GoKawiilCells
We maintained Expi293F cells (Thermo Fisher Scientific, A14527) in Expi293 Expression Medium (Thermo Fisher Scientific, A1435101) according to the manufacturer’s instructions. The absence of mycoplasma contamination was verified using the e-Myco PCR detection kit (Bulldog Bio, 25234), with testing performed monthly.
Protein expression and purification
All MPXV protein sequences were derived from MPXV isolate hMPXV/P12/2022 (clade IIb). The sequence encoding full-length MPXV E5 (GenBank: XNX20584.1, residues 1–785), E5(ΔRRM) (GenBank: XNX20584.1, residues 238–785), F8 (GenBank: XNX20538.1, residues 1–1006), F8 with a C-terminal deletion (GenBank: XNX20538.1, residues 1–984) or E4 (GenBank: XNX20583.1, residues 1–218) was cloned into the pCAGGS vector containing a maltose-binding protein (MBP) at the N terminus followed by HRV 3C cleavage site (LEVLFQGP). The sequence encoding wild-type A22 (GenBank: XNX20615.1, residues 1–426) or A22 with the F217A and F263A substitutions (GenBank: XNX20615.1, residues 1–426) were cloned into a pCAGGS vector without tag. Plasmids encoding MPXV polymerase subunits (F8, A22 and E4) were co-transfected into Expi293F cells maintained in suspension using polyethylenimine (PEI) (25000 MW, Polysciences), and E5 was transfected into Expi293F cells grown in suspension using PEI (25000 MW, Polysciences) when the cells reached a density of 2 × 106 cells per ml.
For wild-type MPXV polymerase holoenzyme (F8–A22–E4) and MPXV polymerase holoenzyme mutant purification, after culturing at 37 °C for 72 h, cells were collected by centrifugation at 4,000g for 20 min. Cells were lysed using lysis buffer containing 50 mM HEPES-NaOH, pH 7.5, 300 mM NaCl, 0.5% (v/v) Triton X-100, 5 mM MgCl 2 , 0.5 mM EDTA, 1 mM DTT and protease inhibitor (cOmplete, Mini, EDTA-free protease inhibitor cocktail, Millipore Sigma, 11836170001). Cell debris was removed through centrifugation 50,000g for 2 h on the Ti50.2 rotor. The supernatant was incubated with amylose resin (NEB, E8021S) at 4 °C for 1 h and washed with wash buffer (25 mM HEPES-NaOH, pH 7.5, 300 mM NaCl, 5 mM MgCl 2 , 0.5 mM EDTA and 1 mM DTT). Bound MPXV F8–A22–E4 complex was subjected to on-column digestion overnight with HRV-3C protease (TaKaRa, 7360) at 4 °C. The eluate fractions within the elution buffer (25 mM HEPES-NaOH, pH 7.5, 150 mM NaCl, 2 mM MgCl 2 and 1 mM DTT) were concentrated to 500 μl for further purification using Superdex 200 increase column (Cytiva, 28990944). Fractions containing MPXV polymerase holoenzyme were pooled, concentrated to about 1.5 μg μl−1 and stored for the further structural and functional studies. For exonuclease-defective polymerase mutant, residues Asp166 and Glu168 in F8 were substituted to alanine using site-directed mutagenesis of the wild-type F8 pCAGGS vector, and the same purification strategy was used to purify the polymerase holoenzyme.
For MPXV E5 or E5(ΔRRM) purification, after culturing at 37 °C for 72 h, cells were collected by centrifugation at 4,000g for 20 min. Cells were lysed using lysis buffer containing 50 mM HEPES-NaOH, pH 7.5, 500 mM NaCl, 0.5% (v/v) Triton X-100, 5 mM MgCl 2 , 0.5 mM EDTA, 1 mM DTT and protease inhibitor (cOmplete, Mini, EDTA-free protease inhibitor cocktail, Millipore Sigma, 11836170001). Cell debris was removed through centrifugation 50,000g for 2 h with Ti50.2 rotor. The supernatant was incubated with amylose resin (NEB, E8021S) at 4 °C for 1 h and washed with wash buffer (25 mM HEPES-NaOH, pH 7.5, 500 mM NaCl, 5 mM MgCl 2 , 0.5 mM EDTA and 1 mM DTT). Bound MPXV E5 or E5(ΔRRM) were subjected to on-column digestion overnight with HRV-3C protease (TaKaRa, 7360) at 4 °C. The eluate fractions with the elution buffer (25 mM HEPES-NaOH, pH 7.5, 150 mM NaCl, 2 mM MgCl 2 and 1 mM DTT) were concentrated to 1 ml for further purification using the Superdex 200 increase column (Cytiva, 28990944). The fractions containing MPXV E5 or E5(ΔRRM) hexamers were pooled, concentrated to about 3 μg μl−1 and stored for the further structural and functional studies. For the primase-dead E5 mutant, Asp70 in E5 was substituted to alanine using site-directed mutagenesis of the wild-type E5 pCAGGS vector, and the same purification strategy was used. All purified proteins and complexes were analysed using SDS–PAGE.
MPXV replisome assembly
To determine the structure of the ssDNA-bound MPXV replisome, we used an 80 nucleotide DNA template (5′-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGGCTCCCGCGTCGGAGTCGTTTCGACTCCGACGCGGGAGC-3′) (template 1) (Extended Data Fig. 1c and Supplementary Table 2). The E5 helicase–primase, polymerase (F8–A22–E4) and DNA were mixed at 1:1.2:1.2 molar ratio in a buffer containing 25 mM HEPES-NaOH, pH 7.5, 150 mM NaCl, 2 mM MgCl 2 and 1 mM DTT overnight. The mixture was applied onto the Superdex 200 increase column (Cytiva, 28990944). The fractions containing MPXV replisome were pooled and concentrated to about 0.3 μg μl−1 for subsequent analyses.
To determine the structure of the forked DNA-bound MPXV replisome, we used an 83 nucleotide DNA template (5′-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGTCGGAGTCGTTTCGACTCCGACTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-3′) (template 2) (Extended Data Fig. 5b and Supplementary Table 2). The E5 helicase–primase, polymerase (F8–A22–E4) and DNA were mixed at 1:1.2:1.2 molar ratio in a buffer containing 25 mM HEPES-NaOH, pH 7.5, 150 mM NaCl, 2 mM MgCl 2 and 1 mM DTT overnight. The mixture was applied onto the Superdex 200 increase column (Cytiva, 28990944). The fractions containing MPXV replisome were pooled and concentrated to about 0.3 μg μl−1 for subsequent analyses.
To determine the structure of the MPXV replisome bound to RNA–DNA hybrid, we used a 58 nucleotide DNA oligo (5′-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGCTCCCGCGTCGGAGTCG-3′) and an 18 nucleotide RNA oligo (5′-CGACUCCGACGCGGGAGC-3′) (template 3) (Extended Data Fig. 9b and Supplementary Table 2). The E5 helicase–primase, polymerase (F8–A22–E4) and RNA–DNA hybrid were mixed at 1:1.2:1.2 molar ratio in a buffer containing 25 mM HEPES-NaOH, pH 7.5, 150 mM NaCl, 2 mM MgCl 2 and 1 mM DTT with overnight incubation. The mixture was passed onto the Superdex 200 increase column (Cytiva, 28990944). The fractions containing MPXV replisome were pooled and concentrated to about 0.3 μg μl−1 for subsequent structural analysis.
... continue reading