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

Abstract

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 an in vivo melanoma 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 knockout 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 σ3 viral protein were found to control activation of RIG-I/ IFNindependent genes. Altogether, the study reveals that single amino acid polymorphisms in reovirus genomes can have large impact on host gene expression, by both changing

The reovirus σ3 protein inhibits NF-κB-dependent antiviral signaling

“…Closely related reovirus strains are known to display differences in a variety of phenotypes (29)(30)(31). In previous work we showed that in cells infected with reovirus strain T3A, TNFα fails to induce the expression of NF-κB target genes due to loss of IKKβ and NEMO (13).…”

“…We also demonstrate that in reovirus infected cells, properties of de novo synthesized σ3 impact the expression of NF-κB target genes. Curiously, polymorphic differences in σ3 between T3D L and T3D F (respectively referred to as T3D-PL and T3D-TD in these studies) have recently been demonstrated to regulate host gene expression (30). T3D L σ3 contributes to the greater capacity of T3D L to express RIG-I-and IFNindependent genes in comparison to T3D F .…”

The Paradoxes of Viral mRNA Translation during Mammalian Orthoreovirus Infection

“…Although there is no evidence showing reovirus dsRNA outside of VFs, the dsRNA in rotavirus-infected cells was detected in the cytoplasm outside of viroplasms, the site of rotavirus replication and assembly [ 105 ]. Multiple studies have shown that reovirus infection induces type I and type III IFN production and the up-regulation of multiple IFN-stimulated genes [ 106 , 107 , 108 ]. Viral genome dsRNA or dsRNA formed by secondary structures within viral mRNAs can stimulate a cascade of antiviral events.…”

“…It is important to note differences between the two studies that may explain the differing results. Firstly, the oncolytic strain of T3D is genetically different to the T3D strain used by many labs and importantly has nonsynonomous changes within the S4 gene segment that affect replication and induction of the type I interferon response [ 108 , 123 ]. In addition, the study investigating reovirus-induced host shutoff infected cells at a high multiplicity of infection (80 PFU per cell) [ 114 ], whereas the study using the oncolytic strain of T3D used a lower multiplicity (10 PFU per cell) that does not induce host shutoff [ 122 ].…”

Captivating Perplexities of Spinareovirinae 5′ RNA Caps

“…For instance, given recent discoveries that the reovirus-translating ribosomes segregate in virus factories, did the methods by Skup, Zarbl and Millward also capture these species? Furthermore, given the recognition that reoviruses vary genetically and phenotypically from each other, even among different laboratory strains of serotype 3 Dearing (T3D) MRV, it would be interesting to determine whether uncapped RNAs are common to a broader collection of reovirus isolates [ 41 , 42 , 43 ]. Similarly, is this a unique characteristic of L929 cells or can it be observed in other cell types permissive to reovirus infection?…”