RESUMEN
Background: The SARS-CoV-2 Omicron variant is highly immune evasive but is attenuated in cell and animal models of infection, which many reports attribute to spike mutations. However, the phenotype and contribution to viral fitness of Omicron non-spike mutations remain unknown. Method(s): To study mutations across the entire genome independent of spike, we developed a novel cloning and replicon system capable of generating mutants within 6 hours and obtaining phenotypic results within 3-4 days. Result(s): Using a series of Omicron replicons, we found that ORF1ab harbors critical mutations, especially in the nonstructural protein 6 (NSP6), which lower viral fitness and are currently evolving in Omicron subvariants. In addition, Omicron mutations in several NSPs epistatically interact and are critical for viral replication and polyprotein processing. Conclusion(s): Collectively, we describe a robust replicon technology to study mutations across the genome and our data highlight the need to vigilantly study and monitor non-spike mutations in emerging Omicron subvariants.