Identification of a Ca
            <sup>2+</sup>
            -Binding Domain in the Rubella Virus Nonstructural Protease

Zhou, Yubin; Tzeng, Wen-Pin; Yang, Wei; Zhou, Yumei; Ye, Yiming; Lee, Hsiau‐Wei; Frey, Teryl K.; Yang, Jenny J.

doi:10.1128/jvi.00605-07

Cited by 26 publications

(58 citation statements)

References 74 publications

(79 reference statements)

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“…A dansyl moiety was conjugated to CaM by following the procedures described previously (16,18). RUBCa, the minidomain within the RUBV NS protease (aa 1143-1252 of P150) that contains the predicted CaM binding region, or the mutants RUBCa-R1165Q and RUBCa-R1165D, was expressed as GST fusion proteins and prepared as described in previous studies (4,5).…”

Section: Methodsmentioning

confidence: 99%

“…where f is the relative fluorescence change observed during the experiment, [Ca 2ϩ ] is the concentration of free ionized Ca (4,5). The RUBV NS protease domain (red) is responsible for the self-cleavage of the P200 precursor polypeptide into two mature products, P150 and P90.…”

Section: Methodsmentioning

confidence: 99%

“…(Tb 3ϩ -LRET) experiments were carried out as described previously (4,23). In brief, 10 M Chelex-100-treated protein-peptide complexes were titrated by aliquots of Tb 3ϩ stock in 20 mM PIPES, 150 mM KCl, and 1 mM DTT, pH 6.8 (to prevent precipitation).…”

Section: Methodsmentioning

confidence: 99%

“…RUBV Infectious Clone, RUBV Replicon-based Protease Cleavage Assay, and Co-immunoprecipitation-The infectious RUBV genomic cDNA clone, Robo502-HA, and replicon, RUBrep-HA/GFP, were described previously (4,27,28). Both contain an HA epitope tag at the upstream NotI site in the P150 gene.…”

Section: Methodsmentioning

confidence: 99%

“…Previous studies in our laboratories have shown that this protease, termed the NS protease, is a papain-like protease with catalytic dyad of Cys 1152 and His 1273 (2) (numbering refers to residues in P150). We recently showed that the NS protease contains a 29-residue EF-hand Ca 2ϩ binding motif, which plays a structural role in stabilizing the protease at physiological temperatures (4), as well as a structural Zn 2ϩ binding site coordinated by four cysteines, which is necessary for protease activity (5,6). During a bioinformatic search for possible functional domains in the nonstructural proteins, we detected a putative 1-8-14 class calmodulinbinding domain (CaMBD) in the RUBV NS protease domain by using the calmodulin (CaM) binding site search and analysis tool (7).…”

mentioning

confidence: 99%

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Calcium-dependent Association of Calmodulin with the Rubella Virus Nonstructural Protease Domain

Zhou

Tzeng

Wong

et al. 2010

Journal of Biological Chemistry

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The rubella virus (RUBV) nonstructural (NS) protease domain, a Ca2؉ -and Zn 2؉ -binding papain-like cysteine protease domain within the nonstructural replicase polyprotein precursor, is responsible for the self-cleavage of the precursor into two mature products, P150 and P90, that compose the replication complex that mediates viral RNA replication; the NS protease resides at the C terminus of P150. Here we report the Ca 2؉ -dependent, stoichiometric association of calmodulin (CaM) with the RUBV NS protease. Co-immunoprecipitation and pulldown assays coupled with site-directed mutagenesis demonstrated that both the P150 protein and a 110-residue minidomain within NS protease interacted directly with Ca 2؉ /CaM. The specific interaction was mapped to a putative CaM-binding domain. A 32-mer peptide (residues 1152-1183, denoted as RUBpep) containing the putative CaM-binding domain was used to investigate the association of RUBV NS protease with CaM or its N-and C-terminal subdomains. We found that RUBpep bound to Ca 2؉ /CaM with a dissociation constant of 100 -300 nM. The C-terminal subdomain of CaM preferentially bound to RUBpep with an affinity 12.5-fold stronger than the N-terminal subdomain. Fluorescence, circular dichroism and NMR spectroscopic studies revealed a "wrapping around" mode of interaction between RUBpep and Ca 2؉ /CaM with substantially more helical structure in RUBpep and a global structural change in CaM upon complex formation. Using a site-directed mutagenesis approach, we further demonstrated that association of CaM with the CaMbinding domain in the RUBV NS protease was necessary for NS protease activity and infectivity.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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Calcium-dependent Association of Calmodulin with the Rubella Virus Nonstructural Protease Domain

