Cap binding and immune evasion revealed by Lassa nucleoprotein structure

Abstract

Lassa virus, the causative agent of Lassa fever, causes thousands of deaths annually and is a biological threat agent, for which there is no vaccine and limited therapy. The nucleoprotein (NP) of Lassa virus has essential roles in viral RNA synthesis and immune suppression, the molecular mechanisms of which are poorly understood. Here we report the crystal structure of Lassa virus NP at 1.80 Å resolution, which reveals amino (N)- and carboxy (C)-terminal domains with structures unlike any of the reported viral NPs. The N domain folds into a novel structure with a deep cavity for binding the m7GpppN cap structure that is required for viral RNA transcription, whereas the C domain contains 3'-5' exoribonuclease activity involved in suppressing interferon induction. To our knowledge this is the first X-ray crystal structure solved for an arenaviral NP, which reveals its unexpected functions and indicates unique mechanisms in cap binding and immune evasion. These findings provide great potential for vaccine and drug development.

Figure 1. The crystal structure of LASV NP protein

a, Cartoon diagram of the LASV NP protomer. N domain in cyan with cyan sphere indicating the N terminus, C domain in orange with orange sphere indicating the C terminus. Black sphere shows Mn²⁺, while blue sphere shows Zn²⁺. The dotted lines represent the disordered loops. b, The ring-shaped structure of LASV NP trimer. The first protomer coloured as in (a), the second protomer in blue and the third in magenta. The groove and the interface are indicated by arrows. c, Electrostatic surface potential map of the NP protomer. The entrance of the cap-binding cavity shown as white dotted circle. The blue area represents positively charged residues and the red area represents negatively charged residues. d, Electrostatic surface potential map of the 3′-5′ exoribonuclease cavity. The black sphere represents Mn²⁺.

Figure 2. The C domain of LASV NP protein is a 3′-5′ exoribonuclease

a, Superimposition of the C domain (orange) with human TREX1 protein (green) reveals a high degree of similarity between the two structures.The Mn²⁺ in black in LASV NP and in red in TREX1, the Zn²⁺ in blue. b, The exonuclease catalytic residues of LASV NP and TREX1 are located to identical positions, and shown in orange for NP and in green for TREX1. c, The exoribonuclease activities of the WT and mutant LASV NP with different ssRNAs as substrates. Control 1 contains 10 mM EDTA and no NP. Control 2 contains 10 mM EDTA and NP. d, Comparison of the WT and NP catalytic mutants in degrading the dsRNA substrates, the 5′-hydroxyl dsRNA (upper), double 5′ triphosphorylated dsRNA (middle), and the single 5′ triphosphorylated dsRNA (lower).

Figure 3. The exonuclease activity of NP is important for blocking the IFN induction

Results shown are the average (n=3) with error bars indicating the standard deviations. a, The NP catalytic mutants were expressed at similar levels to the WT in mammalian cells and had similar transcriptional activities in the LASV minigenome assay. b, The NP catalytic mutants were defective in suppressing the Sendai virus-induced IFN induction by a LUC-based IFNbeta promoter assay. c, The NP catalytic mutants were defective in suppressing the IFN production induced by the immunostimulatory RNAs, poly(I:C) and Pichinde virion-associated RNAs.

Figure 4. The cap-binding residues and their roles in viral RNA transcription

Results shown are the average (n=3) with error bars indicating the standard deviations. a, A cap analog dTTP is bound within the deep cavity of the N domain of LASV NP. Original Fo-Fc map for the dTTP in blue contoured 2.5 σ. The F176 and W164 or L172 (L120) residues form a typical cap-binding sandwich structure. The middle cavity binds the triphosphate moiety and the hydrophobic cavity entrance can accommodate the second base of the cap structure. b, The NP mutants were expressed at similar levels as the WT at 15 to 30 ng plasmid (WT-15, WT-30) in the transfected mammalian cells. c. Mutational analyses of the residues within the cap-binding cavity for the transcriptional activity using the LASV minigenome assay.