Identification of residues in the dengue virus type 2 NS2B cofactor that are critical for NS3 protease activation.
Proteolytic processing of the dengue virus polyprotein is mediated by host cell proteases and the virus-encoded NS2B-NS3 two-component protease. The NS3 protease represents an attractive target for the development of antiviral inhibitors. The three-dimensional structure of the NS3 protease domain has been determined, but the structural determinants necessary for activation of the enzyme by the NS2B cofactor have been characterized only to a limited extent. To test a possible functional role of the recently proposed Phix(3)Phi motif in NS3 protease activation, we targeted six residues within the NS2B cofactor by site-specific mutagenesis. Residues Trp62, Ser71, Leu75, Ile77, Thr78, and Ile79 in NS2B were replaced with alanine, and in addition, an L75A/I79A double mutant was generated. The effects of these mutations on the activity of the NS2B(H)-NS3pro protease were analyzed in vitro by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of autoproteolytic cleavage at the NS2B/NS3 site and by assay of the enzyme with the fluorogenic peptide substrate GRR-AMC. Compared to the wild type, the L75A, I77A, and I79A mutants demonstrated inefficient autoproteolysis, whereas in the W62A and the L75A/I79A mutants self-cleavage appeared to be almost completely abolished. With exception of the S71A mutant, which had a k(cat)/K(m) value for the GRR-AMC peptide similar to that of the wild type, all other mutants exhibited drastically reduced k(cat) values. These results indicate a pivotal function of conserved residues Trp62, Leu75, and Ile79 in the NS2B cofactor in the structural activation of the dengue virus NS3 serine protease.
Niyomrattanakit
Pornwaratt
P
Laboratory of Molecular Virology, Institute of Molecular Biology and Genetics, Mahidol University, Salaya Campus, Phutthamonthon 4 Rd., Nakornpathom 73170, Thailand.
Winoyanuwattikun
Pakorn
P
Chanprapaph
Santad
S
Angsuthanasombat
Chanan
C
Panyim
Sakol
S
Katzenmeier
Gerd
G
D000409:Alanine; D000595:Amino Acid Sequence; D003716:Dengue Virus / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D004789:Enzyme Activation; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D020365:RNA Helicases / Q000378:metabolism*; D012697:Serine Endopeptidases / Q000378:metabolism*; D013329:Structure-Activity Relationship; D017361:Viral Nonstructural Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
D000409:Alanine; D020365:RNA Helicases; 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