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Journal of Virology, July 2007, p. 7517-7528, Vol. 81, No. 14
0022-538X/07/$08.00+0     doi:10.1128/JVI.00605-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Identification of a Ca²⁺-Binding Domain in the Rubella Virus Nonstructural Protease

Yubin Zhou,¹ Wen-Pin Tzeng,² Wei Yang,¹ Yumei Zhou,² Yiming Ye,¹ Hsiau-wei Lee,¹ Teryl K. Frey,^2* and Jenny Yang^1*

Department of Chemistry,¹ Department of Biology, Georgia State University, Atlanta, Georgia 30303²

Received 21 March 2007/ Accepted 25 April 2007

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The rubella virus (RUB) nonstructural protein (NS) open reading frame (ORF) encodes a polypeptide precursor that is proteolytically self cleaved into two replicase components involved in viral RNA replication. A putative EF-hand Ca²⁺-binding motif that was conserved across different genotypes of RUB was predicted within the nonstructural protease that cleaves the precursor by using bioinformatics tools. To probe the metal-binding properties of this motif, we used an established grafting approach and engineered the 12-residue Ca²⁺-coordinating loop into a non-Ca²⁺-binding scaffold protein, CD2. The grafted EF-loop bound to Ca²⁺ and its trivalent analogs Tb³⁺ and La³⁺ with K_ds of 214, 47, and 14 µM, respectively. Mutations (D1210A and D1217A) of two of the potential Ca²⁺-coordinating ligands in the EF-loop led to the elimination of Tb³⁺ binding. Inductive coupled plasma mass spectrometry was used to confirm the presence of Ca²⁺ ([Ca²⁺]/[protein] = 0.7 ± 0.2) in an NS protease minimal metal-binding domain, RUBCa, that spans the EF-hand motif. Conformational studies on RUBCa revealed that Ca²⁺ binding induced local conformational changes and increased thermal stability (T_m = 4.1°C). The infectivity of an RUB infectious cDNA clone containing the mutations D1210A/D1217A was decreased by 20-fold in comparison to the wild-type (wt) clone, and these mutations rapidly reverted to the wt sequence. The NS protease containing these mutations was less efficient at precursor cleavage than the wt NS protease at 35°C, and the mutant NS protease was temperature sensitive at 39°C, confirming that the Ca²⁺-binding loop played a structural role in the NS protease and was specifically required for optimal stability under physiological conditions.

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Temporal and spatial changes in the intracellular Ca²⁺ concentration in eukaryotic cells affect the regulation of numerous cellular processes by modulating the activity of Ca²⁺-binding proteins (CaBPs) with varied Ca²⁺-binding affinities in different cellular compartments. Ca²⁺-binding motifs are also present in virus proteins and function in virion assembly and stability (1, 8, 15, 16, 20, 23, 24, 31, 33, 37, 40, 43, 55) and virion-associated activities, such as cell fusion (12, 16) and neuraminidase activity (5, 7). To date, almost all of the reported viral CaBPs are structural proteins, including both capsid proteins and envelope proteins.

The helix-loop-helix EF-hand moiety is one of the most common motifs in proteins of bacteria, archaea, and eukaryotes (28, 73), and by binding to Ca²⁺ this motif may undergo conformational changes enabling Ca²⁺-modulated functions, as seen in the trigger or sensor proteins calmodulin and troponin C (27, 32), or may control the concentration of Ca²⁺ to maintain local Ca²⁺ homeostasis, as reported in buffering proteins such as parvalbumin (29) and calbindin D_9k (56). The coordination of Ca²⁺ in EF-hand motifs is fulfilled by adopting a pentagonal bipyramidal geometry. Though EF-hands have been found abundantly in eukaryotes and bacteria (42, 46, 47, 51, 52, 73), this Ca²⁺-binding motif has seldom been reported in viruses.

