Atomic Structure and Biochemical Characterization of an RNA Endonuclease in the N Terminus of Andes Virus L Protein

<div><p>Andes virus (ANDV) is a human-pathogenic hantavirus. Hantaviruses presumably initiate their mRNA synthesis by using cap structures derived from host cell mRNAs, a mechanism called cap-snatching. A signature for a cap-snatching endonuclease is present in the N terminus of hantavirus L proteins. In this study, we aimed to solve the atomic structure of the ANDV endonuclease and characterize its biochemical features. However, the wild-type protein was refractory to expression in <i>Escherichia coli</i>, presumably due to toxic enzyme activity. To circumvent this problem, we introduced attenuating mutations in the domain that were previously shown to enhance L protein expression in mammalian cells. Using this approach, 13 mutant proteins encompassing ANDV L protein residues 1–200 were successfully expressed and purified. Protein stability and nuclease activity of the mutants was analyzed and the crystal structure of one mutant was solved to a resolution of 2.4 Å. Shape in solution was determined by small angle X-ray scattering. The ANDV endonuclease showed structural similarities to related enzymes of orthobunya-, arena-, and orthomyxoviruses, but also differences such as elongated shape and positively charged patches surrounding the active site. The enzyme was dependent on manganese, which is bound to the active site, most efficiently cleaved single-stranded RNA substrates, did not cleave DNA, and could be inhibited by known endonuclease inhibitors. The atomic structure in conjunction with stability and activity data for the 13 mutant enzymes facilitated inference of structure–function relationships in the protein. In conclusion, we solved the structure of a hantavirus cap-snatching endonuclease, elucidated its catalytic properties, and present a highly active mutant form, which allows for inhibitor screening.</p></div

Nuclease activity of ANDV L_1–200 protein mutants.

<p><b>A</b>, Enzyme was incubated with a radioactively labeled 27mer ssRNA in the presence and absence of 2 mM MnCl₂. Substrate and reaction products were separated on a denaturing polyacrylamide gel. <b>B</b>, Signals for residual uncleaved substrate as shown in panel A were quantified using a phosphorimager. <b>C</b>, Substrate preference of the enzyme. ANDV L_1–200 N167A, K44A, and D97E (as a negative control) were incubated with various substrates of identical length and sequence but different structure: single stranded (ss) and double stranded (ds) RNA and DNA. <b>D</b>, Enzyme activity with structured ssRNA compared to unstructured (polyA) ssRNA. Both substrates were tested as 27mer and 40mer.</p

Stabilization and inhibition of ANDV endonuclease by a known endonuclease inhibitor.

<p><b>A</b>, Thermal stability of ANDV L_1–200 proteins in the presence of 16 mM MnCl₂ and 100 μM 2,4-dioxo-4-phenylbutanoic acid (DPBA) measured in thermofluor assay (T_m, melting temperature). <b>B</b>, RNA nuclease assay with ANDV L_1–200 N167A in the presence of 2 mM MnCl₂ and increasing concentrations of DPBA. The inhibitor was incubated with the enzyme for 15 min prior to the addition of the 27mer ssRNA substrate. The assay was performed for 1 h at 37°C. Substrate and reaction products were separated on a denaturing polyacrylamide gel. <b>C</b>, Signals for residual uncleaved substrate as shown in panel A were quantified using a phosphorimager.</p

Surface charge distribution and active site arrangement of ANDV endonuclease compared to other cap-snatching endonucleases.

<p><b>A</b>, Electrostatic surface potential of the endonucleases of ANDV, LACV, and LCMV. The active site is marked with a black star, the location of helix αe in the ANDV structure is marked with a black arrow, sulfate and glycerol molecules bound in the basic groove of ANDV endonuclease are shown as spheres. The surface potential is shown from -5 KT/e in red to +5 KT/e in blue. <b>B</b>, Superimposition of the side chains of active site residues and the Mn²⁺ ions of ANDV, LACV, and IAV endonucleases. <b>C,</b> Manganese coordination in the active site of ANDV endonuclease. Metal coordinating residues are shown as sticks and coordination with dotted lines. The anomalous difference Fourier map for the manganese atom is shown as orange mesh at 3σ. <b>D,</b> Electron density for a sulfate and a glycerol molecule close to the active site of the ANDV endonuclease (2|Fo|-|Fc| map at 2σ is shown as blue mesh; ligands and coordinating side chains are shown as sticks). <b>E</b>, Tyr32 and Arg35 (in red), which coordinate the glycerol ligand in the ANDV L_1–200 structure, are conserved in hantaviruses, but not present in IAV. LACV only possesses the Arginine. The catalytic His36 and the stabilizing Asp37 are shown in bold.</p

Atomic structure of ANDV L_1–200 K127A and comparison with related cap-snatching endonucleases.

