Comparative nuclease reaction and ion binding experiments for sNSV.

<p><b>A</b>, Tm values derived from TSA experiments shown in bar diagrams for each EN in the presence of 2 mM of each indicated metal ion. <b>B</b>, Stabilization effects of 5 mM MgCl₂ (Mg5), 2 mM MnCl₂ (Mn2), and the super shift induced by 200 μM DPBA inhibitor (Mn2DPBA). <b>C,</b> Comparative nuclease activity assay of the indicated ENs in 2 h reactions at room temperature with G-rich RNA in the presence or absence of metal ions and DPBA inhibitor, all reactions were performed in parallel. <b>D</b>, Comparative nuclease activity assay with three distinct RNA substrates in 2 h reactions at room temperature, non-structured U-rich and G-rich RNA and highly structured <i>Alu</i> SRP RNA.</p

Lassa EN structures, flexibility and metal ion binding.

<p><b>A,</b> Cartoon representation of the Lassa X1 and X3 structures after superposition. The α-helical bundles of X1 and X3 are respectively coloured in light and dark blue. The catalytic loop is highlighted in green and the Mn²⁺ ions of X3 are yellow spheres. The α-helical bundle closes the active site by the indicated rotation (blue arrow). The Lassa X2 structure has the same conformation as X3 (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005636#ppat.1005636.s005" target="_blank">S5A and S5B Fig</a>). <b>B,</b> The same structure superposition as <b>A</b>, with the active site residues as sticks, the X3 Mn²⁺ metal ions as yellow spheres and the ion coordination indicated by dashed green lines. <b>C,</b> Comparison of the Lassa X1 structure (light blue) and the previously reported Lassa EN structure in complex with Mg²⁺ [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005636#ppat.1005636.ref016" target="_blank">16</a>] (light pink, PDB: 4MIW). The Mg²⁺ ions are shown as cyan spheres. <b>D,</b> The same superposition as <b>C,</b> with the active site residues as sticks and the bridging water molecules as small red spheres. The ion coordination and hydrogen bond network is shown by green dashed lines.</p

Crystal structure of Hantaan and Lassa ENs in comparison with LACV and Influenza.

<p>Cartoon representation of the crystal structures of Hantaan, Lassa (form X3), LACV (PDB: 2xi5) and IAV (PDB: 4avq) cap-snatching ENs. The alpha helices are coloured in light pink, the beta strands in light blue and the loops in light grey. The conserved flexible loop involved in the active site is highlighted in green. The same nomenclature is used to label homologous secondary structures. The metal ions are shown as yellow (Mn²⁺) or cyan (Mg²⁺) small spheres. The catalytic metal ions are labelled (Mn1-Mn2/Mg2) indicating the active site position. Note that the orientation of the catalytic ions changes between arenavirus Lassa EN and the rest of ENs.</p

Data refinement and collection statistics.

<p>In parenthesis are indicated the values for the last shell. The additives used in the sample and cryoprotectant solution are also indicated.</p

Mutational analysis of Lassa EN minireplicon transcriptional activity and thermal stability.

<p><b>A,</b> TSA experiments showing the stabilization by metal ions and DPBA. Tm values (bars) are shown for mutated Lassa EN in comparison with the wild-type with no ions, 5 mM MgCl₂, 2 mM MnCl₂ or 200 μM DPBA plus 2 mM MnCl₂, coloured as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005636#ppat.1005636.g004" target="_blank">Fig 4B</a>. <b>B,</b> Nuclease activity assays of Lassa EN mutants in 1 h reactions at room temperature with U-rich and G-rich RNAs and <i>Alu</i> SRP RNA using LACV and Hantaan ENs as positive control. The LCMV EN (NL1 construct [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005636#ppat.1005636.ref013" target="_blank">13</a>]), another His- EN, is also included in the experiment. <b>C,</b> Transcription and/or replication activity of L protein mutants was measured via Ren-Luc reporter gene expression. The Ren-Luc activity is shown in the bar graphs with mean standard deviation [n = 5] of standardized relative light units [sRLU] as a percentage of the wild-type (WT). A defective L protein with a mutation in the catalytic site of the RdRp served as a negative control (neg.). An activity less than 3% of the wild-type is considered as inactive, activities between 3 and 40% are intermediate and mutants with activity above 40% count as wild-type-like. <b>D,</b> Synthesis of antigenome and Ren-Luc mRNA was evaluated by Northern blotting using a radiolabelled riboprobe hybridizing to the Ren-Luc gene. As a marker for gel loading and RNA-transfer the methylene blue stained 28S rRNA is shown. Immunoblot bands of FLAG-tagged L protein mutants give proof of protein expression.</p

