NS3 Serine Protease of Bovine Viral Diarrhea Virus: Characterization of Active Site Residues, NS4A Cofactor Domain, and Protease–Cofactor Interactions

Tautz, Norbert; Kaiser, Astrid; Thiel, Heinz-Jürgen

doi:10.1006/viro.2000.0425

Cited by 65 publications

(73 citation statements)

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“…This clearly validates our in vivo finding that the serine at position 236 confers protease activity to Ynm3. It has been documented that exchange of active site serines to cysteines does not necessarily abolish proteolytic activity, which remains to varying degrees in different proteases (Hahn and Strauss, 1990;Tautz et al, 2000). In Ynm3, exchanging serine at position 236 to alanine significantly enhanced its stability, but exchange to cysteine was not so effective, with only 65% of the protein left in the reaction after 16 h. Together, there is sufficient evidence that the serine residue at position 236 confers catalytic activity to Ynm3.…”

Section: The Serine Protease Activity Of Ynm3 Is Important To Mediatementioning

confidence: 99%

The Yeast HtrA Orthologue Ynm3 Is a Protease with Chaperone Activity that Aids Survival Under Heat Stress

Padmanabhan

Fichtner

Dickmanns

et al. 2009

MBoC

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Ynm3 is the only budding yeast protein possessing a combination of serine protease and postsynaptic density 95/disclarge/zona occludens domains, a defining feature of the high temperature requirement A (HtrA) protein family. The bacterial HtrA/DegP is involved in protective stress response to aid survival at higher temperatures. The role of mammalian mitochondrial HtrA2/Omi in protein quality control is unclear, although loss of its protease activity results in susceptibility toward Parkinson's disease, in which mitochondrial dysfunction and impairment of protein folding and degradation are key pathogenetic features. We studied the role of the budding yeast HtrA, Ynm3, with respect to unfolding stresses. Similar to Escherichia coli DegP, we find that Ynm3 is a dual chaperone-protease. Its proteolytic activity is crucial for cell survival at higher temperature. Ynm3 also exhibits strong general chaperone activity, a novel finding for a eukaryotic HtrA member. We propose that the chaperone activity of Ynm3 may be important to improve the efficiency of proteolysis of aberrant proteins by averting the formation of nonproductive toxic aggregates and presenting them in a soluble state to its protease domain. Suppression studies with ⌬ynm3 led to the discovery of chaperone activity in a nucleolar peptidyl-prolyl cis-trans isomerase, Fpr3, which could partly relieve the heat sensitivity of ⌬ynm3.

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Section: The Serine Protease Activity Of Ynm3 Is Important To Mediatementioning

confidence: 99%

The Yeast HtrA Orthologue Ynm3 Is a Protease with Chaperone Activity that Aids Survival Under Heat Stress

Padmanabhan

Fichtner

Dickmanns

et al. 2009

MBoC

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“…DI9c derivative 27 has been described by Yu et al (37). Mutations 28,29,30, and 31 were created by primer-directed PCR mutagenesis techniques applying oligonucleotides BVD CS 11, L2336R, L2683Rϩ/plus L2683RϪ, and 5ABLSDPs/plus 5ABLSDPas, respectively (Table 1).…”

Section: Cells and Virusesmentioning

confidence: 99%

“…Processing of the nonstructural protein NS2-3 (125 kDa) and release of the 80-kDa C-terminal NS3 portion occur at different extents in various pestivirus strains and were demonstrated to be associated with a certain phenotype of virus infection in tissue culture: whereas viruses that replicate without obvious damage to the host cell express predominantly NS2-3, generation of high amounts of NS3 is strictly correlated with cytopathogenicity, i.e., virusinduced lysis of the cell (reviewed in reference 28). Proteolysis of the remainder of the polyprotein that gives rise to the mature nonstructural proteins NS3 to NS5B is catalyzed by a viral proteinase complex consisting of a serine protease domain within the N terminus of NS3 and the essential cofactor NS4A (27,30,34,35).…”

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confidence: 99%

Genetic Analysis of the Pestivirus Nonstructural Coding Region: Defects in the NS5A Unit Can Be Complemented in trans

