Navigating the Fitness Landscape: Host Density, Epistasis, and Clonal Interference Drive Divergent Evolutionary Pathways in Phage Qβ

Understanding how ecological factors shape viral evolution is essential for predicting adaptation in RNA viruses. In this study, we investigated the evolutionary dynamics of bacteriophage Qβ under varying host densities, focusing on two nonsynonymous mutations-A1930G and C2011A-located in the A1 protein. Using experimental evolution, phenotypic assays, and competition experiments, we found that C2011A is consistently selected at low bacterial densities, enhancing viral entry but reducing burst size. In contrast, A1930G is fixed at high densities, despite similar phenotypic effects, suggesting its advantage arises from interactions with additional mutations. Clonal analysis revealed that compensatory or beneficial mutations modulate the fitness of A1930G, enabling its fixation. The absence of both mutations in the same genome points to negative epistasis, confirmed by the poor performance of the double mutant generated by site-directed mutagenesis. Sequencing of intermediate transfers showed early emergence of A1930G, but its fixation was prevented by clonal interference with C2011A. These findings highlight how host availability, fitness trade-offs, epistasis, and competition among variants shape the adaptive landscape of RNA viruses.