Substrate determinants for cleavage in cis and in trans by the hepatitis C virus NS3 proteinase

Bartenschlager, Ralf; Ahlborn-Laake, L; Yasargil, Kaya; Mous, Jan; Jacobsen, Helmut

doi:10.1128/jvi.69.1.198-205.1995

Cited by 101 publications

(35 citation statements)

References 52 publications

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“…These critical cleavage events in theory could occur through intraor intermolecular (i.e., cis or trans) mechanisms. Biochemical evidence of cis-cleavages at the hepaci-and pestivirus NS3-NS4A and flavivirus NS2B-NS3 has been reported for various virus systems (10,15,47,59). The capture of the post-cis-cleavage state in HCV (29) and in CSFV (this study) has provided valid evidence to further support the existence of cis-cleavage at the NS3-NS4A junction for the Hepacivirus and Pestivirus genera.…”

Section: Discussionsupporting

confidence: 73%

Uncoupling of Protease trans -Cleavage and Helicase Activities in Pestivirus NS3

Zheng

et al. 2017

J Virol

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The nonstructural protein NS3 from the family is a multifunctional protein that contains an N-terminal protease and a C-terminal helicase, playing essential roles in viral polyprotein processing and genome replication. Here we report a full-length crystal structure of the classical swine fever virus (CSFV) NS3 in complex with its NS4A protease cofactor segment (PCS) at a 2.35-Å resolution. The structure reveals a previously unidentified ∼2,200-Å intramolecular protease-helicase interface comprising three clusters of interactions, representing a "closed" global conformation related to the NS3-NS4A -cleavage event. Although this conformation is incompatible with protease-cleavage, it appears to be functionally important and beneficial to the helicase activity, as the mutations designed to perturb this conformation impaired both the helicase activities and virus production Our work reveals important features of protease-helicase coordination in pestivirus NS3 and provides a key basis for how different conformational states may explicitly contribute to certain functions of this natural protease-helicase fusion protein. Many RNA viruses encode helicases to aid their RNA genome replication and transcription by unwinding structured RNA. Being naturally fused to a protease participating in viral polyprotein processing, the NS3 helicases encoded by the family viruses are unique. Therefore, how these two enzyme modules coordinate in a single polypeptide is of particular interest. Here we report a previously unidentified conformation of pestivirus NS3 in complex with its NS4A protease cofactor segment (PCS). This conformational state is related to the protease-cleavage event and is optimal for the function of helicase. This work provides an important basis to understand how different enzymatic activities of NS3 may be achieved by the coordination between the protease and helicase through different conformational states.

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Section: Discussionsupporting

confidence: 73%

Uncoupling of Protease trans -Cleavage and Helicase Activities in Pestivirus NS3

Zheng

et al. 2017

J Virol

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“…During infection and also during the experimental setup of in vitro translation, the increase in local concentration of the substrate that the cis cleavage provides may make it the predominant pathway. This situation is different from the cleavage of the hepatitis C virus (HCV) polyprotein site NS3/ 4A, which is obligatorily cleaved in cis (4,21). The sequence of the NS3/4A site is distinct from those of the other HCV cleavage sites and more tolerant for substitutions, due to additional interactions involved in recognition of the site and due to protein folding (47).…”

Section: Discussionmentioning

confidence: 98%

Molecular Determinants of Substrate Specificity for Semliki Forest Virus Nonstructural Protease

