Innate sensor responses set a supra-unitary threshold for viral replication

Cells resist viral infection by triggering rapid and sensitive innate immune responses, initiated by cytoplasmic viral sensor proteins (CyVSP), retinoic acid-inducible gene I (RIG-I), melanoma differentiation-associated protein 5 (MDA-5), and protein kinase R (PKR). Our single-cell analysis reveals that viral replication requires a supra-unitary viral genome input, significantly more than one, which we define as the virion input threshold for replication (VITREP). This threshold varies depending on viruses and cell types, independently of viral protein synthesis. Through mathematical modeling and experimental testing, we uncover that supra-unitary viral genome quantities activate negative feedback regulators (NFR) of CyVSP, including death-associated protein kinase 1 (DAPK1), Rio kinase 3 (RIOK3), and protein phosphatase 2 regulatory subunit alpha (PPP2R2A), that suppress innate immune responses and allow replication. We validate key predictions of our model, namely that downregulating NFR reduces replication, whereas upregulating NFR or coinfecting cells promotes replication. Together, these findings reveal a fundamental mechanism governing viral infectivity and tissue tropism.