In vitro synthesis of West Nile virus proteins indicates that the amino-terminal segment of the NS3 protein contains the active centre of the protease which cleaves the viral polyprotein after multiple basic amino acids

Wengler, Gerd; Czaya, Gerd; Färber, Petra M.; Hegemann, Johannes H.

doi:10.1099/0022-1317-72-4-851

Cited by 139 publications

(98 citation statements)

References 22 publications

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“…23,24,26,39,40 Host proteases cleave the polyprotein at the following junctions, C/prM, prM/E, E/NS1, whereas the viral protease (NS2B-NS3) is considered to cleave at the NS2A/NS2B, NS2B/NS3, NS3/NS4A, and NS4B/NS5 protein junctions. 41 However, the viral replication can not begin until all single proteins are cleaved and released from the polyprotein precursor. Therefore, inhibition of the viral protease (NS2B-NS3) or host proteases could be an attractive target for the design of effective WNV antiviral therapies.…”

Section: Replication Cyclementioning

confidence: 99%

“…NS3 is a large ~70 kDa protein, that contains two distinct enzymatic domains, a protease domain within the N-terminus [41][42][43][44] and a helicase/ATPase domain within its C-terminus. 41,43 The helicase/ATPase domain participates in viral RNA genome replication in association with RdRP in NS5 by unwinding the double-stranded RNA intermediates, facilitating NS5 polymerase activity. 45 The NS3 protease domain is a serine protease, containing the catalytic triad formed by residues Ser135, His51, and…”

Section: The Biological Roles Of the Nonstructural Viral Proteinsmentioning

confidence: 99%

“…46 The main role of NS3 protease is the catalytic processing of the polyprotein to generate the single mature nonstructural proteins (Figure 1.6). 41 To exhibit the artificial heterocomplex, the NS2B-NS3, is depicted in the enlarged section (figure was adopted from Lescar et al). 50 To generate a stable complex of NS2B and NS3 for enzymatic studies and crystallography, an artificial sequence consisting of a (Gly) 4 -Ser-(Gly) 4 sequence was introduced between both domains.…”

Section: The Biological Roles Of the Nonstructural Viral Proteinsmentioning

confidence: 99%

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Development and Characterization of New Peptidomimetic Inhibitors of the West Nile Virus NS2B–NS3 Protease

et al. 2013

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A series of new substrate analogue inhibitors of the WNV NS2B–NS3 protease containing decarboxylated arginine mimetics at the P1 position was developed. Among the various analogues, trans‐(4‐guanidino)cyclohexylmethylamide (GCMA) was identified as the most suitable P1 residue. In combination with dichloro‐substituted phenylacetyl groups at the P4 position, three inhibitors with inhibition constants of <0.2 μM were obtained. These GCMA inhibitors have a better selectivity profile than the previously described agmatine analogues, and possess negligible affinity for the trypsin‐like serine proteases thrombin, factor Xa, and matriptase. A crystal structure in complex with the WNV protease was determined for one of the most potent inhibitors, 3,4‐dichlorophenylacetyl‐Lys‐Lys‐GCMA (Ki=0.13 μM). The inhibitor adopts a horseshoe‐like conformation, most likely due to a hydrophobic contact between the P4 phenyl ring and the P1 cyclohexyl group, which is further stabilized by an intramolecular hydrogen bond between the P1 guanidino group and the P4 carbonyl oxygen atom. These inhibitors are stable, readily accessible, and have a noncovalent binding mode. Therefore, they may serve as suitable lead structures for further development.

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Section: Replication Cyclementioning

confidence: 99%

Section: The Biological Roles Of the Nonstructural Viral Proteinsmentioning

confidence: 99%

Section: The Biological Roles Of the Nonstructural Viral Proteinsmentioning

confidence: 99%

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Development and Characterization of New Peptidomimetic Inhibitors of the West Nile Virus NS2B–NS3 Protease

et al. 2013

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“…The proteases of DEN, YF, West Nile, Murray Valley, and TBE viruses have all been shown to have activity (5,20,23,26,33). A catalytic triad of amino acids, His-AspSer, is essential for protease activity in vitro and for viral replication (5,20,30,33). The proteolytic function of the NS3 protein is dependent on the presence of NS2B as a cofactor and is associated with the membrane fraction of infected cells (6).…”

mentioning

confidence: 99%

“…The serine protease domain is located at the amino terminus of the protein (2,10). The proteases of DEN, YF, West Nile, Murray Valley, and TBE viruses have all been shown to have activity (5,20,23,26,33). A catalytic triad of amino acids, His-AspSer, is essential for protease activity in vitro and for viral replication (5,20,30,33).…”

