Structural analysis of viral ExoN domains reveals polyphyletic hijacking events

Abstract

Nidoviruses and arenaviruses are the only known RNA viruses encoding a 3'-5' exonuclease domain (ExoN). The proofreading activity of the ExoN domain has played a key role in the growth of nidoviral genomes, while in arenaviruses this domain partakes in the suppression of the host innate immune signaling. Sequence and structural homology analyses suggest that these proteins have been hijacked from cellular hosts many times. Analysis of the available nidoviral ExoN sequences reveals a high conservation level comparable to that of the viral RNA-dependent RNA polymerases (RdRp), which are the most conserved viral proteins. Two highly preserved zinc fingers are present in all nidoviral exonucleases, while in the arenaviral protein only one zinc finger can be identified. This is in sharp contrast with the reported lack of zinc fingers in cellular ExoNs, and opens the possibility of therapeutic strategies in the struggle against COVID-19.

Fig 1. Unrooted tree based on the structural comparisons of DEDD exonucleases.

The dots indicate exonuclease function, which colors stand for: yellow, proofreading; green, cytoplasmic RNA and/or DNA degradation; red, immune activity; dark blue, RNA processing and maturation; fuschia, DNA repair. The single letters indicate the DEDD exonuclease subgroup according to the fifth most conserved residue in the catalytic core: y, DEDDy subgroup; h, DEDDh subgroup. The tertiary structures of the exonucleases and their respective abbreviations used in this analysis were: DNA polymerase I klenow fragment, EcolKLFR; Exonuclease I, EcolEXO1; RB69 gp43 DNA polymerase, Ph69EXON; Phage T7 exonuclease, T7EXON; Phage T4 exonuclease, T4EXON; Phage phi29 exonuclease, ϕ29EXON; Nuclease domain of 3’hExo, HsapERI1; Human ISG20, HsapISG2; TREX2 3’ exonuclease, HsapTREX; RNase D, EcolRIBD; WRN exonuclease, HsapWEXO; Pol III epsilon-hot proofreading complex, EcolPEPS; DNA-directed DNA polymerase, PfurPEXO; Pop2p deadenylation subunit, SpomPOP2; ERI1 exoribonuclease 3, HsapERI3; TREX1 exonuclease, MmusTREX; Cell-death related nuclease 4, CelgCRN4; DNA polymerase delta, ScerPDEL; Pan2 catalytic unit, NcraPAN2; Exonuclease X, EcolEXOX; nsp14 3–5 exoribonuclease, SarsEXON; Nucleoprotein with 3–5 exoribonuclease, LvirNUCL; RNase T, EcolRIBT; Mopeia virus Exonuclease domain, MvirNUCL; Junin virus nucleoprotein, JvirNUCL; Maelstrom of Drosophila melanogaster, DromMAEL; Maelstrom of Bombyx mori, BmorMAEL; Ribosomal RNA processing protein 6, TbruRRP6; RNase AS, MtubRNAS; DNA polymerase epsilon, ScerDEPS; Exonuclease I, MszeEXO1; Small RNA degrading nuclease 1, AthaSDN1; Oligoribonuclease, CpsyOLRN; and REXO2 oligoribonuclease, HsapREX2. For PDB IDs see supplementary data. Viruses figures (SARS and Arenavirus) were made using Keynote and the DNA phage was drawn by hand.

Fig 2. Multiple sequence alignment of the nidovirus ExoN domain.

The Exo I (DE), Exo II (D/E), and Exo III (Dh) conserved sequence motifs are signaled with unfilled circles above them. Zinc binding motif 1 (ZF1, CCCH/C) and zinc-binding motif 2 (ZF2, HCHC) are signaled with filled circles above them. Secondary structure is indicated as red, helix; green, beta strand. Viral sequences abbreviation: PCSN, Planarian cell-secretory nidovirus; APNV, Aplysia californica nidovirus; NDiV, Nam Dinh virus; GoTV, Goat torovirus; DeV1, Decronivirus 1; YHV, Yellow head virus; and TuV1, Turrinivirus 1.

Fig 3. Nidovirus RdRp and ExoN maximum-likelihood phylogenies.

The colors in the names of the viral species correspond to the families they belong to. The Coronaviridae family has been divided in its corresponding genuses: alphacoronavirus (α), betacoronavirus (β), deltacoronavirus (δ), and gammacoronavirus (ɣ). Bootstraps branches with < 50% have been collapsed.

Fig 4. Structural comparisons of DnaQ-like exonucleases.

(A) Structural conservation among the DnaQ-like nucleases. ɑ-helices colored in red; β-sheets in yellow; and connecting elements in green. (B) Structural superposition between Lassa virus NP exonuclease (PDB ID: 3Q7V), orange; and SARS-CoV ExoN domain (PDB ID: 5C8U), green. (C) Structural superposition between Lassa virus NP exonuclease (PDB ID: 3Q7V), orange; and Drosophila melanogaster Maelstrom (PDB ID: 4YBG), blue. Herein, the presence of the ZF (ECHC) in both proteins is highlighted.