Evolutionary inferences from the analysis of mutation dynamics in the SARS-CoV-2 replication-transcription complex

The SARS-CoV-2 virus continues to evolve and remains a significant public health threat, while the worldwide monitoring and sequencing of its genomic variants provide a unique opportunity to study its evolution and better understand its molecular mechanisms. In our work, we analyze its replication-transcription complex (RTC) over a 5.5- year period (December 2019-July 2025). This complex is significantly more conserved (as any alteration impairing its function prevents viral replication) than the S-protein (directly impacting infectivity and immune evasion) but still dynamically evolving part of the genome. The study focuses on high-frequency substitutions, their temporal behavior, co-occurrence, and structural context. Using genomes from GISAID, we identified 22 amino acid point mutations present in at least 1 % of currently available sequences, analyzed their weekly dynamics, revealed three distinct temporal patterns, and enumerated frequent co-occurring groups (pairs, triplets, and larger sets) within the same genomes. We mapped the affected residues onto an RTC 3D structure and reviewed the literature to examine the reported functional consequences. Notably, all these substitutions were single-nucleotide. One of the mutations, nsp12:G671S, showed a unique dynamic feature: it emerged, dominated globally for months, disappeared twice, and in 2025 reappeared for the 3rd time, always accompanied with other mutations in the RTC. Thus, it was interesting to trace its dynamics as an indicator of probable changes. In addition, our analysis of mutation and variant timelines suggests that the Delta variant may have emerged 7-8 months earlier than commonly reported. Taken together, these results provide a consolidated view of recurrent RTC variation, its temporal classes, co-occurrence, and structural context, underscoring the value of systematic surveillance of nsp7-nsp14 alongside analyses focused on structural proteins.