<p><b>A</b>, Ribbon diagram of the ANDV L_1–200 K127A structure. N and C termini are marked and secondary structure elements are annotated in analogy to related published structures. The conserved structural core is colored in orange, the conserved helix-bundle domain in green, and the remaining helical part in yellow. The manganese ion is shown as red sphere. Side chains of the active site residues and bound sulfate and glycerol molecules are shown as sticks. <b>B</b>, Comparison of ANDV L_1–200 K127A with the cap-snatching endonucleases of orthobunyavirus LACV (PDB ID 2XI7), arenavirus LCMV (PDB ID 3JSB), and orthomyxovirus IAV (PDB ID 2W69). Structural elements are colored as in A. <b>C</b>, Top view of ANDV endonuclease showing the presence of a small helix αe (encircled) compared to the LACV structure, where this element is lacking. <b>D</b>, Superimposition of SAXS derived molecular shape with crystal structure (ribbon diagram) confirms the elongated and flat shape of the ANDV endonuclease.</p

Structure–function relationships in the ANDV endonuclease.

<p>Based on the crystal structure of L_1–200 K127A, a role for the 15 amino acid residues, that have been implicated in the activity of the endonuclease in mammalian cells [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005635#ppat.1005635.ref037" target="_blank">37</a>], was inferred. Side chains are shown as sticks and important hydrogen bonds are highlighted with dashed lines. Lys127 is mutated to alanine and therefore not completely represented in the structure. The manganese ion is shown as red sphere.</p

Thermal stability of ANDV L_1–200 protein mutants.

<p><b>A</b>, Representative melting curves for three mutant enzymes. The thermofluor assay was performed in absence of divalent ions (plus 10 mM EDTA), in the presence of manganese (4 mM MnCl₂ or 16 mM MnCl₂), and in the presence of magnesium (16 mM MgCl₂). <b>B</b>, Melting temperatures (T_m) are shown for all mutant enzymes that could be expressed. Protein stability was analyzed in the absence and presence of manganese or magnesium. The data represent mean and standard deviation of four independent thermofluor assays.</p

Development and evaluation of antibody-capture immunoassays for detection of Lassa virus nucleoprotein-specific immunoglobulin M and G

<div><p>Background</p><p>The classical method for detection of Lassa virus-specific antibodies is the immunofluorescence assay (IFA) using virus-infected cells as antigen. However, IFA requires laboratories of biosafety level 4 for assay production and an experienced investigator to interpret the fluorescence signals. Therefore, we aimed to establish and evaluate enzyme-linked immunosorbent assays (ELISA) using recombinant Lassa virus nucleoprotein (NP) as antigen.</p><p>Methodology/Principal findings</p><p>The IgM ELISA is based on capturing IgM antibodies using anti-IgM, and the IgG ELISA is based on capturing IgG antibody–antigen complexes using rheumatoid factor or Fc gamma receptor CD32a. Analytical and clinical evaluation was performed with 880 sera from Lassa fever endemic (Nigeria) and non-endemic (Ghana and Germany) areas. Using the IFA as reference method, we observed 91.5–94.3% analytical accuracy of the ELISAs in detecting Lassa virus-specific antibodies. Evaluation of the ELISAs for diagnosis of Lassa fever on admission to hospital in an endemic area revealed a clinical sensitivity for the stand-alone IgM ELISA of 31% (95% CI 25–37) and for combined IgM/IgG detection of 26% (95% CI 21–32) compared to RT-PCR. The specificity of IgM and IgG ELISA was estimated at 96% (95% CI 93–98) and 100% (95% CI 99–100), respectively, in non-Lassa fever patients from non-endemic areas. In patients who seroconverted during follow-up, Lassa virus-specific IgM and IgG developed simultaneously rather than sequentially. Consistent with this finding, isolated IgM reactivity, i.e. IgM in the absence of IgG, had no diagnostic value.</p><p>Conclusions/Significance</p><p>The ELISAs are not equivalent to RT-PCR for early diagnosis of Lassa fever; however, they are of value in diagnosing patients at later stage. The IgG ELISA may be useful for epidemiological studies and clinical trials due its high specificity, and the higher throughput rate and easier operation compared to IFA.</p></div

Clinical performance characteristics of the IgM ELISA as stand-alone test and in combination with the IgG ELISA depending on prevalence of Lassa fever and pre-existing IgG among all patients tested.

<p>The calculations are based on the data shown in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0006361#pntd.0006361.t003" target="_blank">Table 3</a>. PPV and NPV of the stand-alone IgM ELISA depend only on the Lassa fever prevalence in diagnostics. However, PPV, NPV, sensitivity, and positive likelihood ratio for combined detection of IgM and IgG (i.e. a positive test result means that both IgM and IgG is positive) depend on the prevalence of Lassa fever as well as the prevalence of pre-existing IgG among all patients tested. To simplify calculation, we assumed a ratio of 1:3 between prevalence of Lassa fever and pre-existing IgG, which roughly corresponds to the setting in Nigeria where the study was performed.</p

Sample-to-cut-off (S/CO) values for 880 sera tested in IgM ELISA, RF-based IgG ELISA, and CD32-based IgG ELISA and comparison with IFA results.

<p>The S/CO values obtained with the ELISA are shown as histograms according to sample origin, type of ELISA, and IFA results. The cut-offs for the ELISAs are indicated by vertical dotted lines. The number of samples with OD < CO and OD > CO is indicated in left and right corner, respectively, of each diagram.</p