title: Activation of RNase L is dependent on OAS3 expression during infection with diverse human viruses.
abstract: The 2',5'-oligoadenylate (2-5A) synthetase (OAS)-RNase L system is an IFN-induced antiviral pathway. RNase L activity depends on 2-5A, synthesized by OAS. Although all three enzymatically active OAS proteins in humans--OAS1, OAS2, and OAS3--synthesize 2-5A upon binding dsRNA, it is unclear which are responsible for RNase L activation during viral infection. We used clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein-9 nuclease (Cas9) technology to engineer human A549-derived cell lines in which each of the OAS genes or RNase L is knocked out. Upon transfection with poly(rI):poly(rC), a synthetic surrogate for viral dsRNA, or infection with each of four viruses from different groups (West Nile virus, Sindbis virus, influenza virus, or vaccinia virus), OAS1-KO and OAS2-KO cells synthesized amounts of 2-5A similar to those synthesized in parental wild-type cells, causing RNase L activation as assessed by rRNA degradation. In contrast, OAS3-KO cells synthesized minimal 2-5A, and rRNA remained intact, similar to infected RNase L-KO cells. All four viruses replicated to higher titers in OAS3-KO or RNase L-KO A549 cells than in parental, OAS1-KO, or OAS2-KO cells, demonstrating the antiviral effects of OAS3. OAS3 displayed a higher affinity for dsRNA in intact cells than either OAS1 or OAS2, consistent with its dominant role in RNase L activation. Finally, the requirement for OAS3 as the major OAS isoform responsible for RNase L activation was not restricted to A549 cells, because OAS3-KO cells derived from two other human cell lines also were deficient in RNase L activation.
authors: Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104;
authors: Yize
authors: Y
authors: Li
authors: Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic, Cleveland, OH 44195;
authors: Shuvojit
authors: S
authors: Banerjee
authors: Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104; Key Laboratory of Medical Molecular Virology, Department of Medical Microbiology, School of Basic Medical Sciences, Shanghai Medical College, Fudan University, Shanghai 200032, China.
authors: Yuyan
authors: Y
authors: Wang
authors: Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104;
authors: Stephen A
authors: SA
authors: Goldstein
authors: Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic, Cleveland, OH 44195;
authors: Beihua
authors: B
authors: Dong
authors: Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic, Cleveland, OH 44195;
authors: Christina
authors: C
authors: Gaughan
authors: Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic, Cleveland, OH 44195; silverr@ccf.org weisssr@upenn.edu.
authors: Robert H
authors: RH
authors: Silverman
authors: Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104; silverr@ccf.org weisssr@upenn.edu.
authors: Susan R
authors: SR
authors: Weiss
mesh_terms: D015088:2',5'-Oligoadenylate Synthetase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D018354:Alphavirus Infections / Q000235:genetics / Q000378:metabolism; D064113:CRISPR-Cas Systems; D002460:Cell Line; D004722:Endoribonucleases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation; D055786:Gene Knockout Techniques; D006801:Humans; D007251:Influenza, Human / Q000235:genetics / Q000378:metabolism; D008954:Models, Biological; D012335:RNA, Ribosomal / Q000235:genetics / Q000378:metabolism; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism; D012845:Sindbis Virus; D014615:Vaccinia / Q000235:genetics / Q000378:metabolism; D014777:Virus Diseases / Q000235:genetics / Q000378:metabolism*; D014901:West Nile Fever / Q000235:genetics / Q000378:metabolism
chemical_list: D012335:RNA, Ribosomal; D012367:RNA, Viral; C475085:OAS1 protein, human; C000607948:OAS2 protein, human; D015088:2',5'-Oligoadenylate Synthetase; C577439:OAS3 protein, human; D004722:Endoribonucleases; C031956:2-5A-dependent ribonuclease
keywords: 2-5A; antiviral response; oligoadenylate synthetase 3; ribonuclease L; type I interferon