Analysis of RNA binding by the dengue virus NS5 RNA capping enzyme.
Flaviviruses are small, capped positive sense RNA viruses that replicate in the cytoplasm of infected cells. Dengue virus and other related flaviviruses have evolved RNA capping enzymes to form the viral RNA cap structure that protects the viral genome and directs efficient viral polyprotein translation. The N-terminal domain of NS5 possesses the methyltransferase and guanylyltransferase activities necessary for forming mature RNA cap structures. The mechanism for flavivirus guanylyltransferase activity is currently unknown, and how the capping enzyme binds its diphosphorylated RNA substrate is important for deciphering how the flavivirus guanylyltransferase functions. In this report we examine how flavivirus NS5 N-terminal capping enzymes bind to the 5' end of the viral RNA using a fluorescence polarization-based RNA binding assay. We observed that the K(D) for RNA binding is approximately 200 nM Dengue, Yellow Fever, and West Nile virus capping enzymes. Removal of one or both of the 5' phosphates reduces binding affinity, indicating that the terminal phosphates contribute significantly to binding. RNA binding affinity is negatively affected by the presence of GTP or ATP and positively affected by S-adensyl methoninine (SAM). Structural superpositioning of the dengue virus capping enzyme with the Vaccinia virus VP39 protein bound to RNA suggests how the flavivirus capping enzyme may bind RNA, and mutagenesis analysis of residues in the putative RNA binding site demonstrate that several basic residues are critical for RNA binding. Several mutants show differential binding to 5' di-, mono-, and un-phosphorylated RNAs. The mode of RNA binding appears similar to that found with other methyltransferase enzymes, and a discussion of diphosphorylated RNA binding is presented.
Department of Microbiology, Immunology, and Pathology, Colorado State University, Fort Collins, Colorado, United States of America.
Brittney R
BR
Henderson
Bejan J
BJ
Saeedi
Grace
G
Campagnola
Brian J
BJ
Geiss
D004252:DNA Mutational Analysis; D003716:Dengue Virus / Q000187:drug effects / Q000201:enzymology*; D005416:Flavivirus / Q000187:drug effects / Q000201:enzymology; D005454:Fluorescence Polarization; D006160:Guanosine Triphosphate / Q000494:pharmacology; D007700:Kinetics; D008024:Ligands; D008958:Models, Molecular; D011485:Protein Binding / Q000187:drug effects; D012315:RNA Caps / Q000378:metabolism*; D012367:RNA, Viral / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism*; D012436:S-Adenosylmethionine / Q000494:pharmacology; D017361:Viral Nonstructural Proteins / Q000378:metabolism*; D014764:Viral Proteins / Q000378:metabolism; D015005:Yellow fever virus / Q000187:drug effects
D008024:Ligands; D012315:RNA Caps; D012367:RNA, Viral; D016601:RNA-Binding Proteins; C088682:VP39 protein, Vaccinia virus; D017361:Viral Nonstructural Proteins; D014764:Viral Proteins; D012436:S-Adenosylmethionine; D006160:Guanosine Triphosphate