Rubella virus (RUB), the only member of the genus Rubivirus, in the Togaviridae family, is the causative agent of a disease called rubella or German measles. RUB is an enveloped, single-stranded positive-polarity RNA virus with a genome size of 9,762 nucleotides (21). The RUB genome contains two long open reading frames (ORFs): a 5'-proximal ORF, or NSP-ORF (nucleotides [nt] 41 to 6389 of the genome; 2,116 amino acids [aa]; molecular mass, 240 kDa), encodes the nonstructural proteins (NSP) involved in viral RNA replication; a 3'-proximal ORF, or structural protein (SP)-ORF (nt 6512 to 9700; 1,063 aa; molecular mass, 110 kDa), encodes one capsid protein (C) and two envelope glycoproteins (E1 and E2) (18, 20). Multiple-sequence alignments of the RUB NSP-ORF against other positive-stranded RNA virus genomes revealed a number of conserved domains, namely, methyltransferase (M), protease (P), helicase (H), replicase (R), and a proline-rich region (G), as well as an X domain (X) showing high homology to the Appr-1-p processing enzyme (1, 18). Upon uncoating and release of viral mRNA after entry into host cells, the NSP-ORF is immediately translated into a polypeptide precursor (P200), which is subsequently self cleaved by its protease domain (termed the nonstructural, or NS, protease) at a cleavage site mapped between residues G1301 and G1302 into two mature products (P150 and P90) involved in viral RNA replication in association with late endosomal or lysosomal membranes (30, 38, 43). The NS protease has been reported to be a Zn²⁺-dependent papain-like cysteine protease with C1152 and H1273 constituting the catalytic dyad (6, 23, 34, 36, 37).

In the present study, we report our prediction of a single EF-hand Ca²⁺-binding motif within the NS protease domain of RUB. To confirm this prediction, the binding loop was grafted into a scaffolding protein, CD2, and the minimal metal-binding domain of the NS protease was bacterially expressed. Both were found to bind Ca²⁺ or its trivalent analogs Tb³⁺ and La³⁺. We also found that mutagenesis of critical Ca²⁺-binding residues negatively impacted virus infectivity and rendered the activity of the NS protease temperature sensitive. This is one of the few demonstrations of an EF-hand Ca²⁺-binding motif in a virus-encoded protein.

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Grafting the predicted Ca²⁺-binding loop into CD2. The predicted 12-residue Ca²⁺-binding loop was directly inserted between Gly53 and Ala54 by PCR using an established protocol (70). The mutant with double mutations D5A and D12A (numbered according to the loop position of the inserted Ca²⁺-binding motif) was produced using standard PCR methods. All sequences were verified by automated sequencing on an ABI PRISM-377 DNA sequencer (Applied Biosystems) in the Advanced Biotechnology Core Facilities of Georgia State University.

Molecular cloning of the minimal metal-binding domain. The sequence encoding the putative Ca²⁺-binding domain (RUBCa; aa 1143 to 1252 of the NS ORF) was amplified from the RUB infectious cDNA clone Robo502 (49, 61) by using standard PCR methods. The PCR product was subsequently inserted into the pGEX-2T vector (GE Healthcare) to produce the plasmid pGEX-2T-RUBCa.

Expression and purification of RUBCa, CD2.RUBCa, and its mutant. RUBCa was expressed as a glutathione S-transferase (GST) fusion protein in Escherichia coli BL21(DE3) transformed with the plasmid pGEX-2T-RUBCa in Luria-Bertani medium with 100 mg/liter of ampicillin and grown at 37°C. One hundred micromolars of isopropyl-ß-D-thiogalactopyranoside (IPTG) and 50 µM of ZnCl₂ were added when the optical density at 600 nm reached 0.7 to induce protein expression for another 3 to 4 h. The proteins were purified following the protocols for GST fusion protein purification (70) using glutathione Sepharose 4B beads (GE Healthcare). The protein was cleaved from its GST tag on beads by taking advantage of the thrombin cleavage site and eluted. The eluted fractions containing RUBCa were further purified using Superdex 75 and Hitrap SP columns (GE Healthcare). The molecular weight of RUBCa was confirmed by matrix-assisted laser desorption ionization-time of flight mass spectrometry in the Advanced Biotechnology Core Facilities of Georgia State University. The concentration of RUBCa was measured by its absorption at 280 nm with an extinction coefficient of 19,630 M^–1 cm^–1, which was calculated according to previously described methods (22). The engineered proteins CD2.RUBCa and the mutant CD2.RUBCa.D5A/D12A were expressed and purified according to methods described previously (70). The molecular weights of CD2.RUBCa and the D5A/D12A mutant were also confirmed by matrix-assisted laser desorption ionization-time of flight mass spectrometry. The concentration was determined by using the absorption at 280 nm with an extinction coefficient of 11,700 M^–1 cm^–1 (15).

