Activation of dengue protease autocleavage at the NS2B-NS3 junction by recombinant NS3 and GST-NS2B fusion proteins.
Dengue virus possesses a protease complex made up of the non-structural proteins NS2B and NS3. This protease complex catalyzes autocleavage (cis) at the junction between NS2A and NS2B as well as between NS2B and NS3. It also catalyzes trans cleavage at the junctions between NS3 and NS4A as well as NS4B and NS5. The cis cleavage at the NS2B-NS3 junction has been demonstrated in Escherichia coli by linking a 40-residue hydrophilic segment of NS2B to a NS3 N-terminal protease domain carrying the NS2B-NS3 cleavage site. To explore whether the hydrophilic segment could be further shortened, residues from both N- and C-termini of the NS2B hydrophilic segment were deleted. The results indicate that the four C-terminal's consecutive Glu residues could be deleted, each one leading to a further loss of activity, whereas the N-terminal boundary needed to be absolutely preserved. To examine whether an NS2B peptide could be expressed independently and added to activate the NS3 protease domain, the hydrophilic region of NS2B was fused to the C-terminus of glutathione-S-transferase (GST). This recombinant protein was soluble in bacteria and easily purified by affinity chromatography. Without removing the GST, the fusion protein activated the NS3 protease domain allowing it to function at the adjacent NS2B-NS3 junction. Thus, the findings reported below have produced a feasible alternative for the assay of dengue viral protease and this should facilitate the development of a screening method for inhibitors of dengue protease.
Wu
Chia-Fen
CF
Institute of Biopharmaceutics, National Yang-Ming University, Taipei, Taiwan.
Wang
Shao-Hung
SH
Sun
Chang-Ming
CM
Hu
Shau-Ting
ST
Syu
Wan-Jr
WJ
D015153:Blotting, Western; D003716:Dengue Virus / Q000201:enzymology*; D010450:Endopeptidases / Q000378:metabolism*; D004789:Enzyme Activation; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D007124:Immunoenzyme Techniques; D020365:RNA Helicases; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D012697:Serine Endopeptidases; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000378:metabolism*; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
D010450:Endopeptidases; D005982:Glutathione Transferase; 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