Enhanced sialic acid engagement at physiological temperatures by reovirus σ1 mutants facilitates infection of breast cancer cells with low levels of high-affinity receptors.

[UNLABELLED] Reovirus serotype 3 (T3D) serves as a safe, tractable model for studying fundamental virus-host interactions and is also under investigation as an oncolytic therapy. Many viruses, including reoviruses, use sialic acids as attachment factors in addition to protein receptors; however, the relative contributions of these interactions, particularly at physiological temperatures, remain poorly defined. T3D engages sialic acids and JAM-A proteins through the tail and head domains of the σ1 cell attachment protein, respectively. In this study, murine E0771 breast cancer cells were found to resist reovirus infection due to a deficiency in high-affinity receptors. T3D bound to E0771 cells at 4°C but not 37°C, revealing that wild-type sialic acid interactions are thermally unstable at physiological temperatures. Exogenous JAM-A expression restored stable binding and infection at 37°C. Notably, engineered reovirus variants lacking the σ1 head domain but possessing either an RGD motif or specific mutations in the sialic acid-binding domain overcame the restriction and could bind and infect E0771 cells efficiently at 37°C. Through neuraminidase treatment and sialic acid-deficient and dependent cell models, enhanced interactions by sialic acid-binding domain mutations were empirically confirmed to be mediated by sialic acid. Serial passaging of σ1-truncated virus rapidly selected for mutations that strengthened sialic acid-binding at 37°C. Together, these findings demonstrate that high-affinity receptor requirements can be bypassed by enhancing interactions at physiological temperatures with sialic acids or through RGD interactions. This work provides insight into how viruses adapt to receptor scarcity and offers a strategy to overcome receptor heterogeneity in oncolytic virotherapy.

[IMPORTANCE] Reoviruses are promising oncolytic agents, yet clinical efficacy can be hindered by heterogeneous receptor expression in tumors. This study demonstrates that reovirus can bypass high-affinity receptor requirements by optimizing sialic acid interactions or incorporating RGD motifs. Crucially, the data reveal that wild-type reovirus attachment to sialic acids is thermally unstable at physiological temperature (37°C), a restriction masked by traditional 4°C assays. Specific mutations were found to stabilize these interactions at 37°C, providing a mechanistic basis for viral adaptation to receptor-deficient environments. These findings establish new experimental approaches to study attachment at 37°C, which can be applied broadly to discover unanticipated mutational effects on viral entry. Ultimately, evaluating virus-cell attachment under physiological conditions is essential for accurately predicting viral tropism and facilitates the design of next-generation oncolytic therapies better equipped to overcome receptor scarcity and thermal barriers in complex tumor environments.