Zhou

Tzeng

Wong

et al. 2010

Journal of Biological Chemistry

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Rubella Virus

Frey

Matthews

2011

Encyclopedia of Life Sciences

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Rubella virus is a human virus belonging to the family Togaviridae and the sole member of the rubivirus genus. Rubella virus has a small genome composed of single‐stranded RNA and produces only five proteins. The rubella virus replication cycle takes place entirely within the cytoplasm of the infected cell. Rubella virus establishes a body‐wide or systemic infection that is manifested by mild symptoms including rash, lymph node swelling and low‐grade fever. However, rubella virus is an important human pathogen because it causes a spectrum of serious birth defects known collectively as congenital rubella syndrome (CRS) when a mother lacking immunity is infected during the first trimester of pregnancy. CRS can include blindness, deafness, mental retardation, heart malformations and endocrine dysfunctions. Before development of effective live, attenuated vaccines, rubella virus was the leading teratogenic agent of an infectious nature. Through use of these vaccines, rubella and CRS are controlled in much, but not all, of the world and are targeted for elimination in the Western Hemisphere and Europe. Key Concepts: Rubella virus causes a benign systemic illness characterised by a rash, but is a dangerous teratogen when infection occurs during pregnancy. Fetal infection by rubella virus results in congenital rubella syndrome, a constellation of serious birth defects, particularly deafness, blindness and mental retardation. Rubella virus is classified in the Togaviridae family and is the only member of the rubivirus genus. Rubella virions are pleiomorphic, approximately 70 nm in diameter and consist of a quasispherical capsid containing the genome RNA surrounded by an envelope composed of a lipid bilayer from which protrude glycoprotein spikes. The rubella virus genome is a plus‐polarity RNA, 10 000 nucleotides in length, which encodes only five proteins: P150 and P90, both of which participate in RNA replication in the infected cells, and capsid (C) and envelope glycoproteins 1 and 2 (E1 and E2) which comprise the virion. In the infected cell, rubella virus replication takes place entirely within the cytoplasm. In the infected cell, rubella virus RNA replication, which proceeds through an RNA intermediate, takes place in association with cell membranes. In the infected cell, rubella virion formation takes place in association with the Golgi apparatus and virions are released by exocytosis. Rubella virus isolates worldwide cluster into 13 genotypes that fit into two distinct clades with an overall diversity at the nucleotide level of approximately 10%, a very low level for an RNA virus. Live, attenuated rubella virus vaccines, available since approximately 1970, have been used successfully to control or eliminate rubella in developed countries whereas implementation of these vaccines is progressing, but not complete, in undeveloped countries of the world.

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Probing Ca2+-Binding Capability of Viral Proteins with the EF-Hand Motif by Grafting Approach

Zhou

Xue

Chen

et al. 2012

Methods in Molecular Biology

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Ca(2+) is implicated in almost every step of the life cycle of viruses, including virus entry into host cells, virus replication, virion assembly, maturation, and release. However, due to the lack of prediction algorithms and rigorous validation methods, only limited cases of viral Ca(2+)-binding sites are reported. Here, we introduce a method to predict continuous EF-hand or EF-hand-like motifs in the viral genomes based on their primary sequences. We then introduce a grafting approach, and the use of luminescence resonance energy transfer and Ca(2+) competition assay for experimental verification of predicted Ca(2+)-binding sites. This protocol will be valuable for the prediction and identification of unknown Ca(2+)-binding sites in virus.

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Identification of a Ca ²⁺ -Binding Domain in the Rubella Virus Nonstructural Protease

Cited by 26 publications

References 74 publications

Calcium-dependent Association of Calmodulin with the Rubella Virus Nonstructural Protease Domain

Calcium-dependent Association of Calmodulin with the Rubella Virus Nonstructural Protease Domain

Rubella Virus

Probing Ca2+-Binding Capability of Viral Proteins with the EF-Hand Motif by Grafting Approach

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Identification of a Ca 2+ -Binding Domain in the Rubella Virus Nonstructural Protease

Cited by 26 publications

References 74 publications

Calcium-dependent Association of Calmodulin with the Rubella Virus Nonstructural Protease Domain

Calcium-dependent Association of Calmodulin with the Rubella Virus Nonstructural Protease Domain

Rubella Virus

Probing Ca2+-Binding Capability of Viral Proteins with the EF-Hand Motif by Grafting Approach

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Identification of a Ca ²⁺ -Binding Domain in the Rubella Virus Nonstructural Protease