Mutagenesis of Conserved Residues at the Yellow Fever Virus 3/4A and 4B/5 Dibasic Cleavage Sites: Effects on Cleavage Efficiency and Polyprotein Processing

doi:10.1006/viro.1993.1076

Virology

Volume 192, Issue 2, February 1993, Pages 596-604

https://doi.org/10.1006/viro.1993.1076 Get rights and content

Abstract

Flavivirus proteins are produced by co- and post-translational proteolytic processing of a large polyprotein using both host- and virus-encoded enzymes, The flavivirus serine proteinase, which consists of NS2B and NS3, is responsible for cleavages of at least four dibasic sites in the nonstructural region. In this study, a number of substitutions for the conserved amino acids flanking the 3/4A and 4B/5 dibasic cleavage sites {Arg(P2)-Arg(P1)↓-Gly(P1′')} were examined for their effects on yellow fever virus (YF) polyprotein processing. The substrate for these studies was a truncated YF polyprotein, called sig2A-5₃₅₆, which consists of a signal sequence fused to NS2A and extending through the first 356 amino acids of NS5. At the P1′ position (Gly) of the 4B/5 site, only Ser and Ala were allowed while six other substitutions abolished cleavage. Substitutions of the 4B/5 P1 Arg residue with Lys, Gln, Asn, or His were tolerated while replacement with Glu eliminated cleavage. The 4B/5 P2 position (Arg) was found to be tolerant of substitutions with polar or hydrophobic residues which allowed varying degrees of partial cleavage. Previous studies have shown that cleavage at the 3/4A site is incomplete in YF-infected cells and that the cleavage efficiency at this site is significantly less for the sig2A-5₃₅₆ polyprotein. Replacement of the 3/4A P1 Arg residue with noncharged polar or hydrophobic residues reduced the cleavage efficiency, whereas substitutions with Glu or Pro abolished cleavage. Studies with polyproteins containing one or both of the 3/4A and 4B/5 cleavage sites blocked indicate that there is not an obligatory processing order for cleavages generating the N termini of YF NS4A, NS4B, and NSS.

References (0)

Cited by (41)

