Epistasis between SARS-CoV-2 M and N Proteins Balances Particle Assembly and Immune Evasion

Abstract

Since its emergence in the human population, SARS-CoV-2 has continuously evolved to evade immune responses and robustly establish global circulation. In this process, the structural viral membrane (M) protein has accumulated amino acid changes whose impact on viral particle assembly and innate immune evasion remains incompletely understood. Here, we designed a SARS-CoV-2 replicon system lacking M that assesses the influence of transiently transfected M protein variants on viral particle production independently of viral RNA replication. We found that M protein variants have reduced particle assembly while innate immune antagonism functions are strengthened. Notably, the assembly defect is rescued by co-evolving N protein variants, highlighting how SARS-CoV-2 evolution coordinates between two of its structural proteins to optimize viral infection. Our work underscores the complex evolutionary trajectories of SARS-CoV-2 variants across different viral proteins and informs future therapeutic strategies targeting viral assembly and limiting infection. Author summarySince the onset of the COVID-19, SARS-CoV-2 has been changing continuously in ways that help it spread efficiently and evade human defenses. The viral membrane (M) protein is a key structural component required to form new viral particles. Nevertheless, it has accumulated changes over time with overlooked functions. In this study, we developed an experimental system to examine the impact of these changes on virus assembly and host immune system. We found that recent M variants are better than older variants at suppressing immune responses but less efficient at forming viral particles. This defect seems to be compensated for by coordinated changes in another structural protein, nucleocapsid (N). These findings reveal that SARS-CoV-2 evolution involves trade-offs between different viral characteristics, with changes in one protein balancing changes in another. Understanding these trade-offs provides new insight into how the virus adapts to humans and may help guide therapeutic strategies to limit infection.