Coronaviruses: an RNA proofreading machine regulates replication fidelity and diversity

Abstract

In order to survive and propagate, RNA viruses must achieve a balance between the capacity for adaptation to new environmental conditions or host cells with the need to maintain an intact and replication competent genome. Several virus families in the order Nidovirales, such as the coronaviruses (CoVs) must achieve these objectives with the largest and most complex replicating RNA genomes known, up to 32 kb of positive-sense RNA. The CoVs encode sixteen nonstructural proteins (nsp 1-16) with known or predicted RNA synthesis and modification activities, and it has been proposed that they are also responsible for the evolution of large genomes. The CoVs, including murine hepatitis virus (MHV) and SARS-CoV, encode a 3'-to-5' exoribonuclease activity (ExoN) in nsp14. Genetic inactivation of ExoN activity in engineered SARS-CoV and MHV genomes by alanine substitution at conserved DE-D-D active site residues results in viable mutants that demonstrate 15- to 20-fold increases in mutation rates, up to 18 times greater than those tolerated for fidelity mutants of other RNA viruses. Thus nsp14-ExoN is essential for replication fidelity, and likely serves either as a direct mediator or regulator of a more complex RNA proofreading machine, a process previously unprecedented in RNA virus biology. Elucidation of the mechanisms of nsp14-mediated proofreading will have major implications for our understanding of the evolution of RNA viruses, and also will provide a robust model to investigate the balance between fidelity, diversity and pathogenesis. The discovery of a protein distinct from a viral RdRp that regulates replication fidelity also raises the possibility that RNA genome replication fidelity may be adaptable to differing replication environments and selective pressures, rather than being a fixed determinant.

Figure 1

SARS-CoV genome organization, reverse genetics and nsp14-ExoN motifs and mutations. (A) SARS-CoV genome. ∼29. kb single-strand (+) RNA. ORF1ab replicase gene is shown in gray with nonstructural protein domains (nsp) 1–16 indicated. Putative RNA synthesis proteins are indicated as in text. Nsp14/ExoN is yellow. (B) Schematic shows nsp14 with three ExoN motifs in red and zinc finger motif hatched. (C) Alignment and MHV and SARS-CoV nsp14 motifs, with conserved active site residues in red. Location of alanine substitutions are indicated, with resulting mutant.

Figure 2

Coronavirus subgenomic (sg) mRNA synthesis. See text for discussion. Red arrows show direction of RNA synthesis.

Figure 3

Mutations detected in SARS-CoV and S-ExoN Mutants. (A) SARS-CoV genome with nsp14-ExoN in yellow and diamond indication location of ExoN mutant. (B) Mutations identified in 10 SARS-CoV plaque clones (∼320 knt). Legend above indicates type of mutations. (C) Mutations identified in 10 S-ExoN plaque clones (∼320 knt). (D) Representative S-ExoN plaque clones showing unique and shared mutations and patterns. (E) Spike glycoprotein showing the schematic of spike subunits and domains. Lower schematic shows unique mutations across spike from 10 S-ExoN plaque clones (upright-blue) and from population Solexa Sequencing (inverted-black).

Figure 4

Population and plaque passage of S-ExoN and M-ExoN. (A) Population passage of WT SARS-CoV and S-ExoN (Adapted from Eckerle, et al.7). (B) Parallel plaque passages of 10 plaques each of WT-MHV and M-ExoN. *indicates passage where loss of a plaque lineage occurred.