Yellow fever virus NS2B-NS3 protease: charged-to-alanine mutagenesis and deletion analysis define regions important for protease complex formation and function.
Charged-to-alanine substitutions and deletions within the yellow fever virus NS2B-NS3(181) protease were analyzed for effects on protease function. During cell-free translation of NS2B-3(181) polyproteins, mutations at three charge clusters markedly impaired cis cleavage activity: a single N-terminal cluster in the conserved domain of NS2B (residues ELKK(52-55)) and two in NS3 (ED(21-22), and residue H(47)). These mutations inhibited other protease-dependent cleavages of a transiently expressed nonstructural polyprotein, although differential effects occurred. NS2B and NS3(181) proteins harboring these mutations were impaired in their ability to associate for trans cleavage activity. N-terminal deletions in NS3 also implicated residues ED(21-22) in the association with NS2B. Deletions within NS2B revealed that the conserved domain alone provided minimal cofactor activity, with optimal function requiring both flanking hydrophobic regions. NS2B-3(181)- and NS3(181)-green fluorescent protein fusion proteins were used to determine the intracellular distribution of the protease complex. The former localized in membrane-based vesicular structures, whereas the latter localized poorly. The data suggest that NS2B-NS3 complex formation requires charge interactions involving the N-terminus of the conserved domain of NS2B and 22 N-terminal residues of NS3. A role for the putative transmembrane regions of NS2B in targeting of NS3 to intracellular membranes is also suggested.
Droll
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DA
Department of Molecular Microbiology and Immunology, St. Louis University School of Medicine, 1402 South Grand Avenue, St. Louis, Missouri 63104, USA.
Krishna Murthy
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HM
Chambers
T J
TJ
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000235:genetics / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D020365:RNA Helicases; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D012697:Serine Endopeptidases; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000378:metabolism*; D015005:Yellow fever virus / Q000201:enzymology / Q000378:metabolism*; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
D010450:Endopeptidases; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D020365:RNA Helicases; D011993:Recombinant Fusion Proteins; D012697:Serine Endopeptidases; D017361:Viral Nonstructural Proteins; C080102:NS2B protein, flavivirus; C060028:NS3 protein, flavivirus; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases