IFN-Independent Expression of RIG-I is a Determinant of Heterogeneous IFN-B Expression States in Antiviral Signaling

Abstract

RIG-I plays a major role in antiviral innate immunity by detecting the cytoplasmic viral RNA and triggering the pathway that leads to transcriptional activation of type I interferon (IFN). Type I IFN induces a large set of genes called interferon stimulated genes (ISGs) which coordinate to antagonize viral evasion. We utilized single-molecule fluorescence in situ hybridization (smFISH) to study viral and antiviral gene expression in individual cells. We precisely quantified kinetics of mRNA expression of RIG-I, IFN-b and Sendai virus (SeV) L gene in clonal mammalian cells upon infection with SeV. Contrary to the expectation that RIG-I expression is only up-regulated through positive feedback involving Type I IFN, we found that RIG-I is directly induced by viral infection in the absence of IFN at the early stages of viral infection. Examination of expression kinetics of a subset of ISGs showed the differential regulation of ISGs in response viral infection. MDA5, LGP2, OasL and Viperin were directly activated by SeV infection before IFN-b. On the other hand, activation of some other ISGs was found to be dependent on IFN signaling, such as PKR, MxA, IRF7, TRIM25, and NLRX1. smFISH experiments revealed two distinct populations of cells which would otherwise be obscured in ensemble measurements: IFN-b expressing and IFN-b non-expressing cells. Simultaneous counting of IFN-b, RIG-I and SeV L gene mRNA and correlation analysis in individual cells revealed that transcriptional activation of IFN-b is dependent on the level of RIG-I mRNA in individual cells that is IFN-independently induced at the early stages of viral infection but is independent of the level of viral replication.