Any feedback?
Information on EC 3.4.21.113 - pestivirus NS3 polyprotein peptidase
for references in articles please use BRENDA:EC3.4.21.113Please wait a moment until all data is loaded. This message will disappear when all data is loaded.
EC Tree
3.4.21.113 pestivirus NS3 polyprotein peptidaseSpecify your search results
Word Map
- 3.4.21.113
- polyproteins
- dengue
-
cytopathogenic
-
bluetongue
- ns4a
- ntpase
- flaviviruses
- flaviviridae
- horsesickness
- pestiviruses
- drug development
- medicine
The enzyme appears in viruses and cellular organisms
Reaction Schemes
Leu is conserved at position P1 for all four cleavage sites. Alanine is found at position P1' of the NS4A-NS4B cleavage site, whereas serine is found at position P1' of the NS3-NS4A, NS4B-NS5A and NS5A-NS5B cleavage sites
Synonyms
nonstructural protein ns3, csfv ns3, wnv ns2b/ns3, flavivirus ns3 protease, 
more
topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
hydrolysis of peptide bond
-
-
-
-
CAS REGISTRY NUMBER
COMMENTARY
37259-58-8
-
SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
additional information
?
-
the enzyme is a natural protease-helicase fusion protein, structure-function analysis, overview
-
-
?
additional information
?
-
-
the enzyme is a natural protease-helicase fusion protein, structure-function analysis, overview
-
-
?
additional information
?
-
the S1 pocket of the active site to accommodate the P1 residue (i.e., the immediate N-terminal residue of the cleavage site) is relatively deep and small, with a primarily hydrophobic environment, similar to the S1 pocket of HCV NS3. In contrast, the flavivirus NS3 P1 site is relatively flat and spacious but not as hydrophobic, substrate specificity, overview
-
-
?
additional information
?
-
-
the S1 pocket of the active site to accommodate the P1 residue (i.e., the immediate N-terminal residue of the cleavage site) is relatively deep and small, with a primarily hydrophobic environment, similar to the S1 pocket of HCV NS3. In contrast, the flavivirus NS3 P1 site is relatively flat and spacious but not as hydrophobic, substrate specificity, overview
-
-
?
NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
additional information
?
-
the enzyme is a natural protease-helicase fusion protein, structure-function analysis, overview
-
-
?
additional information
?
-
-
the enzyme is a natural protease-helicase fusion protein, structure-function analysis, overview
-
-
?
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
malfunction
metabolism
upon infection of the host cell the viral RNA genome is translated into a polyprotein that is processed by cellular and viral proteases into the mature structural (SP) and nonstructural (NS) proteins. For members of the genus Pestivirus the array in the polyprotein is the following: NH2-Npro (N-terminal autoprotease), C (capsid protein, core), Erns (envelope protein RNase secreted), E1, E2, p7, NS2-3 (NS2 and NS3), NS4A, NS4B, NS5A, NS5B-COOH. The N-terminal autoprotease Npro generates its own C-terminus and thereby the N-terminus of the capsid protein core (C). Further cleavages releasing the structural proteins C, Erns, E1 and E2 as well as p7 are mediated by proteases residing in the endoplasmatic reticulum (ER). The cleavage between NS2 and NS3 is catalyzed by an autoprotease in NS2. The activity of the NS2 protease is temporally regulated by a cellular cofactor leading to significant amounts of uncleaved NS2-3 in pestivirus infected cells. The cleavages downstream of NS3, NS4A, NS4B and NS5A are catalyzed by the serine protease domain of NS3 which requires NS4A as cofactor for full proteolytic activity and is termed NS3-4A protease
