Brothers in Arms: Structure, Assembly and Function of Arenaviridae Nucleoprotein

Abstract

Arenaviridae is a family of viruses harbouring important emerging pathogens belonging to the Bunyavirales order. Like in other segmented negative strand RNA viruses, the nucleoprotein (NP) is a major actor of the viral life cycle being both (i) the necessary co-factor of the polymerase present in the L protein, and (ii) the last line of defence of the viral genome (vRNA) by physically hiding its presence in the cytoplasm. The NP is also one of the major players interfering with the immune system. Several structural studies of NP have shown that it features two domains: a globular RNA binding domain (NP-core) in its N-terminal and an exonuclease domain (ExoN) in its C-terminal. Further studies have observed that significant conformational changes are necessary for RNA encapsidation. In this review we revisited the most recent structural and functional data available on Arenaviridae NP, compared to other Bunyavirales nucleoproteins and explored the structural and functional implications. We review the variety of structural motif extensions involved in NP-NP binding mode. We also evaluate the major functional implications of NP interactome and the role of ExoN, thus making the NP a target of choice for future vaccine and antiviral therapy.

Keywords: Arenaviridae; Bunyavirales; emerging diseases; exonuclease; nucleoprotein; structure.

Figure 1

Architecture of Arenaviridae nucleoprotein (NP) and Nucleoprotein structures. (A) Annotated schematic of NP architecture. Colour code of annotation is in caption. (B) Structure of full-length NP (PDB 3MWP) represented in ribbon. NP-core domain is in blue and the exonuclease domain (ExoN) in green, the proposed multimerization arm of the NP-core in pink, the hidden RNA binding cleft highlighted in orange circle. (C) Structure of NP-core domain (blue/ kaki) in open and closed conformation focus on the RNA binding cleft. Left panel shows the RNA binding cleft of the 3MWP structure. Right panel presents the corresponding domain of the structure 3T5Q with RNA (red ribbon). Comparison of these two structures shows that in the absence of RNA, the cavity is closed by the α5 and α6 helix shown in kaki as well as by the loop (residues 234–245) shown also in kaki. The α5 and α6 helix as well as the loop are displaced in the case of the 3T5Q structure, permitting the adsorption of the viral RNA. All structural figures and movies were done using UCSF chimera [31].

Figure 2

Structure of the exonuclease domain (ExoN) and conservation of the catalytic site. (A) Annotated structure of the ExoN domain represented in ribbon with ions Mn²⁺ in purple and Zn²⁺ in grey and zoom on the catalytic residues DEDDh shown in sticks with catalytic ions and 3′ end of double-stranded (ds)RNA substrate (cyan) (PDB). Metallic ion Mn²⁺ are marked as 1 and 2, 1 being the ion that is always observed and 2 the ion dynamically brought by the RNA. (B) Weblogo [51] of the DEDDh catalytic site through Arenaviridae.

Figure 3

TEM images and data from freshly purified Mopeia virus (MOPV) NP protein. (A) Ribonucleoprotein complex (RNP) particle and several classses showing heptamer organisation (0.05 mg/mL). A 5 μL drop was applied to a freshly deposited and glow-discharged formvar-carbon-coated grid (Copper 300). The grid was stained with Nano-W^® (Nanoprobes) and transferred into a Tecnai 120 kV Electron Microscope. A total of 100 raw images were recorded with an EAGLE 2k × 2k CCD camera. Images were under-focused at 1–2 μm with a final resolution of 2.8 Å/pix. Boxing, classification, initial model calculation, as well as refinement for 3D reconstruction, was done with the EMAN2 pipeline [53]. Arrows indicate the sides of the measured object. (B) Top: Graph of the Fourier Shell Correlation (FSC) coefficient in function of the spatial frequencies in Å⁻¹, arrow indicating the maximum resolution; Central: 3D reconstruction at 27Å resolution with below corresponding particle classes used (1224 particles).

Figure 4

The schematics of slight increase in complexity of NP assembly. Major secondary structures involved in interprotomer interactions are represented as arrows for β-strand and cylinders for α-helices. The RNA binding cavity is represented as a central shaded part in the middle of the NP-core (blue macaron). Schematics representation from top to bottom of Rift Valley Fever Virus (RVFV)-NP, Hantaan virus (HTNV)-NP, Tomato spotted wilt virus (TSWV)-NP, LaCrosse virus (LACV)-NP, Crimean Congo Haemorrhagic Fever virus (CCHFV)-NP. Schematic representation of RNA binding and NP–NP interactions. RNA is shown as a black line. The main NP–NP interactions between adjacent subunits are indicated. For clarity, Ni interactions with Ni-2, Ni-3, Ni+2 and Ni+3 are absent from the schematic representation.

Figure 5

Structural comparison of known Bunyavirales NP. Surface representation of the different structures of representative Bunyavirales from left to right Phenuiviridae (RVFV, open conformation PDB: 3OUO /Closed conformation PDB: 3LYF), Hantaviridae (HTNV, PDB: 5FSG), Tospoviridae (TSWV, PDB: 5IP1), Peribunyaviridae (SHMV, open conformation PDB: 4DIX /Closed conformation PDB: 4DIU, LACV PDB: 4BHH), Nairoviridae (CCHFV, PDB: 4AQF) and, Arenaviridae (LASV core domain, open conformation PDB: 3T5Q /Closed conformation from PDB: 3MWP). Highlighted are the 3 important parts of the NP core in blue, multimerization arms N-terminal in pink, central in green, and the C-terminal in kaki.

Figure 6

Structural comparison between the NP-core with RNA of LASV (blue, PDB 3T5Q) and the head domain of CCHF (yellow, PDB 3U3I) represented in ribbon. The two structures present a RMSD of 2.58 Å. The clear structural conservation between the two cores suggests that α6 is a valid structural candidate for multimerization.

Figure 7

Pathways of innate immunity and its disruption by NP. Symbols are explained in the bottom caption.