Natural variation in BtSARSr-CoV nsp12 harbors adaptive mutations that enhance viral replication and spillover risk in humans

The non-structural protein 12 (nsp12) is essential for SARS-related coronavirus (SARSr-CoV) replication and a major antiviral target, yet the functional significance of its natural variation in bat reservoirs remains unclear. Here, by analyzing 183 bat SARS-related coronavirus nsp12 sequences, we identified mutation hotspots that overlapped with sites variable in human SARS-CoV-2 variants (14 of 33 sites analyzed, including P323L), suggesting shared functional constraints across species. Using a cell-based reporter assay for high-throughput profiling of nsp12 function, we found that 8 of 21 bat-derived mutations enhanced polymerase activity at 37°C, with several also conferring an advantage at 33°C or 40°C. Four of these mutations (I171V, A185S, R249K, and N611S) increased replication in a SARS-CoV-2 replicon system, and all have emerged independently in circulating lineages (A185S reached detectable global prevalence by 2025). These mutations did not affect remdesivir sensitivity. Structurally, the mutations localize to regions involved in RNA capping or the RNA-dependent RNA polymerase (RdRp) catalytic core. Notably, SARS-CoV-related strains naturally encode multiple such adaptive residues, whereas SARS-CoV-2-related viruses retain ancestral states. Our study demonstrates that bat SARSr-CoV nsp12 variation pre-encodes mutations capable of enhancing human-cell replication, providing a predictive framework for monitoring polymerase changes with zoonotic potential. IMPORTANCE: Bat SARS-related coronaviruses (BtSARSr-CoVs) are closely linked to past human pandemics and represent an ongoing source of zoonotic risk. Proactive evaluation of their spillover potential is therefore critical for pandemic preparedness. Current risk assessments have focused largely on receptor compatibility, with limited experimental investigation of viral replication fitness in human cells. In this study, we demonstrate that specific mutations in the viral RNA polymerase non-structural protein 12 (nsp12), naturally circulating in BtSARSr-CoVs, can enhance viral replication in both bat and human cells. By functionally validating several such mutations that later emerged in SARS-CoV-2, we establish a direct link between reservoir virus diversity and pathways of human adaptation. The high-throughput reporter platform developed here provides a scalable tool for prospectively assessing the replication fitness of nsp12 variants identified through surveillance. Together, these findings enhance our ability to interpret genetic signatures from animal coronaviruses and contribute to a more predictive framework for monitoring variants with zoonotic potential.