Characterization of the Zinc Binding Activity of the Rubella Virus Nonstructural Protease

Liu, X; Yang, Jenny J.; Ghazi, A. Mohamad; Frey, Teryl K.

doi:10.1128/jvi.74.13.5949-5956.2000

Cited by 32 publications

(34 citation statements)

References 33 publications

(24 reference statements)

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“…Two mutants, BM26 and BM29 (5), that had previously been shown to be compromised for RNA binding, were each competent to bind zinc, but when substitutions of all five basic amino acids were combined into the (Ϫ)BM mutant, zinc binding was reduced to nearly undetectable levels. The minor residual signal detected for the (Ϫ)BM mutant could be attributed to the increased charge interaction between acidic residues in this region and the zinc cation, as has also been suggested for residual zinc binding observed with a mutant of the Rubella virus nonstructural protease (19). These results indicate that the basic residues that mediate RNA-binding activity are also critical for zinc binding.…”

Section: Vol 78 2004supporting

confidence: 52%

“…MBP fusions suitable for affinity purification were constructed with the wt ␥b (pMalC2X␥b) and a ␥b mutant containing cysteine (C)-to-serine (S) mutations at amino acid positions 7,9,10,19,23,60,64,71, and 81 and a histidine (H)-to-leucine (L) mutation at amino acid 85 [pMalC2X␥b(Ϫ)C1C2]. For this purpose, the ␥b gene was amplified from the ␥42 cDNA clone of BSMV RNA␥ (23) or RNA␥(Ϫ)C1C2 by using the primers BamMal2 and gb3ЈPst1.…”

Section: Methodsmentioning

confidence: 99%

“…Putative zinc-binding regions of ␥b were assayed for zinc-binding activity by blotting protein derivatives onto nitrocellulose and probing them with radiolabeled 65 ZnCl 2 (26). This assay has been used previously to demonstrate and map the zinc-binding ability of a number of different viral proteins (3,9,19,27). Since the ␥b(Ϫ)C1C2 mutant could not be purified from infected barley in amounts sufficient for use in zinc affinity chromatography, we assessed the zinc binding of wt ␥b and ␥b mutants expressed as MBP fusions in E. coli.…”

Section: Species Of ␥B Corresponding To Sizes Expected For Monomers (mentioning

confidence: 99%

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The N-Terminal 85 Amino Acids of the Barley Stripe Mosaic Virus γb Pathogenesis Protein Contain Three Zinc-Binding Motifs

Bragg

Lawrence

Jackson

2004

J Virol

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Barley stripe mosaic virus RNA␥ encodes ␥b, a cysteine-rich protein that affects pathogenesis. Nine of the eleven cysteines are concentrated in two clusters, designated C1 (residues 1 to 23) and C2 (residues 60 to 85), that are arranged in zinc finger-like motifs. A basic motif (BM) rich in lysine and arginine (residues 19 to 47) resides between the C1 and C2 clusters. We have demonstrated that ␥b binds zinc and that the C1, BM, and C2 motifs have independent zinc-binding activities. To evaluate the requirements for binding, mutations were introduced into each region. Cysteine residues at positions 7, 9, 10, 19, and 23 in the C1 motif were replaced with serines. In the BM, asparagines were substituted for lysines at positions 26 and 35, glutamine for arginine at position 25, and glycines for arginines at positions 33 and 36. The C2 mutations included cysteine replacements with serines at positions 60, 64, 71, and 81, and a histidine-to-leucine change at position 85. These mutations destroyed zinc-binding activity in each of the isolated motifs. ␥b derivatives containing mutations in only two of the motifs retained the ability to bind zinc, whereas a ␥b derivative containing mutations inactivating all three motifs destroyed the ability to bind zinc. Plants inoculated with transcripts containing combinations of the C1, BM, and C2 mutations elicited a "null" phenotype in barley characteristic of ␥b deletion mutants and also delayed the appearance and reduced the size of local lesions in Chenopodium amaranticolor. These results show that zinc binding of each of the motifs is critical for the biological activity of ␥b.Barley stripe mosaic virus (BSMV) encodes a 17-kDa cysteine-rich protein (CRP) protein, designated ␥b, that is expressed from the 3Ј-proximal cistron of RNA␥ (Fig. 1A). The ␥b protein is expressed early and remains at elevated levels throughout the course of BSMV infection (6). Although ␥b is dispensable for both viral replication and movement in the ND18 strain of the virus, in the ␥⌬2.1 deletion mutant, which removes the bulk of the ␥b gene, symptom onset is delayed in barley, and symptoms are attenuated, resulting in a null phenotype characterized by an erratic mosaic pattern (21). Further analysis of this mutant suggests that ␥b has differential effects on RNA accumulation and protein expression and that it may regulate the synthesis of proteins encoded on RNA␤. Interestingly, in the type strain of BSMV, the ␥b protein is essential for systemic symptom development (21), and the requirement for ␥b is linked to a 372-nucleotide (nt) direct repeat in the 5Ј region of RNA␥ that overlaps the start codon and increases the size of the ␥a gene. This gene encodes the polymerase component of the viral replicase and therefore, this strain-specific difference suggests that subtle effects on replication contribute to the phenotypic effects of mutations in the ␥b protein.Nine of the eleven cysteine residues in the ␥b protein are concentrated in two clusters toward the N terminus of the protein. The C1 (amino acid...