ESI-MS. The detection of metal-protein complex by electrospray ionization mass spectrometry (ESI-MS) was performed by using a Waters Micromass Q-Tof micro instrument. The data were acquired in positive-ion mode by syringe pump infusion of the protein solutions at a flow rate of 7 µl/min. The protein sample stock (1 mM) in 10 mM Tris, pH 7.4, was diluted 100-fold into water. Metal ions were added in 5 M excesses to the protein concentration to observe specific binding.

Refolding of RUBCa. RUBCa denatured in 8 M urea was refolded by gradually diluting the sample in the presence of a 2x excess molar concentration of CaCl₂ until the concentration of urea reached 2 M. The mixture was incubated and stirred gently for 16 h at 4°C. The sample was then extensively dialyzed against 10 mM Tris-HCl (pH 7.4) buffer. Considering the presence of six cysteines in the protein, 1 mM reduced and 0.2 mM oxidized glutathione were added into the buffer to facilitate the proper formation of possible disulfide bonds.

ICP-MS. All the glassware, plasticware, and Teflon containers used in the preparation of samples were pretreated with 2% HNO₃ (optima grade; Fisher Scientific). All buffers were pretreated with Chelex-100 (Bio-Rad) to remove the background Ca²⁺ ions. The refolded proteins (20 to 30 µM) were acidified with 2% HNO₃ and analyzed by inductively coupled plasma mass spectrometry (ICP-MS) (Finnigan Element 2). The dialyzed buffer was used as a blank, and the residual Ca²⁺ background of 2 to 5 µM was subtracted from the measurement of protein samples.

CD spectroscopy. The circular dichroism (CD) spectra of proteins were recorded in a Jasco-810 spectropolarimeter at ambient temperature using a quartz cell of 10-mm path length, with the protein concentrations ranging from 2 to 5 µM. All spectra were obtained as the average of at least eight scans with a scan rate of 100 nm/min. The calculation of secondary-structure elements was performed by using DICHROWEB, an online server for protein secondary structure analyses (67). The thermal denaturation was studied using a 1-mm quartz cell with protein concentrations of 15 to 30 µM in 10 mM Tris-HCl (pH 7.4), 100 mM KCl with 1 mM EGTA or 1 mM CaCl₂. The ellipticity was measured from 190 to 260 nm and converted to mean residue molar ellipticity (degrees [deg] cm² dmol^–1 residue [res]^–1). To obtain the thermal transition point, the signal changes at 222 nm were fitted using the equation described previously (39).

Fluorescence spectroscopy. Fluorescence emission spectra were measured using a 1-cm-path-length cell on a PTI lifetime fluorimeter at ambient temperature. For intrinsic tryptophan fluorescence, spectral measurements were carried out at protein concentrations of 2 to 4 µM in 20 mM piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES)-10 mM KCl at pH 6.8 with slit widths of 4 and 8 nm for excitation and emission, respectively. The emission spectra were collected from 300 to 400 nm with an excitation wavelength at 282 nm. 8-Anilino-1-naphthalene sulfonic acid (ANS) fluorescence emission spectra were recorded from 400 to 600 nm, with the excitation wavelength at 390 nm. Protein samples (5 µM) with 1 mM CaCl₂ or 1 mM EGTA were added into the solution containing 40 µM ANS, 20 mM PIPES-10 mM KCl at pH 6.8.

Tyr/Trp-sensitized Tb³⁺ fluorescence energy transfer (Tb³⁺-FRET) experiments were conducted as described previously (69, 71). For the metal competition studies, the solution containing 40 µM of Tb³⁺ and 1.5 µM of protein was set as the starting point. The stock solution of metal ions with the same concentration of Tb³⁺ and protein was gradually added into the initial mixture. The fluorescence intensity was normalized by subtracting the contribution of the baseline slope using logarithmic fitting. The Tb³⁺-binding affinity of protein was obtained by fitting normalized fluorescence intensity data using the equation

(1)

where f is the fractional change, K_d is the dissociation constant for Tb³⁺, and [P]_T and [M]_T are the total concentrations of protein and Tb³⁺, respectively.