Structural insights into reptarenavirus cap-snatching machinery

<div><p>Cap-snatching was first discovered in influenza virus. Structures of the involved domains of the influenza virus polymerase, namely the endonuclease in the PA subunit and the cap-binding domain in the PB2 subunit, have been solved. Cap-snatching endonucleases have also been demonstrated at the very N-terminus of the L proteins of mammarena-, orthobunya-, and hantaviruses. However, a cap-binding domain has not been identified in an arena- or bunyavirus L protein so far. We solved the structure of the 326 C-terminal residues of the L protein of California Academy of Sciences virus (CASV), a reptarenavirus, by X-ray crystallography. The individual domains of this 37-kDa fragment (L-Cterm) as well as the domain arrangement are structurally similar to the cap-binding and adjacent domains of influenza virus polymerase PB2 subunit, despite the absence of sequence homology, suggesting a common evolutionary origin. This enabled identification of a region in CASV L-Cterm with similarity to a cap-binding site; however, the typical sandwich of two aromatic residues was missing. Consistent with this, cap-binding to CASV L-Cterm could not be detected biochemically. In addition, we solved the crystal structure of the corresponding endonuclease in the N-terminus of CASV L protein. It shows a typical endonuclease fold with an active site configuration that is essentially identical to that of known mammarenavirus endonuclease structures. In conclusion, we provide evidence for a presumably functional cap-snatching endonuclease in the N-terminus and a degenerate cap-binding domain in the C-terminus of a reptarenavirus L protein. Implications of these findings for the cap-snatching mechanism in arenaviruses are discussed.</p></div

Atomic structure of isolated CASV L-Cterm domain 2.

<p><b>A)</b> Ribbon diagram of CASV L-Cterm domain 2 structure. Chain A is shown in palegreen, chain B in grey. The N- and C-termini are marked and potential cap-binding aromatic sidechains Y1872 and W1818 are shown as sticks and colored in orange. <b>B)</b> Superimposition of SAXS derived molecular shape of L-Cterm at a concentration of 4.5 mg/ml and ribbon diagram of crystal structure. <b>C)</b> Superimposition of ribbon diagrams of chain A and B from isolated CASV L-Cterm domain 2 crystal structure (magenta and yellow, respectively) and L-Cterm crystal structure (green). Potential cap-binding aromatic sidechains are highlighted with saturate colors. <b>D)</b> Representation of chain B of L-Cterm domain 2 colored by B-factor with the highest observed B being 106 (orange) and the lowest 22 (dark blue).</p

Comparison of CASV L-Cterm structure with influenza PB2 (PDB ID 5FMM) structure.

<p><b>A)</b> Comparison of domain arrangements within PB2 and L-Cterm. Identifiers of the areas within the protein are shown in the bars. Domain 1 (D1) of L-Cterm is separated into three parts (D1-I, D1-II, and D1-III). Linkers to domain 2 or the cap-binding domain are colored in yellow. Residue numbers of the differently colored areas are given below the bars. N- and C-termini are labelled. C-terminal parts of PB2 missing in the figure are indicated by dashed lines. <b>B)</b> Structures of parts I and II of L-Cterm domain 1 (left panel) and the mid-link domains of PB2 (right panel) are shown as ribbon diagrams. Colors are coded as presented in A). Linkers to domain 2 and the cap-binding domain are shown in yellow. N-termini are labelled. <b>C)</b> Comparison of L-Cterm domain 2 (left panel) with PB2 cap-binding domain (right panel) with structures presented as ribbon diagrams. Structurally similar elements have similar colors. Linkers to other domains are colored in yellow. <b>D)</b> Structural comparison of parts II and III of L-Cterm domain 1 (left panel) and link-627 domains of PB2 (right panel). Structures are shown as ribbon diagrams. Colors are coded as in A). β-strands of part III of L-Cterm domain 1 and 627-domain of PB2 are colored in red. C-termini are labelled.</p