Abstract
Flaviviruses are serious human pathogens for which treatments are generally lacking. The proteolytic maturation of the 375-kDa viral polyprotein is one target for antiviral development. The flavivirus serine protease consists of the N-terminal domain of the multifunctional nonstructural protein 3 (NS3) and an essential 40-residue cofactor (NS2B(40)) within viral protein NS2B. The NS2B-NS3 protease is responsible for all cytoplasmic cleavage events in viral polyprotein maturation. This study describes the first biochemical characterization of flavivirus protease activity using full-length NS3. Recombinant proteases were created by fusion of West Nile virus (WNV) NS2B(40) to full-length WNV NS3. The protease catalyzed two autolytic cleavages. The NS2B/NS3 junction was cleaved before protein purification. A second site at Arg(459) decreasing Gly(460) within the C-terminal helicase region of NS3 was cleaved more slowly. Autolytic cleavage reactions also occurred in NS2B-NS3 recombinant proteins from yellow fever virus, dengue virus types 2 and 4, and Japanese encephalitis virus. Cis and trans cleavages were distinguished using a noncleavable WNV protease variant and two types of substrates as follows: an inactive variant of recombinant WNV NS2B-NS3, and cyan and yellow fluorescent proteins fused by a dodecamer peptide encompassing a natural cleavage site. With these materials, the autolytic cleavages were found to be intramolecular only. Autolytic cleavage of the helicase site was insensitive to protein dilution, confirming that autolysis is intramolecular. Formation of an active protease was found to require neither cleavage of NS2B from NS3 nor a free NS3 N terminus. Evidence was also obtained for product inhibition of the protease by the cleaved C terminus of NS2B.
Highlights
Most flaviviruses, including West Nile virus (WNV),2 yellow fever virus (YFV), dengue viruses, and Japanese encephalitis virus (JEV), cause severe human diseases
The putative product inhibition by Lys-Arg-COOϪ was not observed in NS2B79-NS3FL or in NS2B40-KR-NS3FL. This is the first study to examine the activity of the WNV NS2B40-nonstructural protein 3 (NS3) protease in the context of purified, full-length NS3 protein
Previous detailed biochemical studies of flavivirus proteases were of truncations (13–16, 19 –22) that included only the protease region of NS3 (WNV NS3 residues ϳ1–180) but lacked the full helicase region (WNV NS3 residues 180 – 619)
Summary
Most flaviviruses, including West Nile virus (WNV), yellow fever virus (YFV), dengue viruses, and Japanese encephalitis virus (JEV), cause severe human diseases. The activity of NS2B-NS3 protease has been studied in vitro using purified, recombinant protease domains of dengue virus type 2 (DV2) and WNV NS3. These studies employed fusions of the NS2B cofactor peptide to truncated forms of NS3 (NS3pro) that included only the N-terminal protease domain, one-third of the full-length NS3, and excluded the C-terminal helicase domain [13,14,15,16]. We report single polypeptide variants of NS2B-NS3 protease from five flaviviruses To our knowledge, this is the first study addressing flavivirus protease activity using the NS2B40 cofactor
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