The Ca²⁺ competition data were first analyzed using the apparent dissociation constant obtained with equation 1. By assuming that the sample is almost saturated with Tb³⁺ at the starting point of the competition, the Ca²⁺-binding affinity is further obtained by using the equation

(2)

where K_d,Ca and K_d,Tb are the dissociation constants of Ca²⁺ and Tb³⁺, respectively. K_app is the apparent dissociation constant. All the measurements were conducted in triplicate.

¹H NMR spectroscopy. One-dimensional ¹H nuclear magnetic resonance (1D ¹H NMR) spectra were recorded on a Varian 500 MHz NMR spectrometer with a spectral width of 6,600 Hz. Samples of 200 to 250 µM were placed in 20 mM PIPES-10 mM KCl, 10% D₂O at pH 6.8. La³⁺ stock solution was gradually added into the NMR sample tube. The program FELIX98 (MSI) was used to process NMR data, with an exponential line broadening of a 2-Hz window function and the suppression of water signal with a Gaussian deconvolution function with a width of 20.

Cells, infectious clone, and site-directed mutagenesis. The infectious genomic cDNA clone Robo502 was previously described (49, 61). To generate the mutated construct Robo502AA (D1210A and D1217A), a two-round asymmetric PCR strategy was employed to create the mutations in an amplified fragment between unique BsmI and RsrII sites at nt 3243 and 3897 of the RUB genome. The doubly digested fragment was used to replace the corresponding fragment in Robo502. The mutations were also transferred to the replicons RUBrep-FLAG/GFP and RUBrep-HA/GFP (63), which express a P150 tagged with the FLAG or the hemagglutinin (HA) epitope and the reporter protein, green fluorescent protein (GFP), in place of the SP-ORF.

In vitro transcription, transfection, Northern blotting, and Western blotting. Vero cells were obtained from the American Type Culture Collection and maintained at 35°C under 5% CO₂ in Dulbecco's modified Eagle's medium (DMEM; Gibco) supplemented with 5% fetal bovine serum and 10 µg/ml gentamicin. CsCl density gradient-purified plasmids were linearized with EcoRI (New England Biolabs), followed by phenol-chloroform extraction and ethanol precipitation. RNA transcripts were synthesized at 37°C in a 25-µl reaction mixture containing 40 mM Tris-HCl (pH 7.5), 1 µg DNA, 6 mM MgCl₂, 2 mM spermidine, 10 mM dithiothreitol, 1 mM of each nucleoside triphosphate, 1 U of RNasin (Roche Applied Science), 2 mM cap analog m⁷G(5')ppp(5')G (New England Biolabs), and 25 U of SP6 DNA-dependent RNA polymerase (Epicenter Technologies). The transcription reaction mixtures were directly used for cell transfection without DNase treatment. The transcripts were analyzed by electrophoresis of 2-µl aliquots of the reaction mixture in 0.8% agarose gel. The precise yield was determined using a UV/vis spectrometer (Shimadzu). Transfection of the RNA transcripts was done using Vero cells at 80% confluence in 60-mm² plates. Vero cells were washed twice with 3 ml of phosphate-buffered saline and once with 3 ml of Opti-MEMI (Gibco). Ten microliters of the transcripts was subsequently mixed with 500 µl Opti-MEMI and 7 µl Lipofectamine 200 (1 mg/ml). The mixture and 1.5 ml of Opti-MEMI were added to the plates to cover the monolayer of Vero cells. After 4 h of incubation, the transfection mixture was removed and 4 ml of DMEM supplemented with 2% fetal bovine serum was added (transfection is designated passage 0, or P0). Following development of significant cytopathic effect, the medium was harvested and passaged twice in Vero cells (P1 and P2). Virus titers were determined by plaque assay as previously described (48, 65). To determine if the D1210A and D1217A mutations were maintained following transfection and passage, plaques were picked from terminal dilution plaque assay plates of P1 culture fluid and amplified once in Vero cells. Following amplification, total infected cell RNA was extracted and the NS protease region of the genome was amplified by reverse transcription-PCR, and the reverse transcription-PCR product was sequenced (62).

