Construction of dengue virus protease expression plasmid and in vitro protease assay for screening antiviral inhibitors.
Dengue virus serotypes 1-4 (DENV1-4) are mosquito-borne human pathogens of global significance causing ~390 million cases annually worldwide. The virus infections cause in general a self-limiting disease, known as dengue fever, but occasionally also more severe forms, especially during secondary infections, dengue hemorrhagic fever and dengue shock syndrome causing ~25,000 deaths annually. The DENV genome contains a single-strand positive sense RNA, approximately 11 kb in length. The 5'-end has a type I cap structure. The 3'-end has no poly(A) tail. The viral RNA has a single long open reading frame that is translated by the host translational machinery to yield a polyprotein precursor. Processing of the polyprotein precursor occurs co-translationally by cellular proteases and posttranslationally by the viral serine protease in the endoplasmic reticulum (ER) to yield three structural proteins (capsid (C), precursor membrane (prM), and envelope (E) and seven nonstructural (NS) proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). The active viral protease consists of both NS2B, an integral membrane protein in the ER, and the N-terminal part of NS3 (180 amino acid residues) that contains the trypsin-like serine protease domain having a catalytic triad of H51, D75, and S135. The C-terminal part of NS3, ~170-618 amino acid residues, encodes an NTPase/RNA helicase and 5'-RNA triphosphatase activities; the latter enzyme is required for the first step in 5'-capping. The cleavage sites of the polyprotein by the viral protease consist of two basic amino acid residues such as KR, RR, or QR, followed by short chain amino acid residues, G, S, or T. Since the cleavage of the polyprotein by the viral protease is absolutely required for assembly of the viral replicase, blockage of NS2B/NS3pro activity provides an effective means for designing dengue virus (DENV) small-molecule therapeutics. Here we describe the screening of small-molecule inhibitors against DENV2 protease.
Department of Microbiology & Immunology, Georgetown University, Washington, DC, USA.
Huiguo
H
Lai
Tadahisa
T
Teramoto
Radhakrishnan
R
Padmanabhan
D000998:Antiviral Agents / Q000032:analysis* / Q000494:pharmacology; D003716:Dengue Virus / Q000201:enzymology*; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D057075:Enzyme Assays / Q000379:methods*; D006801:Humans; D020128:Inhibitory Concentration 50; D007700:Kinetics; D010957:Plasmids / Q000378:metabolism*; D011480:Protease Inhibitors / Q000032:analysis* / Q000494:pharmacology; D020365:RNA Helicases / Q000037:antagonists & inhibitors / Q000378:metabolism; D012697:Serine Endopeptidases / Q000378:metabolism; D054852:Small Molecule Libraries / Q000032:analysis / Q000494:pharmacology; D013379:Substrate Specificity / Q000187:drug effects; D013696:Temperature; D017361:Viral Nonstructural Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
D000998:Antiviral Agents; D011480:Protease Inhibitors; D020365:RNA Helicases; D012697:Serine Endopeptidases; D054852:Small Molecule Libraries; D017361:Viral Nonstructural Proteins; C060028:NS3 protein, flavivirus; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases