Characterization of SARS-CoV-2 Replication and Transcription Complexes via Structural and Evolutionary Approaches

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) caused around 700M cases and over 7M COVID-19-related deaths recorded worldwide (World Health Organization, March 2025). Aiming to effectively combat this and other disease-causing Coronaviruses (CoV), unprecedented research efforts led to the development of new vaccines and antiviral therapies. Due to emergence of variants of concern (VOCs) with increased transmissibility, immune evasion from vaccination, and potential to resist the available treatments, SARS-CoV-2 continues to represent a major threat to global health. Hence, there is a pressing need to discover new antivirals with broad-spectrum efficacy against multiple SARS-CoV-2 variants and related CoVs. This project aims at establishing the sequence conservation and coevolutionary landscapes of SARS-CoV-2 enzymatic domains critical to viral replication and transcription, such as the polymerase (nsp12-RdRp), exonuclease (nsp14-ExoN), and RNA capping (nsp16/nsp10) ones. Drugs targeting conserved regions of these proteins across VOCs can ultimately maintain efficacy, even in the face of ongoing mutations. Specifically, we employed multiple sequence alignments (MSA), phylogenetic tree reconstructions, and coevolution analysis to identify functionally significant residues across multiple CoVs. We then performed Principal Component Analysis (PCA) to separate large sequence data sets into sub-groups, based on sequence conservation. Finally, by combining our results with superimpositions of experimental structures of these proteins in complex with potential drugs, we identified locations within key functional domains to be further explored as potential drug-binding sites. Our work provides a general framework to assist the design of new drugs with broad antiviral properties against SARS-CoV-2 and other emerging CoVs.