Tradeoffs in viral fitness driven by alternative entry pathways

Enveloped viruses can enter cells either through endocytosis or by direct fusion with the plasma membrane. The latter pathway often leads to syncytia formation, caused by the fusion of infected cells with neighboring receptor-bearing cells. Yet, the implications of these two alternative entry routes for viral fitness and evolution remain poorly understood. To address this, we used a vesicular stomatitis virus-based system engineered to express the SARS-CoV-2 spike. Because deletion of the spike furin cleavage site (ΔFCS) abolishes plasma membrane fusion and restricts entry to the endocytic pathway, we compared Wuhan and ΔFCS spike variants. We found that plasma membrane entry allowed the Wuhan spike to accelerate infection and viral spread through syncytia formation. In contrast, the ΔFCS variant was less cytotoxic and reached higher titers. Coinfection assays showed that the Wuhan spike exerted a dominant negative effect on the ΔFCS mutant, outcompeting it through syncytia-mediated spread. However, when spatial segregation was enforced, the ΔFCS variant was favored instead. These findings suggest that virus-virus interactions shape the relative fitness of alternative entry routes and highlight the potential of social evolution theory to model these interactions.IMPORTANCEUnderstanding how viruses enter host cells is critical for elucidating key aspects of viral infectivity, transmission, and pathogenesis. Here, we investigate the consequences of two alternative viral entry routes: endocytosis and direct entry at the plasma membrane. To this end, we employed a recombinant virus expressing the SARS-CoV-2 spike protein. This artificial system produced clear and striking phenotypes, enabling us to observe distinct differences between the two entry pathways. Plasma membrane entry promoted rapid viral spread through cell-cell fusion, whereas endocytic entry supported sustained virion production with reduced cell death. Notably, a spike variant that utilized the direct entry route dominated during coinfection, promoting extensive cell fusion and suppressing the phenotype of a variant restricted to endocytic entry. These findings clarify the functional trade-offs between viral entry pathways and introduce a novel framework for studying them through the lens of virus-virus interactions and social evolution.