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The Essential Role of Double-Stranded RNA–Dependent Antiviral Signaling in the Degradation of Nonself Single-Stranded RNA in Nonimmune Cells

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The Essential Role of Double-Stranded RNA–Dependent Antiviral Signaling in the Degradation of Nonself Single-Stranded RNA in Nonimmune Cells Published June 20, 2018, doi:10.4049/jimmunol.1800456 The Journal of Immunology The Essential Role of Double-Stranded RNA–Dependent Antiviral Signaling in the Degradation of Nonself Single-Stranded RNA in Nonimmune Cells Sayaka Kimura,* Tomoh Matsumiya,* Yuko Shiba,* Michi Nakanishi,* Ryo Hayakari,* Shogo Kawaguchi,† Hidemi Yoshida,* and Tadaatsu Imaizumi* The recognition of nonself dsRNA by retinoic acid–inducible gene-I (RIG-I) leads to the engagement of RIG-I–like receptor signaling. In addition, nonself dsRNA triggers a robust latent RNase (RNase L) activation and leads to the degradation of ribosomal structures and cell death. In contrast, nonself ssRNA is known to be recognized by TLR 7/8 in immune cells such as plasmacytoid dendritic cells and B cells, but little is known regarding the involvement of nonself ssRNA in antiviral signaling in nonimmune cells, including epithelial cells. Moreover, the fate of intracellular nonself ssRNA remains unknown. To address this issue, we developed a quantitative RT-PCR–based approach that monitors the kinetics of nonself ssRNA cleavage following the transfection of HeLa human cervical carcinoma cells, using model nonself ssRNA. We discovered that the degradation of ssRNA is independent of RIG-I and type I IFN signaling because ssRNA did not trigger RIG-I–mediated antiviral signaling. We also found that the kinetics of self (59-capped) and nonself ssRNA decay were unaltered, suggesting that nonself ssRNA is not recognized by nonimmune cells. We further demonstrated that the cleavage of nonself ssRNA is accelerated when nonself dsRNA is also introduced into cells. In addition, the cleavage of nonself ssRNA is completely abolished by knockdown of RNase L. Overall, our data demonstrate the important role of dsRNA–RNase L in nonself ssRNA degradation and may partly explain the positive regulation of the antiviral responses in nonimmune cells. The Journal of Immunology, 2018, 201: 000–000. ammalian antiviral innate immune responses are the type I IFN–mediated antiviral responses [see (7) for an excellent first line of defense against viruses. Following infec- review]. For example, signals elicited by dsRNA-dependent protein M tion, viruses expose their nucleic acids to the cyto- kinase R (PKR)–eukaryotic initiation factor 2a inhibit initiation of plasm of host cells. In nonimmune cells, such as epithelial cells, protein translation and viral replication (8). retinoic acid–inducible gene-I (RIG-I)–like receptors (RLRs) An additional strategy whereby cells achieve protection against serve as cytoplasmic viral RNA sensors (1). RLRs display distinct infection involves the degradation of viral RNA via latent RNase RNA specificities for individual classes of viruses (2). For example, (RNase L), an enzyme that cleaves both cellular and viral RNAs (9). RIG-I senses relatively short dsRNA (3, 4). In contrast, the RLR RNase L2/2 mice have an increased susceptibility to viral infections melanoma differentiation-associated gene-5 (MDA5) senses long (10), which indicates the fundamental role of RNase L in antiviral dsRNAs. Recognition of nonself RNA is followed by RLR asso- defense responses. Homodimerization of RNase L is required for ciation with the adaptor protein MAVS (mitochondrial antiviral- catalytic activity and is regulated by nonself RNA–mediated acti- signaling protein) (5), which leads to the activation of antiviral vation of OAS (11). A recent study identified OAS3 as the isoform signaling events that result in the production of type I IFNs and the involved in RNase L activation (12). In certain settings, RNase L expression of a variety of IFN-stimulated genes (6), some of which can also deregulate ribosomal function by cleaving rRNA in re- (e.g., 2’,59-oligoadenylate synthase [OAS]) have antiviral proper- sponse to viral infection (13) and can result in translational inhibi- ties. Several groups have investigated the mechanisms involved in tory effects that impact both host and viral protein biosynthesis. These RNase L–mediated effects on host cells may account for the ability of this enzyme to induce apoptosis (14, 15). *Department of Vascular Biology, Institute of Brain Science, Hirosaki University Graduate School of Medicine, Hirosaki 036-8562, Japan; and †Department of Gas- In contrast to dsRNA, little is known about how nonself ssRNA is troenterology and Hematology, Hirosaki University Graduate School of Medicine, controlled in the intracellular innate immune system. In this study, Hirosaki 036-8562, Japan we developed a quantitative PCR–based approach to monitor Received for publication March 26, 2018. Accepted for publication June 1, 2018. nonself ssRNA–mediated antiviral signaling and the kinetics of This work was supported by Grants-in-Aid for Scientific Research (KAKENHI) nonself ssRNA decay using in vitro transcription (IVT)–generated (17K10012 to T.M.), the Takeda Science Foundation (to T.M.), and the Karoji Memorial Fund (to T.M.). non-mammalian ssRNA, followed by gel purification. Our results show that nonself RNA does not trigger intracellular innate im- Address correspondence and reprint requests to Dr. Tomoh Matsumiya, Department of Vascular Biology, Institute of Brain Science, Hirosaki University Graduate School mune responses in nonimmune cells. We also found that the of Medicine, 5 Zaifu-cho, Hirosaki 036-8562, Aomori, Japan. E-mail address: cleavage of nonself ssRNA required dsRNA-mediated activation [email protected] of antiviral responses. Our findings may explain how to execute The online version of this article contains supplemental material. nonself ssRNA in nonimmune cells. Abbreviations used in this article: bla, b-lactamase; cDC, conventional DC; CL, cationic lipid; DC, dendritic cell; GM-DC, GM-CSF–induced DC; IVT, in vitro transcription, in vitro transcribed; luc, luciferase; mbla, capped bla; mluc, capped luc; OAS, 2’,59-oligoadenylate synthase; pDC, plasmacytoid DC; qRT-PCR, quanti- Materials and Methods tative RT-PCR; RIG-I, retinoic acid–inducible gene-I; RLR, RIG-I–like receptor; Cell culture RNase L, latent RNase; siRNA, small interfering RNA; UTR, untranslated region. HeLa cells (Japanese Cancer Resources Bank, Ibaraki, Japan) were Copyright Ó 2018 by The American Association of Immunologists, Inc. 0022-1767/18/$35.00 maintained in a 5% CO2 atmosphere at 37˚C in DMEM (Sigma-Aldrich, www.jimmunol.org/cgi/doi/10.4049/jimmunol.1800456 2 dsRNA CLEAVES NONSELF ssRNA IN NONIMMUNE CELLS St. Louis, MO) supplemented with 10% FBS (Perbio Science, Switzerland) Immunoblot analyses and antibiotics. For the immunoblot analyses, we washed the cells twice with PBS and then Preparation of dendritic cells lysed them in hypotonic lysis buffer (10 mM Tris [pH 7.4], 100 mM NaCl, 1.5 mM MgCl2, and 0.5% NP-40] containing 0.2% protease inhibitors Preparation of bone marrow dendritic cells (DCs) from female ICR mouse (Sigma-Aldrich). The lysates were cleared by centrifugation at 6000 rpm femora (8 wk old) was performed as Kato et al. (16) reported. Briefly, bone for 15 min at 4˚C. Aliquots of the supernatants (10 mg) were subjected to marrow cells were cultured in RPMI 1640 (Sigma-Aldrich) supplemented electrophoresis on 10% SDS-polyacrylamide gels. The proteins were m with 10% FBS, 100 M 2-ME (Sigma-Aldrich), and 10 ng/ml recombinant transferred to polyvinylidene fluoride membranes (Millipore, Billerica, murine GM-CSF (PeproTech, Rocky Hill, NJ) or 100 ng/ml recombinant MA), which were then blocked for 1 h at room temperature in TBST buffer human Flt3 ligand (PeproTech). After 8 d, the cells were collected and (20 mM Tris [pH 7.4], 150 mM NaCl, 0.1% Tween-20) containing 5% used as DCs (Flt3 ligand–induced DCs [Flt-DCs]) or GM-CSF–induced nonfat dry milk (blocking buffer). The membranes were incubated over- DCs (GM-DCs), respectively. To culture GM-DCs, the medium was night at 4˚C with one of the following primary Abs: mouse anti-RIG-I replaced with fresh medium containing GM-CSF every 2 d. All the animal (Enzo Life Sciences, Miami, FL), mouse anti-RNase L (BD Biosciences, experiments in this study were approved by the Animal Research Com- San Jose, CA), or rabbit anti-b-actin (Sigma-Aldrich). After five washes mittee at Hirosaki University and were conducted according to the with TBST, the membranes were further incubated for 1 h at room tem- Guidelines for Animal Experimentation, Hirosaki University, Japan. perature with HRP-labeled bovine anti-rabbit (Santa Cruz Biotechnology, Generation of in vitro-transcribed RNAs Santa Cruz, CA) or goat anti-mouse IgG Abs (Thermo Fisher Scientific) in blocking buffer. The washes were repeated using TBST, and the immu- The sense and antisense ssRNAs for b-lactamase (bla) and bla dsRNA were noreactive proteins were then visualized using Luminata Crescendo generated as described previously (17). Firefly luciferase (luc) ssRNA was Western HRP Substrate (Millipore). synthesized as a template using a T7 RiboMAX Large Scale RNA Pro- duction kit and contained the linearized full-length luc gene harboring the Blue native PAGE T7 promoter (Promega Life Sciences, Madison, WI). The length of the HeLa cells were harvested in NativePAGE sample buffer (Thermo Fisher ∼ 9 IVT-ssRNAs was 900 (bla) and 1800 bp (luc). The 5 -capped bla (mbla) Scientific) containing 1% n-dodecyl-D-maltoside. The lysate was then and luc RNAs were synthesized using a mMESSAGE mMACHINE kit centrifuged at 20,000 3 g at 4 ˚C for 30 min to collect the soluble fraction. (Thermo Fisher Scientific, Waltham, MA). The samples were loaded onto 4–16% Novex Bis-Tris protein gels Transfection (Thermo Fisher Scientific). NativeMark unstained protein standards (Thermo Fisher Scientific) were used as molecular mass markers. The Transient transfections were performed as previously reported (18). Briefly, details of the blue native (BN) PAGE protocols were those provided in the cells were seeded into 12-well culture plates at a density of 5 3 104 cells instruction manual from Thermo Fisher Scientific. Before protein transfer, per well. After 16 h, the cells reached 30–50% confluence and were used the gels were washed with transfer buffer containing 0.05% SDS for for the studies requiring transfection with small interfering RNA (siRNA).
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