Grassmann

Isken

Tautz

et al. 2001

J Virol

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The functional analysis of molecular determinants which control the replication of pestiviruses was considerably facilitated by the finding that subgenomic forms of the positive-strand RNA genome of BVDV (bovine viral diarrhea virus) are capable of autonomous replication in transfected host cells. The prototype replicon, BVDV DI9c, consists of the genomic 5 and 3 untranslated regions and a truncated open reading frame (ORF) encoding mainly the nonstructural proteins NS3, NS4A, NS4B, NS5A, and NS5B. To gain insight into which of these proteins are essential for viral replication and whether they act in cis or in trans, we introduced a large spectrum of in-frame mutations into the DI9c ORF. Tests of the mutant RNAs in terms of their replication capacity and their ability to support translation and cleavage of the nonstructural polyprotein, and whether defects could be rescued in trans, yielded the following results. (i) RNA replication was found to be dependent on the expression of each of the DI9c-encoded mature proteins NS3 to NS5B (and the known associated enzymatic activities). In the same context, a finely balanced molar ratio of the diverse proteolytic processing products was indicated to be crucial for the formation of an active catalytic replication complex. (ii) Synthesis of negative-strand intermediate and progeny positive-strand RNA was observed to be strictly coupled with all functional DI9c ORF derivatives. NS3 to NS5B were hence suggested to play a pivotal role even during early steps of the viral replication pathway. (iii) Mutations in the NS3 and NS4B units which generated nonfunctional or less functional RNAs were determined to be cis dominant. Likewise, lethal alterations in the NS4A and NS5B regions were invariably noncomplementable. (iv) In surprising contrast, replication of functional and nonfunctional NS5A mutants could be clearly enhanced and restored, respectively. In summary, our data provide initial insights into the organization of the pestivirus replication machinery.Bovine viral diarrhea virus (BVDV) types I and II, border disease virus of sheep, and classical swine fever virus constitute the genus Pestivirus, comprising widely distributed pathogens of ruminants and pigs (reviewed in reference 31). Together with the genera Flavivirus and Hepacivirus (hepatitis C viruses [HCVs]), the pestiviruses are classified in the family Flaviviridae (reviewed in reference 24), all members of which are characterized by an enveloped virion that harbors a singlestranded, linear RNA genome of positive polarity. The genomic RNA, which in the case of pestiviruses has a length of approximately 12.5 kb, consists of a single open reading frame (ORF) and untranslated regions (UTRs) at the 5Ј and 3Ј ends, respectively. Following infection, it operates initially as a messenger in the cytoplasm. Translation is mediated by an internal ribosomal entry site (IRES) within the 5Ј UTR (22) and leads to the synthesis of a polyprotein that is co-and posttranslationally processed into a range of viral proteins. The ord...

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“…NS3 is a multifunctional protein and has serine protease, helicase, and NTPase activities (7)(8)(9)(10). NS3 processes all downstream cleavage sites in concert with its cofactor, NS4A (11,12). For pestiviruses, NS3, NS4A, NS4B, NS5A, and NS5B have been shown to assemble into the active viral RNA-replicase together with an unknown number of host factors (4,13).…”

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confidence: 99%

Characterization of the Determinants of NS2-3-Independent Virion Morphogenesis of Pestiviruses

Klemens

Dubrau

Tautz

2015

J Virol

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A peculiarity of the Flaviviridae is the critical function of nonstructural (NS) proteins for virus particle formation. For pestiviruses, like bovine viral diarrhea virus (BVDV), uncleaved NS2-3 represents an essential factor for virion morphogenesis, while NS3 is an essential component of the viral replicase. Accordingly, in natural pestivirus isolates, processing at the NS2-3 cleavage site is not complete, to allow for virion morphogenesis. Virion morphogenesis of the related hepatitis C virus (HCV) shows a major deviation from that of pestiviruses: while RNA replication also requires free NS3, virion formation does not depend on uncleaved NS2-NS3. Recently, we described a BVDV-1 chimera based on strain NCP7 encompassing the NS2-4B*-coding region of strain Osloss (E. Lattwein, O. Klemens, S. Schwindt, P. Becher, and N. Tautz, J Virol 86:427-437, 2012, doi:10.1128/JVI.06133-11). This chimera allowed for the production of infectious virus particles in the absence of uncleaved NS2-3. The Osloss sequence deviates in the NS2-4B* part from NCP7 in 48 amino acids and also has a ubiquitin insertion between NS2 and NS3. The present study demonstrates that in the NCP7 backbone, only two amino acid exchanges in NS2 (E1576V) and NS3 (V1721A) are sufficient and necessary to allow for efficient NS2-3-independent virion morphogenesis. The adaptation of a bicistronic virus encompassing an internal ribosomal entry site element between the NS2 and NS3 coding sequences to efficient virion morphogenesis led to the identification of additional amino acids in E2, NS2, and NS5B that are critically involved in this process. The surprisingly small requirements for approximating the packaging schemes of pestiviruses and HCV with respect to the NS2-3 region is in favor of a common mechanism in an ancestral virus. IMPORTANCEFor positive-strand RNA viruses, the processing products of the viral polyprotein serve in RNA replication as well as virion morphogenesis. For bovine viral diarrhea virus, nonstructural protein NS2-3 is of critical importance to switch between these processes. While free NS3 is essential for RNA replication, uncleaved NS2-3, which accumulates over time in the infected cell, is required for virion morphogenesis. In contrast, the virion morphogenesis of the related hepatitis C virus is independent from uncleaved NS2-NS3. Here, we demonstrate that pestiviruses can adapt to virion morphogenesis in the absence of uncleaved NS2-3 by just two amino acid exchanges. While the mechanism behind this gain of function remains elusive, the fact that it can be achieved by such minor changes is in line with the assumption that an ancestral virus already used this mechanism but lost it in the course of adapting to a new host/infection strategy. T he family Flaviviridae comprises the genera Flavivirus, Hepacivirus, Pestivirus, and Pegivirus (1-3). Pestiviruses, like bovine viral diarrhea virus (BVDV) and classical swine fever virus (CSFV), are important pathogens causing significant economic damage in livestock industries (3)....

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NS3 Serine Protease of Bovine Viral Diarrhea Virus: Characterization of Active Site Residues, NS4A Cofactor Domain, and Protease–Cofactor Interactions

Cited by 65 publications

References 65 publications

The Yeast HtrA Orthologue Ynm3 Is a Protease with Chaperone Activity that Aids Survival Under Heat Stress

The Yeast HtrA Orthologue Ynm3 Is a Protease with Chaperone Activity that Aids Survival Under Heat Stress

Genetic Analysis of the Pestivirus Nonstructural Coding Region: Defects in the NS5A Unit Can Be Complemented in trans

Characterization of the Determinants of NS2-3-Independent Virion Morphogenesis of Pestiviruses

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