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Section: Vol 78 2004supporting

confidence: 52%

Section: Methodsmentioning

confidence: 99%

Section: Species Of ␥B Corresponding To Sizes Expected For Monomers (mentioning

confidence: 99%

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The N-Terminal 85 Amino Acids of the Barley Stripe Mosaic Virus γb Pathogenesis Protein Contain Three Zinc-Binding Motifs

Bragg

Lawrence

Jackson

2004

J Virol

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“…From predictions based on computer alignment with sequences from other viruses, P150 contains (from N terminus to C terminus) a methyltransferase domain, a Y domain, a proline hinge domain, an X domain, and a protease domain that catalyzes the cleavage of the NS precursor; P90 contains helicase and RNA-dependent RNA polymerase (RDRP) domains (12). Of these, the activities of the protease and helicase domains have been confirmed experimentally (3,8,15,16,19,20,22,34,35). While it has been hypothesized that the X domain functions in trans cleavage mediated by the protease (15), putative functions for the Y and proline hinge domains have not been proposed.…”

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confidence: 99%

Complementation of a Deletion in the Rubella Virus P150 Nonstructural Protein by the Viral Capsid Protein

Tzeng

Frey

2003

J Virol

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Rubella virus (RUB) replicons with an in-frame deletion of 507 nucleotides between two NotI sites in the P150 nonstructural protein (⌬NotI) do not replicate (as detected by expression of a reporter gene encoded by the replicon) but can be amplified by wild-type helper virus (Tzeng et al., Virology 289:63-73, 2001). Surprisingly, virus with ⌬NotI was viable, and it was hypothesized that this was due to complementation of the NotI deletion by one of the virion structural protein genes. Introduction of the capsid (C) protein gene into ⌬NotI-containing replicons as an in-frame fusion with a reporter gene or cotransfection with both ⌬NotI replicons and RUB replicon or plasmid constructs containing the C gene resulted in replication of the ⌬NotI replicon, confirming the hypothesis that the C gene was the structural protein gene responsible for complementation and demonstrating that complementation could occur either in cis or in trans. Approximately the 5 one-third of the C gene was necessary for complementation. Mutations that prevented translation of the C protein while minimally disturbing the C gene sequence abrogated complementation, while synonymous codon mutations that changed the C gene sequence without affecting the amino acid sequence at the 5 end of the C gene had no effect on complementation, indicating that the C protein, not the C gene RNA, was the moiety responsible for complementation. Complementation occurred at a basic step in the virus replication cycle, because ⌬NotI replicons failed to accumulate detectable virus-specific RNA.Rubella virus (RUB) is the sole member of the genus Rubivirus in the family Togaviridae (for a review, see reference 7). The RUB virion consists of a genomic, single-stranded RNA enclosed in a quasispherical capsid composed of multiple copies of the viral capsid protein, C, which is in turn surrounded by a lipid bilayer envelope in which are embedded two virus glycoproteins, E1 and E2. The RUB genome is 9,762 uncleotides (nt) in length, of positive-polarity, and contains two long open reading frames (ORFs). The 5Ј-proximal ORF, or nonstructural protein ORF (NS-ORF), is translated from the genome RNA into a 240-kDa precursor that is proteolytically cleaved at a single site by a virus-encoded protease into two products: an N-terminal product of 150 kDa (P150) and a C-terminal product of 90 kDa (P90). The 3Ј-proximal ORF, or structural protein ORF (SP-ORF), which is translated from a subgenomic (SG) RNA, encodes the virion proteins in the order 5Ј-C-E2-E1-3Ј; processing of these proteins is mediated by the cellular enzyme signal endopeptidase.The RUB NS proteins function in viral RNA replication. From predictions based on computer alignment with sequences from other viruses, P150 contains (from N terminus to C terminus) a methyltransferase domain, a Y domain, a proline hinge domain, an X domain, and a protease domain that catalyzes the cleavage of the NS precursor; P90 contains helicase and RNA-dependent RNA polymerase (RDRP) domains (12). Of these, the activities of the proteas...

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“…A virus encoded metalloproteinase had not been identified when a review article (Dougherty and Semler, 1993) was written although serine, cysteine (or thiol) and aspartic (or acidic) proteases were known. Since then at least three virus-encoded metalloproteinases (including the Zn binding site HEXXH in enhancin) have been discovered (Lepore et al, 1996;Liu et al, 2000;Peng et al, 1999;Suzuki et al, 1999). To this growing list can now be added the phage-encoded proteinase of clostridial polyprotein NT, given the proposed new recognition.…”

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confidence: 99%

Botulinum neurotoxins: Perspective on their existence and as polyproteins harboring viral proteases

DasGupta

2006

J. Gen. Appl. Microbiol.

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Characterization of the Zinc Binding Activity of the Rubella Virus Nonstructural Protease

Cited by 32 publications

References 33 publications

The N-Terminal 85 Amino Acids of the Barley Stripe Mosaic Virus γb Pathogenesis Protein Contain Three Zinc-Binding Motifs

The N-Terminal 85 Amino Acids of the Barley Stripe Mosaic Virus γb Pathogenesis Protein Contain Three Zinc-Binding Motifs

Complementation of a Deletion in the Rubella Virus P150 Nonstructural Protein by the Viral Capsid Protein

Botulinum neurotoxins: Perspective on their existence and as polyproteins harboring viral proteases

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