title: Predicted structure and domain organization of rotavirus capping enzyme and innate immune antagonist VP3.
abstract: Rotaviruses and orbiviruses are nonturreted Reoviridae members. The rotavirus VP3 protein is a multifunctional capping enzyme and antagonist of the interferon-induced cellular oligoadenylate synthetase-RNase L pathway. Despite mediating important processes, VP3 is the sole protein component of the rotavirus virion whose structure remains unknown. In the current study, we used sequence alignment and homology modeling to identify features common to nonturreted Reoviridae capping enzymes and to predict the domain organization, structure, and active sites of rotavirus VP3. Our results suggest that orbivirus and rotavirus capping enzymes share a domain arrangement similar to that of the bluetongue virus capping enzyme. Sequence alignments revealed conserved motifs and suggested that rotavirus and orbivirus capping enzymes contain a variable N-terminal domain, a central guanine-N7-methyltransferase domain that contains an additional inserted domain, and a C-terminal guanylyltransferase and RNA 5'-triphosphatase domain. Sequence conservation and homology modeling suggested that the insertion in the guanine-N7-methyltransferase domain is a ribose-2'-O-methyltransferase domain for most rotavirus species. Our analyses permitted putative identification of rotavirus VP3 active-site residues, including those that form the ribose-2'-O-methyltransferase catalytic tetrad, interact with S-adenosyl-l-methionine, and contribute to autoguanylation. Previous reports have indicated that group A rotavirus VP3 contains a C-terminal 2H-phosphodiesterase domain that can cleave 2'-5' oligoadenylates, thereby preventing RNase L activation. Our results suggest that a C-terminal phosphodiesterase domain is present in the capping enzymes from two additional rotavirus species. Together, these findings provide insight into a poorly understood area of rotavirus biology and are a springboard for future biochemical and structural studies of VP3.
  
  
  OBJECTIVE
  Rotaviruses are an important cause of severe diarrheal disease. The rotavirus VP3 protein caps viral mRNAs and helps combat cellular innate antiviral defenses, but little is known about its structure or enzymatic mechanisms. In this study, we used sequence- and structure-based alignments with related proteins to predict the structure of VP3 and identify enzymatic domains and active sites therein. This work provides insight into the mechanisms of rotavirus transcription and evasion of host innate immune defenses. An improved understanding of these processes may aid our ability to develop rotavirus vaccines and therapeutics.
authors.affiliation: Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA guglielmikm@niaid.nih.gov.
authors.forename: Kristen M
authors.initials: KM
authors.lastname: Ogden
authors.affiliation: Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Snyder
authors.affiliation: Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: Allison F
authors.initials: AF
authors.lastname: Dennis
authors.affiliation: Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: John T
authors.initials: JT
authors.lastname: Patton
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D036022:Capsid Proteins / Q000235:genetics* / Q000276:immunology*; D020134:Catalytic Domain / Q000235:genetics / Q000276:immunology; D002460:Cell Line; D007113:Immunity, Innate / Q000276:immunology*; D008969:Molecular Sequence Data; D018163:Orbivirus / Q000235:genetics / Q000276:immunology; D010802:Phylogeny; D017434:Protein Structure, Tertiary / Q000235:genetics*; D012401:Rotavirus / Q000235:genetics* / Q000276:immunology*; D012400:Rotavirus Infections / Q000276:immunology* / Q000821:virology; D016415:Sequence Alignment; D061987:Sf9 Cells; D018411:Spodoptera; D014158:Transcription, Genetic / Q000235:genetics / Q000276:immunology; D014771:Virion / Q000235:genetics / Q000276:immunology
chemical_list: D036022:Capsid Proteins; C442546:VP3 protein, Rotavirus