physiological function
additional information
malfunction
altering either of the autocleavage sites (Leu192/Met193 and Leu159/Lys160) by Leu deletion or mutation greatly inhibits RNA replication in a CSFV replicon system and results in a loss of genome RNA infectivity
malfunction
the gain of function mutation 3/V132A as well as the mutations in the NS4A-kink region expose the TEV cleavage site suggesting a destabilization of the NS3/4A-kink interaction. Single mutations at the NS3/4A-kink interface allow for polyprotein processing and RNA replication. The gain of function mutation 3/V132A can be functionally substituted by the single mutations 4A/L45A or 4A/Y47A in NS2-3-independent virion morphogenesis. The replication-deficient NS3/4A double mutant 4A/L45-Y47-AA is functional in viral packaging when supplied in trans. Analysis of NS3 and NS2 mutations combined, overview
physiological function
cleavages downstream of NS3, NS4A, NS4B and NS5A, former parts of the viral polyprotein, are catalyzed by the serine protease domain of NS3 which requires NS4A as cofactor for full proteolytic activity and is termed NS3-4A protease. A special feature of pestiviruses is the existence of significant amounts of uncleaved NS2-3 in the infected cell and its essential role in virion formation, temporal restriction of NS2-3 processing by the NS2 autoprotease, mostly restricted to the early phase of infection. NS2-3 translated at later time points is only inefficiently processed leading to the accumulation of uncleaved NS2-3 which temporally correlates with the onset of virion morphogenesis. Downregulation of NS2-3 processing plays a crucial role for the non-cytopathogenic (ncp) biotype of pestiviruses in cell culture
physiological function
NS3 is a multifunctional enzyme and a natural fusion of an N-terminal chymotrypsin-like serine protease and a C-terminal nucleotide triphosphatase (NTPase)/helicase. All Flaviviridae NS3 proteases require a segment of another viral protein, NS4A for pestiviruses, as the structurally integrated essential cofactor, termed protease cofactor segment (PCS) to fulfill the protease function, and for pestiviruses the protease is responsible for the cleavage of all the linkages in the NS3-NS4A-NS4B-NS5A-NS5B region of the viral polyprotein. The enzyme is a natural protease-helicase fusion protein, structure-function analysis, overview. Analysis of the mechanism of NS3 helicase regulation by its fusion partner protease
additional information
structure-function analysis suggests that NS3/4A can adopt two different conformations in the infected cell, a closed form that is used in RNA replication complexes and a more open conformation functional in viral assembly. Furthermore, the NS2-3/4A complex, required for virion assembly of prototype pestiviruses, displays a similar open conformation. CSFV NS3/4A complex crystal structure analysis, overview
additional information
the catalytic triad is formed by residues H69, D97, and S163. The intramolecular interface between protease and helicase in CSFV NS3 is featured by three clusters of interactions, overview
additional information
-
the catalytic triad is formed by residues H69, D97, and S163. The intramolecular interface between protease and helicase in CSFV NS3 is featured by three clusters of interactions, overview
UNIPROT
ENTRY NAME
ORGANISM
NO. OF AA
NO. OF TRANSM. HELICES
MOLECULAR WEIGHT[Da]
SOURCE
SEQUENCE
LOCALIZATION PREDICTION
The reliability class of the localization prediction ranges from 1 (very reliable) to 5 (not reliable).
