Functional determinants of NS2B for activation of Japanese encephalitis virus NS3 protease.
Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus, causing severe central nerve system diseases without specific treatments. The NS2B-NS3 protease of flaviviruses mediates several cleavages on the flavivirus polyprotein, being believed to be a target for antiviral therapy. NS2B is the cofactor of the viral serine protease, correlating with stabilization and substrate recognition of the NS3 protease. In this study, we investigate the functional determinants in the JEV NS2B for the activation of the NS3 protease. Cis- and trans-cleavage assays of the deletions at the N-terminal of NS2B demonstrated that the NS2B residues Ser(46) to Ile(60) were the essential region required for both cis and trans activity of the NS3 protease. In addition, alanine substitution at the residues Trp53, Glu55, and Arg56 in NS2B significantly reduced the cis- and trans-cleavage activities of the NS3 protease. Sequence alignment and modeled structures suggested that functional determinants at the JEV NS2B residues Ser46 to Ile60, particularly in Trp53, Glu55 and Arg56 could play an important configuration required for the activity of the flavivirus NS3 protease. Our results might be useful for development of inhibitors that block the interaction between NS2B and NS3.
Lin
Cheng-Wen
CW
Department of Medical Laboratory Science and Biotechnology, China Medical University, Taichung 404, Taiwan, ROC. cwlin@mail.cmu.edu.tw
Huang
Hong-Da
HD
Shiu
Shi-Yi
SY
Chen
Wei-June
WJ
Tsai
Ming-Hong
MH
Huang
Su-Hua
SH
Wan
Lei
L
Lin
Ying-Ju
YJ
D004664:Encephalitis Virus, Japanese / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism; D011499:Protein Processing, Post-Translational / Q000502:physiology*; D020365:RNA Helicases / Q000737:chemistry / Q000378:metabolism; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000378:metabolism; D013329:Structure-Activity Relationship; D017361:Viral Nonstructural Proteins / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
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