Predicted putative CBD of SNV L is less similar to LACV L CBD.

Structural comparison of the putative cap-binding domain within the C-terminal region of the hantavirus SNV L protein as predicted by AlphaFold2 [34] and the known structure of the phenuivirus LACV L cap-binding domain (from PDB 7ORL). The side chains that may be involved in forming the aromatic sandwich to bind the cap are shown as orange sticks. The black dotted line corresponds to the distance between these two side chains as measured in PyMol. (TIFF)</p

Alignment of <i>Hantaviridae</i> and <i>Peribunyaviridae</i> full-length L protein sequences.

The L protein sequences of New World hantaviruses Sin Nombre virus (SNV, KT885044.1), Andes virus (ANDV, QRY27107.1), Bayou virus (BAYV, K7N869), and Black creek canal virus (BCCV, V5IVB1); Old World hantaviruses Hantaan virus (HTNV, ABD28179.1), Puumala virus (PUUV, ABN51178.1), Tula virus (TULV, A0A481S3H6), and Seoul virus (SEOV, A0A0B5JFL8), and peribunyaviruses Bunyamwera virus (BUNV, A0A0A7KU93), and La crosse virus (LACV, A5HC98) were aligned using Clustal Omega [47]. Manual adjustments were made and the alignment was visualized with ESPript 3 [48] Shown is a comparison of the secondary structure elements of SNV L and LACV L (PDB 6Z6G). (PDF)</p

Putative cap-binding domain predicted within the C terminus of SNV L.

(A) The structure of the C-terminal region of SNV L was predicted using AlphaFold2, visualized in PyMol and searched for a structural motif resembling the published cap-binding domains of bunyaviruses and orthomyxoviruses. The predicted structural model is colored based on the confidence as determined by AlphaFold2’s predicted local distance difference test (pLDDT score). A residue with a pLDDT score greater than 90 (blue) indicates high estimated accuracy of the position of its backbone and sidechain rotamers. A pLDDT score above 70 (cyan) suggested that the prediction of the backbone is confident. (B) Structural comparison of the putative cap-binding domain within the predicted C-terminal region of the hantavirus SNV L protein and the known structure of the phenuivirus RVFV L cap-binding domain (PDB 6QHG). The black dotted line indicates the distance measurement between the two aromatic amino acid side chains shown as orange sticks, that may be involved in forming the aromatic sandwich to bind the cap.</p

Image processing strategy to obtain SNV L bound to 5′ 1–18 RNA cryo-EM map.

(A) Display of a representative micrograph after MotionCor2. The scale bar corresponds to 200 nm. (B) Representative 2D class averages. (C) 3D class averages of the 2nd 3D classification. The surrounded 3D class contains the particles selected for the last 3D reconstruction. The presence of the 5’ 1–18 RNA forming the hook is indicated. The percentage of particles and the corresponding number is indicated below each class (D) Final reconstruction is displayed. Electron density map is colored according to the local resolution. Fourier Shell Correlation curves (FSC) and angular distribution of particles used in the final reconstruction are displayed. (TIFF)</p

<i>In vitro</i> endonuclease actictivity with different RNA substrates.

A 10 μL reaction containing 0.5 μM full-length SNV L K124A, radiolabeled RNA substrate, and 0.1 mM (low MnCl2) or 10 mM (high) MnCl2 or MgCl2 in reaction buffer was incubated at 37°C for one hour. The reactions were stopped by adding 10 μL of 2x RNA loading dye (98% formamide, 18 mM EDTA, 0.025 mM SDS, xylene cyanol, bromophenol blue) and heated to 95°C for five minutes. RNA was separated on denaturing PAGE (25% acrylamide, 7 M urea, 0.5 x TBE) and visualized by autoradiography. The control lane shows a reaction without the addition of SNV L K124A. (TIFF)</p

Cryo-EM data collection, refinement and validation statistics.

Cryo-EM data collection, refinement and validation statistics.</p

Primer-elongation RdRp activity of SNV L K124A.

(A) SNV L segment 3′ RNA 1-18P RNA (3′-P-AUCAUCAUACGAGGCUCU-5′) was incubated with a tenfold excess of the indicated primers for 15 minutes prior to the addition of L protein and NTPs supplemented with [α]32P-GTP. Reactions were incubated at 30°C for one hour. Reaction products were separated by denaturing PAGE and signals were visualized via phosphor screen autoradiography. The lane marked with (-) shows an unprimed reaction. (B) SNV L was incubated with SNV L segment 5′ RNA 1-13P (5′-UAGUAGUAGACUC-P-3′) as indicated for 15 minutes prior to the addition of equimolar concentrations of SNV L segment 3′ RNA 1-18P and primers as indicated. The assay was carried out as described above. The products marked with asterisks were likely caused by the underlined binding site for the AGU primer on the 5′ RNA 1-13P. (C) Primer elongation activity of SNV L RdRp in the absence of different nucleotides. The radioactively labeled AGU primer was used for product detection instead of [α]32P-GTP. The dotted lines indicate cutting of the lanes for presentation purposes. The contrast of the marker lane in (C) was adjusted as the intensity of the marker was higher than that of the product bands. (D) Sequence of the template RNA and potential primer annealing sites including the resulting product length.</p

Density corresponding to RNA residues 2–12 of SNV L 5 1–18 RNA.

The RNA was built de novo in Coot [45] and was displayed as sticks in PyMol with the density displayed as mesh, contoured at 2.0 sigma within 1.5 Å of the displayed atoms. (TIFF)</p

Comparison of bunyavirus L protein CBD sequences.

The L protein CBD sequences of hantaviruses Sin Nombre virus (SNV, KT885044.1) and Hantaan virus (HTNV, ABD28179.1), phenuiviruses Rift Valley fever virus (RVFV, A2SZS3) and Severe fever with thrombocytopenia syndrome virus (SFTSV, I0DF35), and peribunyaviruses Bunyamwera virus (BUNV, A0A0A7KU93), and La crosse virus (LACV, A5HC98) were aligned using Clustal Omega [47]. Manual adjustments were made and the alignment was visualized with ESPript 3 [48] Shown is a comparison of the secondary structure elements of the predicted SNV L CBD and LACV L CBD (PDB 6Z6G). (B) The sequence identity and similarity between the different CBD within the alignment shown in (A) were analysed using the Sequence Identity And Similarity tool (http://imed.med.ucm.es/Tools/sias.html). (TIFF)</p

SNV and LACV L protein 5′ RNA binding sites.

Comparison between 5′ RNA binding to (A) SNV L and (B) LACV L (PDB 5AMQ). The 5′ RNA binding sites are shown as cartoon representations with domains colored according to Fig 6, and RNA is shown as orange sticks. Amino acid side chains potentially involved in protein-RNA binding are shown as sticks.</p