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Literature summary for 3.4.21.113 extracted from

  • 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 (2017), PLoS Pathog., 13, e1006134 .
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

Cloned (Comment) Organism
recombinant expression of His-tagged wild-type and mutant enzymes in Escherichia coli Classical swine fever virus

Crystallization (Commentary)

Crystallization (Comment) Organism
purified recombinant soluble CSFV NS4A37NS3 complex, X-ray diffraction structure determination and analysis at 3.05-3.21 A resolution Classical swine fever virus

Protein Variants

Protein Variants Comment Organism
K232A site-directed mutagenesis, helicase active site mutant Classical swine fever virus
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 Classical swine fever virus
S163A site-directed mutagenesis, protease active site mutant, proteolytically inactive Classical swine fever virus
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 Classical swine fever virus

Organism

Organism UniProt Comment Textmining
Classical swine fever virus Q5U8X5 CSFV, a pestivirus
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Posttranslational Modification

Posttranslational Modification Comment Organism
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 Classical swine fever virus

Purification (Commentary)

Purification (Comment) Organism
recombinant His-tagged wild-type and mutant enzymes from Escherichia coli by nickel affinity chromatography, followed by tag cleavage through TEV protease Classical swine fever virus

Synonyms

Synonyms Comment Organism
CSFV NS3
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Classical swine fever virus
NS3-4A protease
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Classical swine fever virus

General Information

General Information Comment Organism
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 Classical swine fever virus
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 Classical swine fever virus
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 Classical swine fever virus
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 Classical swine fever virus