title: ADP-ribosylhydrolase activity of Chikungunya virus macrodomain is critical for virus replication and virulence.
abstract: Chikungunya virus (CHIKV), an Old World alphavirus, is transmitted to humans by infected mosquitoes and causes acute rash and arthritis, occasionally complicated by neurologic disease and chronic arthritis. One determinant of alphavirus virulence is nonstructural protein 3 (nsP3) that contains a highly conserved MacroD-type macrodomain at the N terminus, but the roles of nsP3 and the macrodomain in virulence have not been defined. Macrodomain is a conserved protein fold found in several plus-strand RNA viruses that binds to the small molecule ADP-ribose. Prototype MacroD-type macrodomains also hydrolyze derivative linkages on the distal ribose ring. Here, we demonstrated that the CHIKV nsP3 macrodomain is able to hydrolyze ADP-ribose groups from mono(ADP-ribosyl)ated proteins. Using mass spectrometry, we unambiguously defined its substrate specificity as mono(ADP-ribosyl)ated aspartate and glutamate but not lysine residues. Mutant viruses lacking hydrolase activity were unable to replicate in mammalian BHK-21 cells or mosquitoAedes albopictuscells and rapidly reverted catalytically inactivating mutations. Mutants with reduced enzymatic activity had slower replication in mammalian neuronal cells and reduced virulence in 2-day-old mice. Therefore, nsP3 mono(ADP-ribosyl)hydrolase activity is critical for CHIKV replication in both vertebrate hosts and insect vectors, and for virulence in mice.
authors.affiliation: Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD 21205.
authors.forename: Robert Lyle
authors.initials: RL
authors.lastname: McPherson
authors.affiliation: W. Harry Feinstone Department of Molecular Microbiology and Immunology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD 21205.
authors.forename: Rachy
authors.initials: R
authors.lastname: Abraham
authors.affiliation: W. Harry Feinstone Department of Molecular Microbiology and Immunology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD 21205.
authors.forename: Easwaran
authors.initials: E
authors.lastname: Sreekumar
authors.affiliation: Department of Pharmacology, University of Washington, Seattle, WA 98195.
authors.forename: Shao-En
authors.initials: SE
authors.lastname: Ong
authors.affiliation: Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD 21205.
authors.forename: Shang-Jung
authors.initials: SJ
authors.lastname: Cheng
authors.affiliation: W. Harry Feinstone Department of Molecular Microbiology and Immunology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD 21205.|Department of Molecular and Comparative Pathobiology, School of Medicine, Johns Hopkins University, Baltimore, MD 21205.
authors.forename: Victoria K
authors.initials: VK
authors.lastname: Baxter
authors.affiliation: Department of Bio-organic Synthesis, Leiden University, Einsteinweg 55, 2333 CC Leiden, The Netherlands.
authors.forename: Hans A V
authors.initials: HA
authors.lastname: Kistemaker
authors.affiliation: Department of Bio-organic Synthesis, Leiden University, Einsteinweg 55, 2333 CC Leiden, The Netherlands.
authors.forename: Dmitri V
authors.initials: DV
authors.lastname: Filippov
authors.affiliation: W. Harry Feinstone Department of Molecular Microbiology and Immunology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD 21205; anthony.leung@jhu.edu dgriffi6@jhmi.edu.
authors.forename: Diane E
authors.initials: DE
authors.lastname: Griffin
authors.affiliation: Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD 21205; anthony.leung@jhu.edu dgriffi6@jhmi.edu.|Department of Oncology, School of Medicine, Johns Hopkins University, Baltimore, MD 21205.
authors.forename: Anthony K L
authors.initials: AK
authors.lastname: Leung
mesh_terms: D000246:Adenosine Diphosphate Ribose / Q000378:metabolism*; D000330:Aedes / Q000821:virology; D000595:Amino Acid Sequence; D000818:Animals; D000831:Animals, Newborn; D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D065632:Chikungunya Fever / Q000821:virology; D002646:Chikungunya virus / Q000235:genetics / Q000378:metabolism* / Q000472:pathogenicity; D002522:Chlorocebus aethiops; D007303:Insect Vectors / Q000821:virology; D009699:N-Glycosyl Hydrolases / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014709:Vero Cells; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000378:metabolism*; D014774:Virulence / Q000235:genetics; D014779:Virus Replication / Q000235:genetics
chemical_list: D017361:Viral Nonstructural Proteins; D000246:Adenosine Diphosphate Ribose; D009699:N-Glycosyl Hydrolases; C046422:ADP-ribosylarginine hydrolase
keywords: ADP-ribosylation; macrodomain; mass spectrometry; viral replication; virulence