Functional Characterization of cis and trans Activity of the Flavivirus NS2B-NS3 Protease

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 2Gly 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. Most flaviviruses, including West Nile virus (WNV),2 yellow fever virus (YFV), dengue viruses, and Japanese encephalitis virus (JEV), cause severe human diseases. The plus-sense RNA genome of flaviviruses is a single open reading frame encoding a polyprotein precursor of ϳ3400 amino acids, consisting of three structural proteins (C, prM, and E) and seven nonstructural replication proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) (Fig. 1A) (1-3). Signal sequences direct the polyprotein into the host endoplasmic reticulum (ER) membrane so that NS1 and the exogenous domains of prM and E are in the lumen; C protein, NS3 and NS5 are cytoplasmic; and proteins NS2A, NS2B, NS4A, and NS4B are predominantly trans-membrane. Post-translational processing of the polyprotein, which is required for virus replication, is performed by a viral NS3 protease (4, 5) in the cytoplasm and by host proteases in the ER lumen. NS3 protease activity is dependent upon association with an NS2B cofactor (NS2B 40 ), a central 40-amino acid hydrophilic domain within the largely hydrophobic NS2B protein (6 -8). The viral NS2B-NS3 protease cleaves the viral polyprotein precursor at the NS2A/NS2B, NS2B/NS3, NS3/ NS4A, and NS4B/NS5 junctions (Fig. 1A), as well as at internal sites within C, NS2A, N...

Development of Virus-Like-Particle Vaccine and Reporter Assay for Zika Virus

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“…In either case, internal cleavages occur within the helicase domain of NS3 and yield C-terminally truncated molecules. While a single study suggested that cellular proteases mediate the internal NS3 cleavage in HCV (36), it was demonstrated that an autocatalytic protease activity of NS2B/ NS3 or NS3/NS4A is responsible for internal NS3 processing (9,13,37,38).…”

Optimization of Assay Conditions for Dengue Virus Protease: Effect of Various Polyols and Nonionic Detergents

“…1 In vivo, the lipophilic NS2B-cofactor is associated with the membrane of the endoplasmic reticulum (ER), whereas the NS3 is located in the cytosplasma. [2][3][4] For optimum activity, the NS3 depends on association with the cofactor domain. 5,6 Because of its importance in the viral life cycle, the protease is an interesting target for development of new antiviral drugs.…”