mentioning

confidence: 99%

Langat Flavivirus Protease NS3 Binds Caspase-8 and Induces Apoptosis

Prikhod'ko¹,

Prikhod’ko

Pletnev³

et al. 2002

J Virol

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The flavivirus NS3 protein plays an important role in the cleavage and processing of the viral polyprotein and in the synthesis of the viral RNA. NS3 recruits NS2B and NS5 proteins to form complexes possessing protease and replicase activities through protease and nucleoside triphosphatase/helicase domains. We have found that NS3 also induces apoptosis. Expression of the Langat (LGT) virus NS3 protein resulted in a cleavage of cellular DNA and reduced the viability of cells. Coexpression of NS3 with apoptotic inhibitors (CrmA and P35) and addition of caspase peptide substrates (Z-VAD-FMK and Z-IETD-FMK) to NS3-transfected cells blocked NS3-induced apoptosis. In cotransfection experiments, NS3 bound to caspase-8 and enhanced caspase-8-mediated apoptosis. NS3 and caspase-8 colocalized in the cytoplasm of transfected cells. Deletion analysis demonstrated that at least two regions of NS3 contribute to its apoptotic activities. The protease and helicase domains are each able to bind to caspase-8, while the protease domain alone induces apoptosis. The protease domain and tetrahelix region of the helicase domain are required for NS3 to augment caspase-8-mediated apoptosis. Thus, the LGT virus NS3 protein is a multifunctional protein that binds to caspase-8 and induces apoptosis.The Flavivirus genus includes mostly arthropod-borne pathogens, many of which are widely distributed throughout the world and cause human diseases that vary in severity. The four serotypes of dengue (DEN) virus, Japanese encephalitis (JE) virus, and yellow fever (YF) virus cause millions of cases of disease annually in the tropics and subtropics. Tick-borne encephalitis (TBE) flaviviruses grouped within the TBE complex (Russian spring-summer encephalitis or TBE, Kyasanur forest disease, Langat [LGT], Louping ill, Negishi, Omsk hemorrhagic fever, and Powassan viruses) are mainly found in the Northern Hemisphere and, except for LGT virus, cause thousands of human cases every year, which can have a mortality as high as 20 to 30%.LGT virus does not naturally infect humans and has been studied as a potential live virus vaccine to protect against encephalitis caused by viruses of the TBE complex (4).LGT virus, like other flaviviruses, is a small lipid-enveloped, positive-sense RNA virus. During infection, a single polycistronic viral RNA is translated into a polyprotein precursor that is co-and posttranslationally processed by host and viral proteases to produce the virion and replicase components. The structural proteins of flaviviruses, capsid (C), premembrane (preM), and envelope (E), are encoded in the 5Ј quarter of the genome, and signal peptidase appears to mediate their cleavages during elongation of the polyprotein precursor through the translocating channel of the rough endoplasmic reticulum. Nonstructural proteins NS2A and -B, NS3, NS4A and -B, and NS5 are cleaved by viral serine protease NS2B-NS3 after a sequence that usually contains two basic residues followed by a short side chain residue (reviewed in reference 19).NS3 protein exhibits p...

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Organization of theFlavivirusRNAreplicase complex

2017

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Flaviviruses, such as Dengue, Japanese encephalitis, West Nile, Yellow Fever, and Zika viruses, are serious human pathogens that cause significant morbidity and mortality globally each year. Flaviviruses are single-stranded, positive-sense RNA viruses, and encode two multidomain proteins, NS3 and NS5, that possess all enzymatic activities required for genome replication and capping. NS3 and NS5 interact within virus-induced replication compartments to form the RNA genome replicase complex. Although the individual enzymatic activities of both proteins have been extensively studied and are well characterized, there are still gaps in our understanding of how they interact to efficiently coordinate their respective activities during positive strand RNA synthesis and capping. Here, we discuss what is known about the structures and functions of the NS3 and NS5 proteins and propose a preliminary NS3:NS5:RNA interaction model based on a large body of literature about how the viral enzymes function, physical restraints between NS3 and NS5, as well as critical steps in the replication process.

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In vitro synthesis of West Nile virus proteins indicates that the amino-terminal segment of the NS3 protein contains the active centre of the protease which cleaves the viral polyprotein after multiple basic amino acids

Cited by 139 publications

References 22 publications

Development and Characterization of New Peptidomimetic Inhibitors of the West Nile Virus NS2B–NS3 Protease

Development and Characterization of New Peptidomimetic Inhibitors of the West Nile Virus NS2B–NS3 Protease

Langat Flavivirus Protease NS3 Binds Caspase-8 and Induces Apoptosis

Organization of theFlavivirusRNAreplicase complex

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