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English⧼Colon⧽ English⧼Colon⧽ a Histogram of LACV-L mini-replicon activity. The percentage of Renilla/Luciferase activity of the constructs containing the N-terminal hexa-histidine tag (N-term tag), the C-terminal hexa-histidine tag (C-term tag), and the California insertion tag (Cl tag) are compared to LACV-L activity of the construct without tag (WT). Tag positions are shown on LACV-L (PDB: 6Z6G) as dotted lines. The polymerase core is colored in light gray with the Cl in orange, the cap-binding domain (CBD) in gold, the endonuclease (ENDO) in green, the mid domain in beige and the zinc-binding domain (ZBD) in brown. n = 3 biologically independent experiments. Error bars are mean ± standard deviation. Source data are provided as a Source Data file. b In vitro replication activity of LACV-LCItag_H34K using different combinations of 5′- and 3′-vRNAs. The assays are done in presence of either the 4 NTPs (AUGC), 3 NTPs (AUG) or 3 NTPs during 4 h, subsequently supplemented with CTP for 30 min (AUG+C). 5′-vRNA containing nucleotides 1 to 17 (5′ vRNA 1-17) or 5′-vRNA with base pair mutation (5′ 1-17BPm) is used. 3′-vRNA contain either nucleotides 1 to 25 (3′ vRNA 1-25) or 1 to 30 (3′ vRNA 1–30). Products have either the expected size or are extended in 5′ with GU or GGU (GU/GGU 5′ elongated products). Replication products and their respective lengths are displayed on the right side of the gel. The decade molecular weight marker (MW) is shown. Source data are provided as a Source Data file. This experiment was repeated independently 3 times with similar results. c Sequence and secondary structures of the RNA bound to LACV-LCItag_H34K for each replication state. 5′-vRNAs, 3′-vRNAs and replication products are respectively colored in pink/gray, gold and cyan. Bases present in the sequences but not seen in the structures are shown in transparent. d Cartoon representation of each LACV-LCItag_H34K replication structure. LACV-L domains are colored as in a. RNAs displayed as surfaces are colored as in a, c. The pre-initiation state corresponds to the PDB 6Z6G.