SARS-CoV-2 Enzyme NSP15 Controls Viral Evolution by Regulating RNA Recombination

Coronaviruses evolve through two main mechanisms: point mutations and RNA recombination. While their proofreading exonuclease keeps mutation rates low, RNA recombination allows large-scale genomic reshuffling that can generate new variants, immune-evasive strains, and defective viral genomes (DVGs). Understanding what controls recombination is critical for anticipating how SARS-CoV-2 and its successors may evolve.

This study systematically compared RNA recombination frequency across four human coronaviruses — SARS-CoV-2, MERS-CoV, HCoV-229E, and HCoV-OC43 — using a random-primed next-generation sequencing (ClickSeq) approach and the ViReMa bioinformatic pipeline to map recombination junctions. SARS-CoV-2 displayed roughly 1.8-fold higher junction frequency than the other three viruses. Critically, all four coronaviruses preferentially recombined at uridine-rich RNA sequences flanking both donor and acceptor recombination sites, suggesting a shared mechanistic basis tied to uridine-cleaving enzymes.

This uridine preference led the investigators to focus on NSP15, a highly conserved coronavirus endoribonuclease (EndoU) that cleaves RNA specifically at uridines. Using reverse genetics, they engineered a catalytically inactive SARS-CoV-2 carrying the NSP15-H234A point mutation. In Vero E6 and Calu-3 respiratory cells, H234A virus replicated significantly less efficiently than wild-type. The attenuation was IFN-dependent: pretreating cells with type I interferon dramatically suppressed H234A but not wild-type, confirming NSP15's role in immune evasion. In a mouse model of COVID-19, H234A-infected animals had lower viral titers but paradoxically similar clinical disease, explained by exaggerated inflammatory responses — higher cytokine and chemokine production — that compensated for reduced viral burden.

NGS analysis of H234A-infected cells revealed dramatic changes in recombination architecture. Sub-genomic mRNA (sgmRNA) production — essential for expressing viral structural proteins — was substantially reduced in H234A virus. Conversely, non-canonical recombination events generating deletions and micro-deletions were increased, leading to a larger and more diverse population of defective viral genomes. In vivo, the H234A virus population showed reduced recombination diversity overall but strong selection pressure favoring specific DVG populations, suggesting that loss of NSP15 reshapes the intra-host viral quasispecies in ways that could influence both immune activation and transmission fitness.

Together, the data reveal NSP15 as a bifunctional regulator: it promotes productive RNA recombination needed for sgmRNA synthesis while simultaneously suppressing aberrant recombination that produces DVGs. By cleaving uridine-rich recombination hotspots, NSP15 appears to sculpt the topology of coronavirus RNA recombination. This positions NSP15 not only as an immune evasion factor but as a master regulator of viral genome integrity and transcriptional output, making it an attractive antiviral target.