Simultaneous uncoupled expression and purification of the Dengue virus NS3 protease and NS2B co-factor domain
2016, Protein Expression and Purification
Citation Excerpt :
The viral protease is highly conserved throughout the Flavivirus genus and is required for cleaving at the NS2A/NS2B, NS2B/NS3, NS3/NS4A and NS4B/NS5 junctions, in addition to cleaving internal linkages within NS4A and the capsid C protein [6–10]. Disruption of proteolytic function has been shown to prevent correct processing of the polyprotein and block viral replication and infection [2,8,10–13]. Consequently, the viral NS2B–NS3 protease is considered to be a highly attractive target for inhibition and drug design.
Dengue Virus (DENV) infection is responsible for the world's most significant insect-borne viral disease. Despite an increasing global impact, there are neither prophylactic nor therapeutic options available for the effective treatment of DENV infection. An attractive target for antiviral drugs is the virally encoded trypsin-like serine protease (NS3pro) and its associated cofactor (NS2B). The NS2B–NS3pro complex is responsible for cleaving the viral polyprotein into separate functional viral proteins, and is therefore essential for replication. Recombinant expression of an active NS2B–NS3 protease has primarily been based on constructs linking the C-terminus of the approximately 40 amino acid hydrophilic cofactor domain of NS2B to the N-terminus of NS3pro via a flexible glycine linker. The resulting complex can be expressed in high yield, is soluble and catalytically active and has been used for most in vitro screening, inhibitor, and X-ray crystallographic studies over the last 15 years. Despite extensive analysis, no inhibitor drug candidates have been identified yet. Moreover, the effect of the artificial linker introduced between the protease and its cofactor is unknown. Two alternate methods for bacterial expression of non-covalently linked, catalytically active, NS2B–NS3pro complex are described here along with a comparison of the kinetics of substrate proteolysis and binding affinities of substrate-based aldehyde inhibitors. Both expression methods produced high yields of soluble protein with improved substrate proteolysis kinetics and inhibitor binding compared to their glycine-linked equivalent. The non-covalent association between NS2B and NS3pro is predicted to be more relevant for examining inhibitors that target cofactor-protease interactions rather than the protease active site. Furthermore, these approaches offer alternative strategies for the high yield co-expression of other protein assemblies.
The flavivirus NS2B-NS3 protease-helicase as a target for antiviral drug development
2015, Antiviral Research
Citation Excerpt :
Polyprotein processing occurs co- and post-translationally in ER-derived intracellular membranes (Chambers et al., 1990a, 1991). NS3 may thus help bring NS4A–NS4B–NS5 in close proximity to NS2BNS3pro for the efficient cleavage and release of individual viral proteins (Amberg et al., 1994; Arias et al., 1993; Cahour et al., 1992; Lin et al., 1993a,b). In addition, NS2B anchors NS3 in the membrane, which is a prerequisite for viral replication complex maturation.
The flavivirus NS3 protein is associated with the endoplasmic reticulum membrane via its close interaction with the central hydrophilic region of the NS2B integral membrane protein. The multiple roles played by the NS2B–NS3 protein in the virus life cycle makes it an attractive target for antiviral drug discovery. The N-terminal region of NS3 and its cofactor NS2B constitute the protease that cleaves the viral polyprotein. The NS3 C-terminal domain possesses RNA helicase, nucleoside and RNA triphosphatase activities and is involved both in viral RNA replication and virus particle formation. In addition, NS2B–NS3 serves as a hub for the assembly of the flavivirus replication complex and also modulates viral pathogenesis and the host immune response. Here, we review biochemical and structural advances on the NS2B–NS3 protein, including the network of interactions it forms with NS5 and NS4B and highlight recent drug development efforts targeting this protein. This article forms part of a symposium in Antiviral Research on flavivirus drug discovery.
A plasmid-based reporter system for live cell imaging of dengue virus infected cells
2015, Journal of Virological Methods
Citation Excerpt :
The 4B5-EGFP reporter also showed detection of cellular infection with each of the DENV serotypes. Previous reports have analyzed sequence variation within the NS4B–NS5 junction between various flaviviruses (Lin et al., 1993; Bera et al., 2007; Shiryaev et al., 2007). The amino acids in the P1, P2 and P1′ positions are similar for all four DENV serotypes as well as Yellow Fever virus, however, amino acids occupying P2′ and P3′ are different (Shiryaev et al., 2007).
Cell culture models are used widely to study the effects of dengue virus (DENV) on host cell function. Current methods of identification of cells infected with an unmodified DENV requires fixation and permeablization of cells to allow DENV-specific antibody staining. This method does not permit imaging of viable cells over time. In this report, a plasmid-based reporter was developed to allow non-destructive identification of DENV-infected cells. The plasmid-based reporter was demonstrated to be broadly applicable to the four DENV serotypes, including low-passaged strains, and was specifically cleaved by the viral protease with minimal interference on viral production. This study reveals the potential for this novel reporter system to advance the studies of virus–host interactions during DENV infection.
Flavivirin
2013, Handbook of Proteolytic Enzymes
Functional characterization of cis and trans activity of the flavivirus NS2B-NS3 protease
2007, Journal of Biological Chemistry
Citation Excerpt :
The situation in the viral polyprotein is more complex. In extensive studies of YFV protease specificity, Rice and co-workers (11, 29, 31) reported high specificity (Arg and Lys) at the P1 position for the NS2A/NS2B and NS2B/NS3 junctions, but more relaxed specificity at the NS3/NS4A (Arg, Lys, Ser, Thr, Ala, Met, and Leu) and NS4B/NS5 (Arg, Lys, Gln, Asn, and His) junctions. We detected no cleavage at Lys15-Gly16 of NS3, as was observed in the variant of the isolated WNV NS2B-NS3 protease domain used for the crystal structure (24).
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 (NS2B₄₀) 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) NS2B₄₀ to full-length WNV NS3. The protease catalyzed two autolytic cleavages. The NS2B/NS3 junction was cleaved before protein purification. A second site at Arg⁴⁵⁹↓Gly⁴⁶⁰ 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.
Site-directed mutagenesis and kinetic studies of the West Nile virus NS3 protease identify key enzyme-substrate interactions
2005, Journal of Biological Chemistry
The flavivirus West Nile virus (WNV) has spread rapidly throughout the world in recent years causing fever, meningitis, encephalitis, and fatalities. Because the viral protease NS2B/NS3 is essential for replication, it is attracting attention as a potential therapeutic target, although there are currently no antiviral inhibitors for any flavivirus. This paper focuses on elucidating interactions between a hexapeptide substrate (Ac-KPGLKR-p-nitroanilide) and residues at S1 and S2 in the active site of WNV protease by comparing the catalytic activities of selected mutant recombinant proteases in vitro. Homology modeling enabled the predictions of key mutations in WNV NS3 protease at S1 (V115A/F, D129A/E/N, S135A, Y150A/F, S160A, and S163A) and S2 (N152A) that might influence substrate recognition and catalytic efficiency. Key conclusions are that the substrate P1 Arg strongly interacts with S1 residues Asp-129, Tyr-150, and Ser-163 and, to a lesser extent, Ser-160, and P2 Lys makes an essential interaction with Asn-152 at S2. The inferred substrate-enzyme interactions provide a basis for rational protease inhibitor design and optimization. High sequence conservation within flavivirus proteases means that this study may also be relevant to design of protease inhibitors for other flavivirus proteases.

View all citing articles on Scopus

View full text

Regular ArticleMutagenesis of Conserved Residues at the Yellow Fever Virus 3/4A and 4B/5 Dibasic Cleavage Sites: Effects on Cleavage Efficiency and Polyprotein Processing

Abstract

Regular Article
Mutagenesis of Conserved Residues at the Yellow Fever Virus 3/4A and 4B/5 Dibasic Cleavage Sites: Effects on Cleavage Efficiency and Polyprotein Processing