Effect of mutations on exonuclease activity.

<p>(a) Wild-type, full-length NP or the immunosuppressive domain of NP (NPΔ340) containing one of several point mutants were incubated with 18 bp blunt-ended dsRNA for 15 min, and products were analyzed by PAGE. NP residues that contact the digested strand are indicated with *; NP residues that contact the non-digested strand are indicated with #.’ (b) Time course of dsRNA digestion by wild-type (WT), and Y429A and Y429L mutant NPs. (c) Digestion of 18 bp hybrid DNA:RNA double-stranded oligonucleotides. The labeled strand is denoted by *.</p

Protein-RNA contacts outside of the active site.

<p>(a) The substrate strand, which leads into the active site, forms hydrogen bonds to the NP through the phosphate backbone and by contacts to G8. D426 hydrogen bonds to its nitrogenous base, and the main chain nitrogen of G392 makes hydrogen bonds to the 2′ and 3′OH of its ribose sugar. (b) The non-substrate strand forms hydrogen bonds between the 2′OH of C1 and Q425, and the 2′OH of C4 and D465. There is also a base stacking interaction between C1 and Y429.</p

Location of the basic loop.

<p>Crystal structures of three DEDDh exonucleases are aligned. ε186 (PDB code 1J54 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044211#pone.0044211-Hamdan1" target="_blank">[6]</a>) is illustrated in yellow, Trex2 (PDB code 1Y97 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044211#pone.0044211-Perrino1" target="_blank">[9]</a>) in magenta, and Lassa NP in green. All of these exonucleases contain a basic loop motif. In ε186, the basic motif (K141, R142, R151) lies above and to the right of the active site. In Trex 2, the basic loop is in the same general position, but all residues except for R152 are disordered and indicated by a dotted line. In Lassa NP, no such loop exists in this location and instead strand β5 and helix α5 are directly connected by an IDIAL hydrophobic sequence (residues I482 to L486). Instead, Lassa NP has a basic loop motif in a projecting “arm” located to the left of the active site rather than above and to the right. This loop contains K516, K517, K518 and R519, although the side chains are disordered in this structure.</p

Structure of the active site.

<p>Carbons of Lassa NP residues are colored green. (a) The phosphate backbone of the terminal 3′ nucleotide (G8) is coordinated by a divalent cation (modeled here as a Mg ion, MgB) and by H528. MgB is further coordinated by D389. Due to mutation of E391 to alanine, the second catalytic divalent ion is not bound in the active site. (b) Comparison of the active site of Lassa NP E391A to the active sites of the DEDDh exonucleases ISG-20 (cyan, PDB code 1WLJ <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044211#pone.0044211-Horio1" target="_blank">[5]</a>) and ε186 (yellow, PDB code 1J54 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044211#pone.0044211-Hamdan1" target="_blank">[6]</a>), which were determined in complex with mononucleotides. Both cations are apparent in the structures of ISG-20 and ε186. Amino acid numbering of the shared DEDDh motif is for Lassa NP. (c) A model of wild-type Lassa NP in complex with the terminal G8 mononucleotide. The crystallized ion MgB is drawn in grey. The position of E391 and the cation in site A are based on their positions in the unbound structure (PDB code 3Q7B <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044211#pone.0044211-Hastie1" target="_blank">[3]</a>). Hydrogen bonds are colored red; coordination bonds are colored black; an arrow indicates the position of the next nucleotide in the digested strand. Expected water molecules, based on structural similarity to other DEDDh exonucleases, are drawn in pale blue.</p

Modeling of different forms of DNA.

<p>Idealized B-form dsDNA (blue) and A-form ssDNA (magenta) were modeled and aligned with the dsRNA bound to Lassa NP (green). The 5′ terminal residue of the non-substrate strand (C1) in the B-form dsDNA clashes with Lassa NP, while A-form ssDNA does not.</p

Electron density for the dsRNA.

<p>(a) Final 2Fo-Fc, contoured at +2.0σ, of the dsRNA bound to Lassa NP. The substrate strand, which leads into the active site is colored blue and the non-substrate strand is colored yellow. (b) Stereo view of a simulated-annealing composite omit map of final RNA density, contoured at +2.0σ.</p

Data collection, phasing and refinement statistics.

*<p>Values in parentheses are for highest-resolution shell. _ENREF_3_ENREF_16_ENREF_17_ENREF_18_ENREF_19_ENREF_11_ENREF_13_ENREF_14_ENREF_9_ENREF_15_ENREF_13_ENREF_3</p

Suspected cases of LF evaluated at the KGH Lassa Laboratory and numbers of patients admitted to the KGH Lassa Ward, 2008–12.

<p>Non-admitted patients include those where only blood samples were submitted for screening from referral health-posts, patients dying en route to the hospital (DOA = dead on arrival), and patients not meeting the LF suspected case criteria (<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002748#pntd-0002748-t001" target="_blank">Table 1</a>). Characteristics of study patients are compiled in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002748#pntd.0002748.s002" target="_blank">Table S1</a>.</p

CFRs in suspected LF cases presenting to the KGH Lassa Ward by serostatus, 2008–12.

<p>Panel A: CFR by serostatus. The presence of LASV Ag and anti-LASV IgM in serum of patients with verifiable outcomes was assessed by recombinant Ag− and IgM− capture ELISA, respectively. Panel B: Alternative calculation of CFRs. Ag+/IgM± plus Ag−/IgM+ compared to Ag−/IgM−. Statistical significance was determined using a logistic regression model predicting CFR (<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002748#pntd.0002748.s004" target="_blank">Table S3</a>). NS = not significant.</p

Geographic distribution of patients presenting to the KGH with LASV antigenemia and anti-LASV IgM serpositivity, 2008–12.

<p>Confirmed cases of LF as assessed by LASV Ag in serum or cases anti-LASV IgM are shown by year of presentation, district of residence and frequency of cases. Panel A: Patients presenting in 2008–9. Panel B: Patients presenting in 2010. Panel C: Patients presenting in 2011. Panel D: Patients presenting in 2012.</p