3Gctd-ssDNA (pdb: 6bux). L1, L3, and L7 regions are labelled. The space-filled representations above highlight the proximity of L1/L3/L7 to the ssDNA binding pocket. (B) Protein sequence alignment of A3Bctd, A3A, and A3Gctd. Identical residues are colored with blue boxes, and the loop 1, 3, and 7 regions are highlighted in yellow. Sequence alignment was done using Clustal Omega multiple sequence alignment tool.

Fig. S6. Summary of key interactions between BORF2 and A3Bctd.
(A) Zoom-in of residues involved in interaction between BORF2 and A3Bctd with alternative views shown in the middle and right. BORF2 residues are colored blue and red, and A3Bctd residues are orange. Residues are depicted as sticks. Cryo-EM map represented by blue mesh. (B) A zoom-in of the BORF2 SHI (blue/red) and A3Bctd L7 region (orange). Ribbon and stick schematic on the left, and the same view with a surface-filled representation of A3Bctd on the right. (C) A zoom-in of BORF2 residue 481 (blue) and A3Bctd L1 region (orange). Ribbon and stick schematic on the left, and the same view with a surface-filled representation of A3Bctd on the right. (D) BORF2 (anti-FLAG) co-IP experiments with the indicated A3-eGFP constructs including key A3Bctd mutants. The two A3A constructs were non-informative in this experiment but still included in the images here to avoid gel-cropping and allow visualization of the negative control reactions (empty +/-BORF2-FLAG). See Fig. 3D for additional BORF2 mutants.

Fig. S8. N-terminal ATP cone-domain is absent in EBV BORF2 despite regulatory functions in other class1a RNRs.
(A) Protein schematic of EBV BORF2, human RNR a-subunit, and E. coli RNR a-subunit with key domains indicated. (B) Surface representations of the active and inactive forms of the E. coli RNR (pdb: 6w4x and pdb: 3uus, respectively). The a-subunit is depicted in green and gray, and the b-subunit in pink and maroon. The active form is an a2/b2 complex and dATP binding triggers the formation of an inactive a4/b4 ring complex. The 90º rotation shows the canonical a/a dimer that exists in both complexes including dATP (spheres) bound to the cone domain. (C) Surface representation of the dATP bound human RNR ring complex (pdb: 6aui). This complex is comprised of two different dimeric forms of the RNR a domain -a canonical dimer (right) and an ATP cone-mediated a-a dimer (below).   Surface representation and ribbon schematic of full-length A3B (purple and grey; downloaded from the AlphaFold Structure Database https://alphafold.ebi.ac.uk/) overlaid on the BORF2-A3Bctd cryo-EM structure (colored blue and orange as in other figures). The full-length A3B model is shaded such that the N-terminal domain (ntd) of A3B is purple and the C-terminal domain (ctd) is grey and placed over the actual A3Bctd structure in orange.

helices.
Overlay of the BORF2 cryo-EM structure (darker blue) with the AlphaFold-predicted BORF2 structure (lighter blue). Right: close-up view of the predicted canonical dimerization region.