Lulla

Tints

et al. 2006

J Virol

100

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The C-terminal cysteine protease domain of Semliki Forest virus nonstructural protein 2 (nsP2) regulates the virus life cycle by sequentially cleaving at three specific sites within the virus-encoded replicase polyprotein P1234. The site between nsP3 and nsP4 (the 3/4 site) is cleaved most efficiently. Analysis of Semliki Forest virus-specific cleavage sites with shuffled N-terminal and C-terminal half-sites showed that the main determinants of cleavage efficiency are located in the region preceding the cleavage site. Random mutagenesis analysis revealed that amino acid residues in positions P4, P3, P2, and P1 of the 3/4 cleavage site cannot tolerate much variation, whereas in the P5 position most residues were permitted. When mutations affecting cleavage efficiency were introduced into the 2/3 and 3/4 cleavage sites, the resulting viruses remained viable but had similar defects in P1234 processing as observed in the in vitro assay. Complete blockage of the 3/4 cleavage was found to be lethal. The amino acid in position P1 had a significant effect on cleavage efficiency, and in this regard the protease markedly preferred a glycine residue over the tyrosine natively present in the 3/4 site. Therefore, the cleavage sites represent a compromise between protease recognition and other requirements of the virus life cycle. The protease recognizes at least residues P4 to P1, and the P4 arginine residue plays an important role in the fast cleavage of the 3/4 site. Semliki Forest virus (SFV)is an enveloped positive-strand RNA virus of the genus Alphavirus, family Togaviridae. Alphavirus replication relies on the production of replicase proteins in the form of polyprotein precursor(s), which are then coand posttranslationally processed (40). This finally leads to the generation of individual replicase protein subunits and ensures the formation of proper interactions between the cleavage products of a single polyprotein (35). However, it is not known whether multiple polyproteins are required to build up an individual replication complex. A large body of evidence indicates that the polyprotein expression and cleavage strategy regulates viral RNA replication (20,24,25,37,46). The fulllength nonstructural (ns) polyprotein, designated P1234, is translated directly from the viral genomic RNA and becomes cleaved immediately after or during its synthesis. Its primary cleavage products, polyprotein P123 and nsP4, form the early replication complexes synthesizing negative-strand RNA. P123 is first cleaved to yield nsP1 and P23, giving an intermediate complex, nsP1-P23-nsP4, which is capable of both negativestrand and positive-strand synthesis (20,24,46) but which appears to be very short lived in infected cells. P23 is rapidly cleaved into nsP2 and nsP3, and the replication complex is rearranged into a stable form making exclusively positive-sense RNA with the negative strand as a template.

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“…Thus, a consensus sequence would read D/E-X-X-X-X-C/T↓S/A-X-X-X (where 'X' is variable). Mutagenesis studies of the serine proteinase-dependent sites have revealed that: first, the acidic P6 residue is dispensable; second, except for the structure-disturbing Pro, the P1' position is tolerant towards substitutions; and third, the P1-Cys is the dominant determinant for cleavage efficiency [84,92,113,117,118]. Alteration of its sulphydryl function and/or bulkiness or chirality resulted in a 20-to 12 500-fold loss of cleavage efficiency [105,115,116].…”

Section: Substrate Specificity Of the Ns3/4a Proteinasementioning

confidence: 99%

“…Alteration of its sulphydryl function and/or bulkiness or chirality resulted in a 20-to 12 500-fold loss of cleavage efficiency [105,115,116]. Interestingly, the severity of substitutions on processing increased with the distance from the proteinase, with the NS3/4A site being much more tolerant than the NS4A/B, NS4B/5A and NS5A/B sites [92,117]. This result probably reflects the different mechanisms operating at the various sites.…”

Section: Substrate Specificity Of the Ns3/4a Proteinasementioning

confidence: 99%

The NS3/4A proteinase of the hepatitis C virus: unravelling structure and function of an unusual enzyme and a prime target for antiviral therapy

Bartenschlager

1999

Journal of Viral Hepatitis

128

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The hepatitis C virus (HCV) is a major causative agent of transfusion-acquired and sporadic non-A, non-B hepatitis worldwide. Infections most often persist and lead, in approximately 50% of all patients, to chronic liver disease. As is characteristic for a member of the family Flaviviridae, HCV has a plus-strand RNA genome encoding a polyprotein, which is cleaved co- and post-translationally into at least 10 different products. These cleavages are mediated, among others, by a virally encoded chymotrypsin-like serine proteinase located in the N-terminal domain of non-structural protein 3 (NS3). Activity of this enzyme requires NS4A, a 54-residue polyprotein cleavage product, to form a stable complex with the NS3 domain. This review will describe the biochemical properties of the NS3/4A proteinase, its X-ray crystal structure and current attempts towards development of efficient inhibitors.

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Substrate determinants for cleavage in cis and in trans by the hepatitis C virus NS3 proteinase

Cited by 101 publications

References 52 publications

Uncoupling of Protease trans -Cleavage and Helicase Activities in Pestivirus NS3

Uncoupling of Protease trans -Cleavage and Helicase Activities in Pestivirus NS3

Molecular Determinants of Substrate Specificity for Semliki Forest Virus Nonstructural Protease

The NS3/4A proteinase of the hepatitis C virus: unravelling structure and function of an unusual enzyme and a prime target for antiviral therapy

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