The Multi-Functional Reovirus Σ3 Protein Is a Virulence Factor That Suppresses Stress Granule Formation to Allow Viral Replicat
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bioRxiv preprint doi: https://doi.org/10.1101/2021.03.22.436456; this version posted March 22, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 The multi-functional reovirus σ3 protein is a 2 virulence factor that suppresses stress granule 3 formation to allow viral replication and myocardial 4 injury 5 6 Yingying Guo1, Meleana Hinchman1, Mercedes Lewandrowski1, Shaun Cross1,2, Danica 7 M. Sutherland3,4, Olivia L. Welsh3, Terence S. Dermody3,4,5, and John S. L. Parker1* 8 9 1Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, 10 Ithaca, New York 14853; 2Cornell Institute of Host-Microbe Interactions and Disease, 11 Cornell University, Ithaca, New York 14853; Departments of 3Pediatrics and 12 4Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, 13 Pittsburgh, PA 15224; and 5Institute of Infection, Inflammation, and Immunity, UPMC 14 Children’s Hospital of Pittsburgh, PA 15224 15 16 17 Running head: REOVIRUS SIGMA3 PROTEIN SUPPRESSES STRESS GRANULES 18 DURING INFECTION 19 20 * Corresponding author. Mailing address: Baker Institute for Animal Health, College 21 of Veterinary Medicine, Cornell University, Hungerford Hill Road; Ithaca, NY 14853. 22 Phone: (607) 256-5626. Fax: (607) 256-5608. E-mail: [email protected] 23 Word count for abstract: 261 24 Word count for text: 12282 1 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.22.436456; this version posted March 22, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 25 Abstract 26 The mammalian orthoreovirus double-stranded (ds) RNA binding protein σ3 is a multifunctional 27 protein that promotes viral protein synthesis and facilitates viral entry and assembly. The 28 dsRNA-binding capacity of σ3 correlates with its capacity to prevent dsRNA-mediated activation 29 of protein kinase R (PKR). However, the effect of σ3 binding to dsRNA during viral infection 30 remains largely unknown. To identify functions of σ3 dsRNA-binding activity during reovirus 31 infection, we engineered a panel of 13 σ3 mutants and screened them for the capacity to bind 32 dsRNA. Six mutants were defective in dsRNA binding, and mutations in these constructs cluster 33 in a putative dsRNA-binding region on the surface of σ3. Two recombinant viruses expressing 34 these σ3 dsRNA-binding mutants, K287T and R296T, display strikingly different phenotypes. In 35 a cell-type dependent manner, K287T, but not R296T, replicates less efficiently than wild-type 36 (WT) virus. In cells in which K287T virus demonstrates a replication deficit, PKR activation 37 occurs and abundant stress granules (SGs) are produced at late times post-infection. In 38 contrast, the R296T virus retains the capacity to suppress activation of PKR and does not form 39 SGs at late times post-infection. These findings indicate that σ3 inhibits PKR independently of 40 its capacity to bind dsRNA. In infected mice, K287T produces lower viral titers in the spleen, 41 liver, lungs, and heart relative to WT or R296T. Moreover, mice inoculated with WT or R296T 42 viruses develop myocarditis, whereas those inoculated with K287T do not. Overall, our results 43 indicate that σ3 functions to suppress PKR activation and subsequent SG formation during viral 44 infection and that these functions correlate with virulence in mice. 2 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.22.436456; this version posted March 22, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 45 Introduction 46 Viral infection induces host cell responses that act to inhibit viral replication. A strong catalyst for 47 these host responses is the synthesis of double-stranded RNA (dsRNA) during viral replication. 48 Binding of dsRNA to innate immune sensors such as Toll-like receptor 3, retinoic acid-inducible 49 gene I (RIG-I), and melanoma differentiation-association protein 5 (MDA5) initiates signaling 50 that leads to secretion of type I and type III interferons (IFNs) [1]. IFNs then bind to their cognate 51 cell-surface receptors to induce signaling and transcription of a large suite of IFN-stimulated 52 antiviral genes [2]. 53 Viral dsRNA produced during infection also can bind to other cellular innate immune sensors. 