Comparative structural and functional analysis of Bunyavirus and Arenavirus cap-snatching Endonucleases

Segmented negative strand RNA viruses of the arena-, bunya- and orthomyxovirus families uniquely carry out viral mRNA transcription by the cap-snatching mechanism. This involves cleavage of host mRNAs close to their capped 5′ end by an endonuclease (EN) domain located in the N-terminal region of the viral polymerase. We present the structure of the cap-snatching EN of Hantaan virus, a bunyavirus belonging to hantavirus genus. Hantaan EN has an active site configuration, including a metal co-ordinating histidine, and nuclease activity similar to the previously reported La Crosse virus and Influenza virus ENs (orthobunyavirus and orthomyxovirus respectively), but is more active in cleaving a double stranded RNA substrate. In contrast, Lassa arenavirus EN has only acidic metal co-ordinating residues. We present three high resolution structures of Lassa virus EN with different bound ion configurations and show in comparative biophysical and biochemical experiments with Hantaan, La Crosse and influenza ENs that the isolated Lassa EN is essentially inactive. The results are discussed in the light of EN activation mechanisms revealed by recent structures of full-length influenza virus polymerase

Mutational analysis of Hantaan EN <i>in vitro</i> EN activity and thermal stability.

<p><b>A,</b> TSA experiments showing EN stabilization by metal ions and DPBA. Tm values (bars) are shown for mutated Hantaan ENs in comparison with the wild-type with either no ions, 5 mM MgCl₂, 2 mM MnCl₂ or 200 μM DPBA plus 2 mM MnCl₂ and are 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 Hantaan EN mutants with U-rich and G-rich RNAs and <i>Alu</i> SRP RNA in 2 h reactions at room temperature. <b>C,</b> Raw data representation of FRET based EN assays comparing the activity of Hantaan wild-type and mutated ENs at 1 μM protein concentration.</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

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

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

Structure of Hantaan and Lassa ENs in comparison with LACV and Influenza active sites and ion coordination.

<p>Structure of the active sites from ENs showed in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005636#ppat.1005636.g001" target="_blank">Fig 1</a>. The conserved catalytic residues are shown as sticks and the metal ions as yellow (Mn²⁺) or cyan (Mg²⁺) spheres and their coordination by dashed green lines. In all cases the helix αd and the strand βb are labelled. <b>A,</b> Hantaan EN active site. <b>B,</b> IAV EN active site. In this structure (PDB: 4avq) a Mg²⁺ ion substitute the Mn2 found in other published Influenza endonuclease structures. The H41 side chain rotamer was corrected from the original PDB to properly coordinate Mn1 with NE2. <b>C,</b> LACV EN active site. <b>D,</b> Lassa EN active site (form X3). The residue E3, coordinating Mn2 from a symmetry related molecule, is shown in black sticks.</p

Fluorescent FRET based EN assays.

<p>Activity rates at increasing protein concentration of <b>A</b>, Influenza (circles), LACV (diamonds), Hantaan (squares) and <b>B</b>, Lassa (circles) ENs. The experiments were performed with 2 mM MnCl₂ and 500 μM of fluorescent RNA. The velocity of fluorescent increase is plotted in the Y axes and the protein concentration in the X axes. <b>C</b>, Fluorescence based endonuclease assay curves. In red is shown the fold increase of Hantaan endonuclease at 1μM protein concentration with 2 mM MnCl₂ (blue line) 5 mM MgCl₂ (red line) and no ions (black line). <b>D</b>, Same experiment is shown for EN reactions with Lassa EN at 60μM protein concentration and <b>E,</b> for LACV EN at 1 μM protein concentration. <b>F,</b> A figure summary showing in logarithmic scale a bar diagram of panel A and B slopes values (blue bars) and the EN activity rates at 5 mM MgCl₂ (red bars) from endonuclease experiments shown in panels C, D or E normalizing the data to IAV activity with Mn²⁺.</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

Influenza Polymerase Can Adopt an Alternative Configuration Involving a Radical Repacking of PB2 Domains.

International audienceInfluenza virus polymerase transcribes or replicates the segmented RNA genome (vRNA) into respectively viral mRNA or full-length copies and initiates RNA synthesis by binding the conserved 3' and 5' vRNA ends (the promoter). In recent structures of promoter-bound polymerase, the cap-binding and endonuclease domains are configured for cap snatching, which generates capped transcription primers. Here, we present a FluB polymerase structure with a bound complementary cRNA 5' end that exhibits a major rearrangement of the subdomains within the C-terminal two-thirds of PB2 (PB2-C). Notably, the PB2 nuclear localization signal (NLS)-containing domain translocates ∼90 Å to bind to the endonuclease domain. FluA PB2-C alone and RNA-free FluC polymerase are similarly arranged. Biophysical and cap-dependent endonuclease assays show that in solution the polymerase explores different conformational distributions depending on which RNA is bound. The inherent flexibility of the polymerase allows it to adopt alternative conformations that are likely important during polymerase maturation into active progeny RNPs