The reliability class of the localization prediction ranges from 1 (very reliable) to 5 (not reliable). SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
additional information
additional information
the NS3-NS4APCS protease contains a typical dual beta-barrel core observed in the chymotrypsin-like serine proteases, and the PCS participates in the folding of both the N- and C-terminal barrels (N-barrel and C-barrel). The N-terminal 14 residues intertwine with NS4A residues 29 to 41 to form a long anti-parallel beta-type structural element that bridges the N- and C-barrels of the protease. NS3 protease protein structure comparisons, overview
additional information
-
the NS3-NS4APCS protease contains a typical dual beta-barrel core observed in the chymotrypsin-like serine proteases, and the PCS participates in the folding of both the N- and C-terminal barrels (N-barrel and C-barrel). The N-terminal 14 residues intertwine with NS4A residues 29 to 41 to form a long anti-parallel beta-type structural element that bridges the N- and C-barrels of the protease. NS3 protease protein structure comparisons, overview
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
proteolytic modification
proteolytic modification
the viral RNA genome contains one large open reading frame (ORF) encoding a polyprotein of about 4,000 amino acids. The polyprotein is co- and posttranslationally processed into at least 12 mature proteins by viral and host proteases through cis (intramolecular) or trans (intermolecular) mechanisms. Core, Erns, E1, and E2 are the structural proteins that become part of the mature virion, and the eight nonstructural proteins Npro, p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B are involved in polyprotein processing, viral genome replication, and virus morphogenesis. cis-Cleavage events are essential to generate structurally independent proteases that may carry out trans-cleavages more efficiently than proteases in the form of polyprotein. Limitations are also applied to cis-cleavages, as the intramolecular recognition of the cleavage site requires the protease protein to adopt certain conformations that may not always achievable. Two minor autocleavage sites, Leu192/Met193 and Leu159/Lys160, are detected within the NS3 protease module
proteolytic modification
upon infection of the host cell, the viral RNA genome is translated into a polyprotein that is processed by cellular and viral proteases into the mature structural (SP) and nonstructural (NS) proteins. For members of the genus Pestivirus the array in the polyprotein is the following: NH2-Npro (N-terminal autoprotease), C (capsid protein, core), Erns (envelope protein RNase secreted), E1, E2, p7, NS2-3 (NS2 and NS3), NS4A, NS4B, NS5A, NS5B-COOH. The N-terminal autoprotease Npro generates its own C-terminus and thereby the N-terminus of the capsid protein core (C). Further cleavages releasing the structural proteins C, Erns, E1 and E2 as well as p7 are mediated by proteases residing in the endoplasmatic reticulum (ER). The cleavage between NS2 and NS3 is catalyzed by an autoprotease in NS2. The activity of the NS2 protease is temporally regulated by a cellular cofactor leading to significant amounts of uncleaved NS2-3 in pestivirus infected cells
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
purified full-length CSFV NS3 with its NS4A PCS covalently tethered to its N terminus through a flexible linker, X-ray diffraction structure determination and analysis at 2.35 A resolution
purified recombinant soluble CSFV NS4A37NS3 complex, X-ray diffraction structure determination and analysis at 3.05-3.21 A resolution
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
K232A
site-directed mutagenesis, helicase active site mutant
S163A
site-directed mutagenesis, protease active site mutant, proteolytically inactive
V132A
mutation of residue 132 of the NS3 protease domain, the gain of function mutation in NS3 maps to a hydrophobic surface patch which interacts with the NS4A-kink region
additional information
construction of recombinant full-length NS3 (residues 1 to 683) followed by the 8 N-terminal residues of NS4A, the NS3 sequence is preceeded by residues 21 to 57 of the protease cofactor NS4A
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
recombinant His-tagged wild-type and mutant enzymes from Escherichia coli by nickel affinity chromatography, followed by tag cleavage through TEV protease
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
recombinant expression of His-tagged wild-type and mutant enzymes in Escherichia coli
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Zheng, F.; Lu, G.; Li, L.; Gong, P.; Pan, Z.
Uncoupling of protease trans-cleavage and helicase activities in pestivirus NS3
J. Virol.
91
e01094-17
2017
Classical swine fever virus (Q5U8X5), Classical swine fever virus
Dubrau, D.; Tortorici, M.A.; Rey, F.A.; Tautz, N.
A positive-strand RNA virus uses alternative protein-protein interactions within a viral protease/cofactor complex to switch between RNA replication and virion morphogenesis
PLoS Pathog.
13
e1006134
2017
Classical swine fever virus (Q5U8X5)
EXTERNAL LINKS (specific for EC-Number 3.4.21.113)
Transporter Classification Database (TCDB): 1.A.53.1.5
Use of this online version of BRENDA is free under the CC BY 4.0 license. See terms of use for full details.
Release 2023.1 (January 2023)
Legal
Curated at
Member of
