title: The 2'-5'-oligoadenylate synthetase 3 enzyme potently synthesizes the 2'-5'-oligoadenylates required for RNase L activation.
abstract: The members of the oligoadenylate synthetase (OAS) family of proteins are antiviral restriction factors that target a wide range of RNA and DNA viruses. They function as intracellular double-stranded RNA (dsRNA) sensors that, upon binding to dsRNA, undergo a conformational change and are activated to synthesize 2'-5'-linked oligoadenylates (2-5As). 2-5As of sufficient length act as second messengers to activate RNase L and thereby restrict viral replication. We expressed human OAS3 using the baculovirus system and purified it to homogeneity. We show that recombinant OAS3 is activated at a substantially lower concentration of dsRNA than OAS1, making it a potent in vivo sensor of dsRNA. Moreover, we find that OAS3 synthesizes considerably longer 2-5As than previously reported, and that OAS3 can activate RNase L intracellularly. The combined high affinity for dsRNA and the capability to produce 2-5As of sufficient length to activate RNase L suggests that OAS3 is a potent activator of RNase L. In addition, we provide experimental evidence to support one active site of OAS3 located in the C-terminal OAS domain and generate a low-resolution structure of OAS3 using SAXS.
  
  
  OBJECTIVE
  We are the first to purify the OAS3 enzyme to homogeneity, which allowed us to characterize the mechanism utilized by OAS3 and identify the active site. We provide compelling evidence that OAS3 can produce 2'-5'-oligoadenylates of sufficient length to activate RNase L. This is contrary to what is described in the current literature but agrees with recent in vivo data showing that OAS3 harbors an antiviral activity requiring RNase L. Thus, our work redefines our understanding of the biological role of OAS3. Furthermore, we used a combination of mutagenesis and small-angle X-ray scattering to describe the active site and low-resolution structure of OAS3.
authors.affiliation: Centre for Structural Biology, Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark.
authors.forename: Mikkel Søes
authors.initials: MS
authors.lastname: Ibsen
authors.affiliation: Centre for Structural Biology, Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark.
authors.forename: Hans Henrik
authors.initials: HH
authors.lastname: Gad
authors.affiliation: Centre for Structural Biology, Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark.
authors.forename: Karthiga
authors.initials: K
authors.lastname: Thavachelvam
authors.affiliation: Centre for Structural Biology, Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark.
authors.forename: Thomas
authors.initials: T
authors.lastname: Boesen
authors.affiliation: Unité Interactions Moléculaires Flavivirus-Hôtes, Institut Pasteur, Paris, France.
authors.forename: Philippe
authors.initials: P
authors.lastname: Desprès
authors.affiliation: Centre for Structural Biology, Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark rh@mb.au.dk.
authors.forename: Rune
authors.initials: R
authors.lastname: Hartmann
mesh_terms: D015088:2',5'-Oligoadenylate Synthetase / Q000378:metabolism*; D000227:Adenine Nucleotides / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D016367:Baculoviridae / Q000235:genetics; D020134:Catalytic Domain; D004722:Endoribonucleases / Q000378:metabolism*; D004789:Enzyme Activation; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009843:Oligoribonucleotides / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011487:Protein Conformation; D012330:RNA, Double-Stranded / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D053838:Scattering, Small Angle; D016415:Sequence Alignment
chemical_list: D000227:Adenine Nucleotides; D009843:Oligoribonucleotides; D012330:RNA, Double-Stranded; D011994:Recombinant Proteins; C023505:2',5'-oligoadenylate; D015088:2',5'-Oligoadenylate Synthetase; C577439:OAS3 protein, human; D004722:Endoribonucleases; C031956:2-5A-dependent ribonuclease