title: Processing of the yellow fever virus nonstructural polyprotein: a catalytically active NS3 proteinase domain and NS2B are required for cleavages at dibasic sites.
abstract: The vaccinia virus-T7 transient expression system was used to further examine the role of the NS3 proteinase in processing of the yellow fever (YF) virus nonstructural polyprotein in BHK cells. YF virus-specific polyproteins and cleavage products were identified by immunoprecipitation with region-specific antisera, by size, and by comparison with authentic YF virus polypeptides. A YF virus polyprotein initiating with a signal sequence derived from the E protein fused to the N terminus of NS2A and extending through the N-terminal 356 amino acids of NS5 exhibited processing at the 2A-2B, 2B-3, 3-4A, 4A-4B, and 4B-5 cleavage sites. Similar results were obtained with polyproteins whose N termini began within NS2A (position 110) or with NS2B. When the NS3 proteinase domain was inactivated by replacing the proposed catalytic Ser-138 with Ala, processing at all sites was abolished. The results suggest that an active NS3 proteinase domain is necessary for cleavage at the diabasic nonstructural cleavage sites and that cleavage at the proposed 4A-4B signalase site requires prior cleavage at the 4B-5 site. Cleavages were not observed with a polyprotein whose N terminus began with NS3, but cleavage at the 4B-5 site could be restored by supplying the the NS2B protein in trans. Several experimental results suggested that trans cleavage at the 4B-5 site requires association of NS2B and the NS3 proteinase domain. Coexpression of different proteinases and catalytically inactive polyprotein substrates revealed that trans cleavage at the 2B-3 and 4B-5 sites was relatively efficient when compared with trans cleavage at the 2A-2B and 3-4A sites.
authors.affiliation: Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri 63110-1093.
authors.forename: T J
authors.initials: TJ
authors.lastname: Chambers
authors.forename: A
authors.initials: A
authors.lastname: Grakoui
authors.forename: C M
authors.initials: CM
authors.lastname: Rice
mesh_terms: D000818:Animals; D002213:Capsid / Q000235:genetics*; D002460:Cell Line; D004279:DNA, Viral / Q000235:genetics* / Q000302:isolation & purification; D015870:Gene Expression; D016366:Open Reading Frames; D010957:Plasmids; D011499:Protein Processing, Post-Translational; D020365:RNA Helicases; D012697:Serine Endopeptidases; D013379:Substrate Specificity; D014158:Transcription, Genetic; D014162:Transfection; D014758:Viral Core Proteins / Q000235:genetics*; D017361:Viral Nonstructural Proteins; D014764:Viral Proteins / Q000378:metabolism*; D015005:Yellow fever virus / Q000235:genetics* / Q000378:metabolism; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
chemical_list: D004279:DNA, Viral; D020365:RNA Helicases; D012697:Serine Endopeptidases; D014758:Viral Core Proteins; D017361:Viral Nonstructural Proteins; D014764:Viral Proteins; C060028:NS3 protein, flavivirus; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases