Structural basis for the assembly of the DNA polymerase holoenzyme from a monkeypox virus variant

Abstract

The ongoing global pandemic caused by a variant of the monkeypox (or mpox) virus (MPXV) has prompted widespread concern. The MPXV DNA polymerase holoenzyme, consisting of F8, A22, and E4, is vital for replicating the viral genome and represents a crucial target for the development of antiviral drugs. However, the assembly and working mechanism for the DNA polymerase holoenzyme of MPXV remains elusive. Here, we present the cryo-electron microscopy (cryo-EM) structure of the DNA polymerase holoenzyme at an overall resolution of 3.5 Å. Unexpectedly, the holoenzyme is assembled as a dimer of heterotrimers, of which the extra interface between the thumb domain of F8 and A22 shows a clash between A22 and substrate DNA, suggesting an autoinhibition state. Addition of exogenous double-stranded DNA shifts the hexamer into trimer exposing DNA binding sites, potentially representing a more active state. Our findings provide crucial steps toward developing targeted antiviral therapies for MPXV and related viruses.

Fig. 1.. Biochemical characteristics of DNA replication machine of MPXV.

(A) Domain organization of F8L. Mutations in the 2022 WA strain are marked by red lines. (B) Representative SEC purification of the F8-A22-E4 complex. SDS-PAGE was visualized by Coomassie blue staining, and fractions for cryo-EM analysis were marked by red line. (C) Overall surface presentation of domain-colored cryo-EM structures of DNA replication machine of monkeypox. F8, A22, and E4 in protomer A are colored gold, blue, and cyan, respectively, and the other protomer is colored green, purple, and pink.

Fig. 2.. The multiple interaction interfaces in the F8-A22-E4 hexameric complex.

(A) Overall structure of the F8-A22-E4 complex. Interaction interfaces between F8 and A22 in one protomer are lined out by red and blue boxes, which are shown in (B). The interacted residues in F8 and E4 are lined out by black box and shown in (C). (D) Structural comparison with E9 DNA polymerase from VACV (PDB ID: 5N2E) shows that the thumb domain is regulated by the interaction of A22′. F8, E4, A22, and A22′ are colored gold, cyan, blue, and purple, respectively. E9 is colored yellow green.

Fig. 3.. The models of different DNA replication machines.

(A) Model of DNA polymerase holoenzyme from MPXV. (B) Archaeal and phage DNA polymerase. The model was drawn according to polymerase structure (PDB ID: 1CLQ) and PCNA structure (PDB ID: 1B8H). (C) HSV DNA polymerase. The model was drawn according to UL42 structure (PDB ID: 1DML) and UL30 structure (PDB ID: 2GV9). (D) Saccharomyces cerevisiae polymerase. The model was drawn according to a complex structure (PDB ID:7KC0). (E) Structural comparison with DNA polymerase delta of yeast (PDB ID: 3IAY) and D4-A20 complex of VACV (PDB ID: 4YIG) shows that there would be a clash between DNA and F8 or A22′. F8, A22, A22′, and E4 are colored gold, blue, purple, and cyan, respectively. D4 and A20 of VACV are colored pink and magenta. DNA polymerase delta of yeast is colored dark sea green. The template and primer chains of DNA are colored red and blue.

Fig. 4.. The putative working model of the F8-A22-E4 complex.

(A) The oligomeric state shift of the F8-A22-E4 complex from hexamer to trimer can be induced by incubation with dsDNA. The peaks of hexamer and trimer are marked by blue line and red line, respectively, on SEC analysis. The major peak of the F8-A22-E4 complex shifts backward when incubated with dsDNA. (B) Left: Map and model of trimeric form complex. The map of trimeric form complex is colored light salmon. The model styled cartoon and F8, A22, and E4 are colored gold, blue, and cyan, respectively. Middle and right: Map comparison of the trimeric and hexameric form complex shows that A22 undergoes marked conformational change during oligomer state shift. (C) Proposed model for autoinhibition, activation, and the DNA replication mechanism catalyzed by the F8-A22-E4 complex.

Fig. 5.. Analysis of drug inhibitors on E9 and F8.

(A) Molecular docking of CDVpp binding mode in E9 and F8, respectively. The interaction residues are shown as sticks. E9 and F8 are colored split pea and yellow, respectively. The structure on the right is the comparative analysis of dCTP (light magenta) and CDVpp in E9 and F8, respectively. (B) Molecular docking of Ara-CTP in E9 and F8. The interaction residues are shown as sticks. The alignment analysis on the right is also the comparative analysis of dCTP (light magenta) and Ara-CTP in E9 and F8, respectively.