Structure of the Lassa virus nucleoprotein reveals a dsRNA-specific 3' to 5' exonuclease activity essential for immune suppression - PubMed (original) (raw)

Structure of the Lassa virus nucleoprotein reveals a dsRNA-specific 3' to 5' exonuclease activity essential for immune suppression

Kathryn M Hastie et al. Proc Natl Acad Sci U S A. 2011.

Abstract

Lassa fever virus, a member of the family Arenaviridae, is a highly endemic category A pathogen that causes 300,000-500,000 infections per year in Western Africa. The arenaviral nucleoprotein NP has been implicated in suppression of the host innate immune system, but the mechanism by which this occurs has remained elusive. Here we present the crystal structure at 1.5 Å of the immunosuppressive C-terminal portion of Lassa virus NP and illustrate that, unexpectedly, its 3D fold closely mimics that of the DEDDh family of exonucleases. Accompanying biochemical experiments illustrate that NP indeed has a previously unknown, bona fide exonuclease activity, with strict specificity for double-stranded RNA substrates. We further demonstrate that this exonuclease activity is essential for the ability of NP to suppress translocation of IFN regulatory factor 3 and block activation of the innate immune system. Thus, the nucleoprotein is a viral exonuclease with anti-immune activity, and this work provides a unique opportunity to combat arenaviral infections.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Structure of the C-terminal, immunosuppressive domain of LASV NP. (A) Cartoon representation of LASV NPΔ340. The basic arm includes residues Lys516, Lys517, Lys518, and Arg519. (B) A single zinc is coordinated by Glu399, His506, Cys509, and Cys529.

Fig. 2.

Superimposition of LASV NP and known DEDD exonucleases. (A) Structural comparison of NPΔ340 and two known DEDDh exonucleases. NPΔ340 is colored green, ISG-20 (PDB ID 1WLJ; ref. 27) is colored cyan, and the E. coli DNA pol IIIε (PDB ID 2GUI; ref. 28) is colored yellow. Inset shows a close-up view of the superimposed DEDDh residues of the active site. Numbered residues reflect those of LASV NP. (B) Electrostatic surface potential calculated with APBS (the Adaptive Poisson-Boltzmann Solver) software (49) shows that each exonuclease has an acidic active site and highlights the basic arm of LASV NP. Positive surface is colored blue; negative surface is colored red with limits ± 10 kT/e.

Fig. 3.

Ribonucleolytic activity of LASV NP. (A) Substrate specificity. NPΔ340 was incubated with different 32P-labeled substrates, and the reaction products were analyzed by denaturing PAGE and autoradiography. (Left) Migration patterns of each nucleic acid substrate alone (in the absence of NPΔ340 exonuclease). (Right) Migration of each substrate when incubated with NPΔ340 for 15 min. A schematic for each substrate is also shown, with the location of the 32P label indicated by an asterisk. Sequences for each substrate can be found in

Table S2

. (B) Time course of exonuclease activity. The 18-bp blunt-ended dsRNA is increasingly digested by wild-type NPΔ340 from 0 to 15 min. (C) Comparison of ribonucleolytic activity of the N terminus of NP (NP1–340) and full-length NP (NP-FL). The 18-bp blunt-ended dsRNA is digested by full-length NP and NPΔ340, whereas NP1–340 does not have exonuclease activity. (D) Effects of mutations to active site and proximal residues on ribonucleolytic activity. Wild-type and NPΔ340 point mutants were incubated with 18-bp blunt-ended dsRNA for 15 min, and products were analyzed by PAGE. QuadA designates a quadruple alanine mutation in residues K516K517K518R519. Note that all point mutations to residues in or near the exonuclease active site and the Zn coordination site, save R393, abrogate exonuclease activity, leaving the dsRNA undigested. The QuadA mutation to the basic arm partially diminishes exonuclease activity.

Fig. 4.

Cartoon representation of NPΔ340 and location of residues important for the exonuclease and anti-IFN activities. Residues corresponding to the DEDDh motif are colored magenta, two Arg residues proximal to the active site are colored yellow, residues involved in coordination of the Zn2+ atom are colored blue, and residues corresponding to the basic arm are colored orange.

Fig. 5.

Sendai virus-mediated activation of an IRF-3 promoter. Full-length NP (NP-FL), the N-terminal domain of NP (NP1–340), the C-terminal domain of NP (NPΔ340), and amino acid substitutions of full-length NP were assayed for inhibition of Sendai virus-mediated activation of an IRF-3–dependent promoter. Values displayed reflect relative luminescence units corrected to an uninfected, empty vector control. E(−) and E(+) indicate an uninfected empty vector control and an infected empty vector control, respectively. Note that the wild-type function of NP is to block IRF-3 translocation and therefore block reporter luminescence.

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Structure of the Lassa virus nucleoprotein reveals a dsRNA-specific 3' to 5' exonuclease activity essential for immune suppression - PubMed (original) (raw)