Naturally occurring N-terminal mutations in SARS-CoV-2 nsp1 impact innate immune modulation but do not affect virus virulence

The non-structural protein 1 (nsp1) of SARS-CoV-2 plays a key role in host innate immune evasion. We identified two deletion variants (Δ82-85 and Δ83-86) in the N-terminal region of the nsp1 of a SARS-CoV-2 BA.5.2.1 variant recovered from a human patient. Analysis of the sequence databases revealed a frequency of 0.5% of these mutations amongst available SARS-CoV-2 sequences. Structural analysis of the deletion mutant nsp1Δ82-85 and nsp1Δ83-86 revealed a distortion in the protein pocket when compared to the wild-type nsp1 which may affect protein function. To evaluate the functional relevance of these mutations, we cloned the mutant BA.5.2.1 nsp1Δ82-85 and nsp1Δ83-86 and wild-type nsp1 proteins in expression plasmids and performed luciferase reporter-based assays to assess activation of the interferon and nuclear factor kappa B (NF-κB) signalling pathways. Both nsp1Δ82-85 and nsp1Δ83-86 mutants showed marked decreased ability to inhibit the interferon beta (IFN-β) and NF-κB pathway activation. To assess the relevance of these deletions in the context of SARS-CoV-2 infection, we generated recombinant viruses carrying the wild type BA.5.2.1 nsp1 or the BA.5.2.1 nsp1Δ82-85 and nsp1Δ83-86 deletions in the backbone of WA1 strain.