Fidelity of Ribonucleotide Incorporation by the SARS-CoV-2 Replication Complex

The SARS-CoV-2 coronavirus has caused a global pandemic. Despite the initial success of vaccines at preventing infection, genomic variation has led to the proliferation of variants capable of higher infectivity. Mutations in the SARS-CoV-2 genome are the consequence of replication errors, highlighting the impor-tance of understanding the determinants of SARS-CoV-2 replication fidelity. The RNA-dependent RNA polymerase (RdRp) is the central catalytic subunit for SARS-CoV-2 RNA replication and genome tran-scription. Here, we report the fidelity of ribonucleotide incorporation by SARS-CoV-2 RdRp (nsp12), along with its co-factors nsp7/nsp8, using steady-state kinetic analysis. Our analysis suggests that in the absence of the proofreading subunit (nsp14), the nsp12/7/8 complex has a surprisingly low base substi-tution fidelity (10-1-10-3). This is orders of magnitude lower than the fidelity reported for other coron-aviruses (10-6-10-7), highlighting the importance of proofreading for faithful SARS-CoV-2 replication. We performed a mutational analysis of all reported SARS-CoV-2 genomes and identified mutations in both nsp12 and nsp14 that appear likely to lower viral replication fidelity through mechanisms that include impairing the nsp14 exonuclease activity or its association with the RdRp. Our observations provide novel insight into the mechanistic basis of replication fidelity in SARS-CoV-2 and the potential effect of nsp12 and nsp14 mutations on replication fidelity, informing the development of future antiviral agents and SARS-CoV-2 vaccines.(c) 2023 Published by Elsevier Ltd.

Automated summary

Genomic variations in SARS-CoV-2 have led to the emergence of more infectious variants. The replication fidelity of the virus is greatly affected when lacking the proofreading subunit, resulting in lower accuracy. Mutations in the nsp12 and nsp14 genes are likely to reduce replication fidelity, providing insight for future antiviral drugs and vaccine development.