Closely related reovirus lab strains induce opposite expression of RIG-I/IFN-dependent versus -independent host genes, via mechanisms of slow replication versus polymorphisms in dsRNA binding σ3 respectively.

Author summary Mammalian orthoreovirus serotype 3 Dearing (T3D reovirus) is being explored as a cancer therapy. Laboratories worldwide use independent strains of T3D, that we previously demonstrated to have different oncolytic potencyin vivo; but what impact does the small genomic diversity between T3D reovirus strains have on host responses? Herein, we discover that host response varies significantly between T3D lab strains; for example, the T3D(TD)strain causes higher IFN induction and expression of IFN-dependent cytokines, while the T3D(PL)strain conversely causes higher induction of RIG-I/IFN-independent genes and cytokines. Mechanistically, higher IFN signalling is caused by gene polymorphisms in S4, M1, or L3 reovirus genes that decelerate virus replication kinetics, while higher non-IFN gene induction is attributed to polymorphisms in the reovirus S4 gene-encoded dsRNA binding protein sigma 3. Unexpectedly, IFN induction by the slow T3D(TD)strain phenodominated during T3D(TD)/ T3D(PL)coinfections, suggesting distinct behaviours of incoming virions. Moreover, IFN responses had minimal effects on the first round of reovirus replication. Overall, the data reveals how single amino acid changes can profoundly and even inversely affect host cell response to reovirus. The prospect of manipulating reovirus replication kinetics and sigma 3 could help optimize the host response for greatest oncolytic and immunotherapeutic potency. The Dearing isolate of Mammalian orthoreovirus (T3D) is a prominent model of virus-host relationships and a candidate oncolytic virotherapy. Closely related laboratory strains of T3D, originating from the same ancestral T3D isolate, were recently found to exhibit significantly different oncolytic properties. Specifically, the T3D(PL)strain had faster replication kinetics in a panel of cancer cells and improved tumor regression in anin vivomelanoma model, relative to T3D(TD). In this study, we discover that T3D(PL)and T3D(TD)also differentially activate host signalling pathways and downstream gene transcription. At equivalent infectious dose, T3D(TD)induces higher IRF3 phosphorylation and expression of type I IFNs and IFN-stimulated genes (ISGs) than T3D(PL). Using mono-reassortants with intermediate replication kinetics and pharmacological inhibitors of reovirus replication, IFN responses were found to inversely correlate with kinetics of virus replication. In other words, slow-replicating T3D strains induce more IFN signalling than fast-replicating T3D strains. Paradoxically, during co-infections by T3D(PL)and T3D(TD), there was still high IRF3 phosphorylation indicating a phenodominant effect by the slow-replicating T3D(TD). Using silencing and knock-out of RIG-I to impede IFN, we found that IFN induction does not affect the first round of reovirus replication but does prevent cell-cell spread in a paracrine fashion. Accordingly, during co-infections, T3D(PL)continues to replicate robustly despite activation of IFN by T3D(TD). Using gene expression analysis, we discovered that reovirus can also induce a subset of genes in a RIG-I and IFN-independent manner; these genes were induced more by T3D(PL)than T3D(TD). Polymorphisms in reovirus sigma 3 viral protein were found to control activation of RIG-I/ IFN-independent genes. Altogether, the study reveals that single amino acid polymorphisms in reovirus genomes can have large impact on host gene expression, by both changing replication kinetics and by modifying viral protein activity, such that two closely related T3D strains can induce opposite cytokine landscapes.