54 Two well-characterized dsRNA-binding sensors are protein kinase R (PKR) and 2'-5'- 55 oligoadenylate synthase I (OAS). Following binding to dsRNA, these sensors activate signaling 56 pathways that lead to inhibition of translation initiation and degradation of mRNA [3,4]. PKR is 57 one of four cellular kinases, general amino acid control nonderepressible 2 (GCN2), heme- 58 regulated eIF2α kinase (HRI), PKR, and PKR-like endoplasmic reticulum kinase (PERK), that 59 can phosphorylate the α subunit of eukaryotic initiation factor 2 (eIF2) [5,6]. Phosphorylation of 60 eIF2α leads to sequestration of the GDP-GTP exchange factor eIF2B in an inactive GDP-bound 61 complex with eIF2. This sequestration rapidly decreases the concentration of the 62 eIF2•GTP•methionyl-initiator tRNA ternary complex (TC). As a consequence, translation 63 initiation is inhibited and the integrated stress response is activated [5]. 64 Another consequence of PKR activation is the assembly of cytoplasmic ribonucleoprotein (RNP) 65 stress granules (SGs) [7]. Cellular and viral mRNAs that stall on 43S ribosomes because of 66 decreased availability of TCs are sequestered in SGs [8]. Cytosolic pathogen recognition 67 receptors such as PKR, MDA5, and RIG-I concentrate in SGs and, thus, SGs are hypothesized 68 to act as signaling hubs for cellular antiviral responses [9]. Many viruses have evolved 69 mechanisms to block or overcome the activation of PKR and prevent SG formation. For 3 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.22.436456; this version posted March 22, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 70 example, influenza A virus nonstructural protein 1 (NS1) binds to PKR and blocks its activation, 71 thereby preventing PKR-mediated phosphorylation of eIF2α and SG formation [10]; poliovirus 72 strains block SG formation by 3C-mediated cleavage of the SG-nucleating protein, GTPase- 73 activating protein-binding protein 1 (G3BP1) [11]. SGs are not the only type of RNP-containing 74 granules that can form in cells in response to dsRNA. While SGs are the most well 75 characterized, RNP granules that are RNase L-dependent and PKR-independent also form in 76 cells treated with poly(I:C) (a dsRNA mimic). These granules are termed RNase L-dependent 77 bodies (RLBs) and are proposed to be induced during viral infection [12]. Whether RLBs are 78 induced during reovirus infection is not known. 79 Mammalian ortheoreoviruses (reoviruses) are nonenveloped viruses that package a genome of 80 10 segments of dsRNA enclosed in a double-layered capsid. The outer-capsid layer serves to 81 attach the virion to host cells and mediates viral penetration of cell membranes. Outer-capsid 82 proteins are removed during viral entry to deposit a viral core particle in the cytoplasm. 83 Transcription of the viral genome begins when the RNA-dependent RNA polymerase packaged 84 in the core particle is activated after removal of the outer-capsid proteins. Viral mRNAs are 85 extruded through pores at the five-fold axes of symmetry. During passage through turrets, the 86 viral mRNAs are capped by guanyltransferase and methyltransferase enzymes [13]. 87 Because reoviruses contain a dsRNA genome, they may have evolved mechanisms to prevent 88 dsRNA-mediated activation of host innate defense mechanisms. One mechanism in which 89 reoviruses likely avoid activating these sensors is by synthesizing their dsRNA genome 90 segments in a viral core particle following encapsidation of single-stranded viral mRNAs 91 representing each genome segment [14]. Reoviruses also encode a protein that can bind 92 dsRNA, the viral outer-capsid protein, σ3 [15]. Overexpression of wild-type (WT) σ3, but not a 93 dsRNA-binding-deficient mutant of σ3, rescues the inhibition of translation induced by PKR 94 overexpression [16]. In addition, overexpression of σ3 rescues replication of E3L-deficient 4 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.22.436456; this version posted March 22, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 95 vaccinia virus and VAI-deficient adenoviruses, demonstrating that σ3 can complement other 96 PKR-inhibiting viral proteins [17,18]. These findings led to the hypothesis that σ3 absorbs 97 dsRNA in infected cells, thereby preventing PKR activation and global translational inhibition. 98 However, this model does not explain why some strains of reoviruses replicate less efficiently in 99 PKR-deficient mouse embryonic fibroblasts (MEFs) [19], where dsRNA-activation of PKR 100 cannot occur, or why reoviruses replicate less efficiently in MEFs that only express a non- 101 phosphorylatable form of eIF2α, findings that suggest reovirus replication benefits from 102 activation of the integrated stress response [20].