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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 ?3 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 T3DPL strain had faster replication kinetics in a panel of cancer cells and improved tumor regression in an in vivo melanoma model, relative to T3DTD. In this study, we discover that T3DPL and T3DTD also differentially activate host signalling pathways and downstream gene transcription. At equivalent infectious dose, T3DTD induces higher IRF3 phosphorylation and expression of type I IFNs and IFN-stimulated genes (ISGs) than T3DPL. 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 T3DPL and T3DTD, there was still high IRF3 phosphorylation indicating a phenodominant effect by the slow-replicating T3DTD. 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, T3DPL continues to replicate robustly despite activation of IFN by T3DTD. 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 T3DPL than T3DTD. Polymorphisms in reovirus ?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.

SUBMITTER: Mohamed A

PROVIDER: S-EPMC7529228 | biostudies-literature | 2020 Sep

REPOSITORIES: biostudies-literature

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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.

Mohamed Adil A Konda Prathyusha P Eaton Heather E HE Gujar Shashi S Smiley James R JR Shmulevitz Maya M

PLoS pathogens 20200921 9

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 T3DPL strain had faster replication kinetics in a panel of cancer cells and improved tumor regression in an in vivo melanoma model, relative to T3DTD. In this study, we dis ...[more]

PMID: 32956403

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Project description:Little is known about how genetic variations in viruses affect their success as therapeutic agents. The type 3 Dearing strain of Mammalian orthoreovirus (T3D) is undergoing clinical trials as an oncolytic virotherapy. Worldwide, studies on reovirus oncolysis use T3D stocks propagated in different laboratories. Here, we report that genetic diversification among T3D stocks from various sources extensively impacts oncolytic activity. The T3D strain from the Patrick Lee laboratory strain (TD3PL) showed significantly stronger oncolytic activities in a murine model of melanoma than the strain from the Terence Dermody laboratory (T3DTD). Overall in vitro replication and cytolytic properties of T3D laboratory strains were assessed by measuring virus plaque size on a panel of human and mouse tumor cells, and results were found to correlate with in vivo oncolytic potency in a melanoma model. T3DPL produced larger plaques than T3DTD and than the T3D strain from the ATCC (T3DATCC) and from the Kevin Coombs laboratory (T3DKC). Reassortant and reverse genetics analyses were used to decipher key genes and polymorphisms that govern enhanced plaque size of T3DPL Five single amino acid changes in the S4, M1, and L3 genome segments of reovirus were each partially correlated with plaque size and when combined were able to fully account for differences between T3DPL and T3DTD Moreover, polymorphisms were discovered in T3DTD that promoted virus replication and spread in tumors, and a new T3DPL/T3DTD hybrid was generated with enhanced plaque size compared to that of T3DPL Altogether, single amino acid changes acquired during laboratory virus propagation can have a large impact on reovirus therapeutic potency and warrant consideration as possible confounding variables between studies.IMPORTANCE The reovirus serotype 3 Dearing (T3D) strain is in clinical trials for cancer therapy. We find that closely related laboratory strains of T3D exhibit large differences in their abilities to replicate in cancer cells in vitro, which correlates with oncolytic activity in a in a murine model of melanoma. The study reveals that five single amino acid changes among three reovirus genes strongly impact reovirus therapeutic potency. In general, the findings suggest that attention should be given to genomic divergence of virus strains during research and optimization for cancer therapy.

Project description:Grass carp reovirus (GCRV), the most virulent aquareovirus, causes epidemic hemorrhagic disease and tremendous economic loss in freshwater aquaculture industry. VP56, a putative fibrin inlaying the outer surface of GCRV-II and GCRV-III, is involved in cell attachment. In the present study, we found that VP56 localizes at the early endosome, lysosome, and endoplasmic reticulum, recruits the cytoplasmic viral RNA sensor retinoic acid-inducible gene I (RIG-I) and binds to it. The interaction between VP56 and RIG-I was detected by endogenous coimmunoprecipitation (co-IP), glutathione S-transferase (GST) pulldown, and subsequent liquid chromatography-tandem mass spectrometry (LC-MS/MS) and was then confirmed by traditional co-IPs and a novel far-red mNeptune-based bimolecular fluorescence complementation system. VP56 binds to the helicase domain of RIG-I. VP56 enhances K48-linked ubiquitination of RIG-I to degrade it by the proteasomal pathway. Thus, VP56 impedes the initial immune function of RIG-I by dual mechanisms (blockade and degradation) and attenuates signaling from RIG-I recognizing viral RNA, subsequently weakening downstream signaling transduction and interferon (IFN) responses. Accordingly, host antiviral effectors are reduced, and cytopathic effects are increased. These findings were corroborated by RNA sequencing (RNA-seq) and VP56 knockdown. Finally, we found that VP56 and the major outer capsid protein VP4 bind together in the cytosol to enhance the degradation of RIG-I and more efficiently facilitate viral replication. Collectively, the results indicated that VP56 allies VP4, recruits, blocks, and degrades RIG-I, thereby attenuating IFNs and antiviral effectors to facilitate viral evasion more effectively. This study reveals a virus attacking target and an escaping strategy from host antiviral immunity for GCRV and will help understand mechanisms of infection of reoviruses. IMPORTANCE Grass carp reovirus (GCRV) fibrin VP56 and major outer capsid protein VP4 inlay and locate on the outer surface of GCRV-II and GCRV-III, which causes tremendous loss in grass carp and black carp industries. Fibrin is involved in cell attachment and plays an important role in reovirus infection. The present study identified the interaction proteins of VP56 and found that VP56 and VP4 bind to the different domains of the viral RNA sensor retinoic acid-inducible gene I (RIG-I) in grass carp to block RIG-I sensing of viral RNA and induce RIG-I degradation by the proteasomal pathway to attenuate signaling transduction, thereby suppressing interferons (IFNs) and antiviral effectors, facilitating viral replication. VP56 and VP4 bind together in the cytosol to more efficiently facilitate viral evasion. This study reveals a virus attacking a target and an escaping strategy from host antiviral immunity for GCRV and will be helpful in understanding the mechanisms of infection of reoviruses.

Dataset Information

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 ?3 respectively.

Publications

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.

Similar Datasets

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