The expression of reporter gene GFP in living, transfected cells was monitored by a Zeiss Axioplan upright microscope with epifluorescence capability and photographed with a Zeiss Axiocam. Northern blotting was used to detect replicon-specific RNA species by following previously established protocols (63). To detect in vivo NS protease activity, Vero cells transfected with RUBrep-HA/GFP, RUBrepAA-HA/GFP, or RUBrepNS*-HA/GFP (containing a C1152A mutation of the catalytic site) were incubated at 35°C or 39°C. Six hours posttransfection, the cells were lysed and resolved by 8% sodium dodecyl sulfate-8% polyacrylamide gel electrophoresis (SDS-8% PAGE), electroblotted onto nitrocellulose membranes, and probed with anti-HA monoclonal antibodies (Sigma) according to a previously established procedure (63).

Computer-aided sequence and structural analyses. The Ca²⁺-binding motif signatures developed in our laboratory (http://www.chemistry.gsu.edu/faculty/Yang/Calciomics.htm) as well as the pattern PS00018 from PROSITE (http://us.expasy.org/cgi-bin/nicedoc.pl?PDOC00018) were used to scan through the genome of RUB for any potential EF-hand Ca²⁺-binding site (73). Sequence alignments were conducted by using the program ClustalW. The secondary structure was predicted based on the consensus prediction results using programs PSIPRED, JPRED, and PHD (10, 41, 54). The homology modeling of the engineered protein and the protease was constructed using the comparative structure modeling server SWISS-MODEL (57). CD2 (Protein Databank [pdb] code 1hng) and the leader protease of foot-and-mouth disease virus (FMDV) (pdb code 1qmy) were used as the templates for structure modeling based on their available high-resolution structures (15, 24). Prediction of Ca²⁺-binding sites in the modeled structure was conducted by using the program GG, a computational algorithm developed in our laboratory on the basis of the geometric description, graph theory, and key structural and chemical features associated with the calcium binding in proteins (11).

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Predicting an EF-hand Ca²⁺-binding motif and homology modeling of the RUB NS protease. By taking advantage of the sequence alignment of currently available EF-hand proteins and considering the structural context of the Ca²⁺-binding loop, we generated a series of patterns for the prediction of EF-hand proteins (73). Using this prediction method (http://www.chemistry.gsu.edu/faculty/Yang/Calciomics.htm), we detected one putative EF-hand Ca²⁺-binding motif (aa 1197 to 1225) within the RUB NS protease intertwined with the partially characterized cysteine-rich Zn²⁺-binding motif (Fig. 1A) (37). This putative Ca²⁺-binding motif shared high homology with well-known EF-hand proteins such as calmodulin and calcyphosine (Fig. 1B). In canonical EF-hand Ca²⁺-binding motifs, the bidentate ligand residue at loop position 12 is either a Glu (95%) or an Asp (5%). Considering its longer side chain and closer distance to the metal ion, a Glu at this position (as seen in calmodulin) ensures a stronger binding affinity for Ca²⁺ than does an Asp. The predicted motif in the RUB NS protease contained an Asp at loop position 12, similar to the first EF-hand in calcyphosine, regulatory light chain of myosin (3, 66), and sarcoplasmic CaBP (9) that exhibit relatively weaker Ca²⁺-binding affinities. Prediction of the secondary structure elements revealed that the RUB NS protease Ca²⁺-binding loop was flanked by two helices, as observed in most EF-hand CaBPs (47). The predicted Ca²⁺-binding motif is highly conserved among the eight genotypes of RUB for which sequence of this region is available (72), including all of the Ca²⁺-binding coordination ligands (Fig. 1B).

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FIG. 1. Genomic organization of RUB and identification of a potential EF-hand Ca2+-binding motif. (A) Schematic representation of the RUB genome organization and domain locations. M, methyltransferase; G, proline-rich hinge; P, protease; H, helicase; R, RNA-dependent RNA polymerase; C, capsid protein; E1 and E2, envelope glycoproteins. The X domain represents a conserved domain with unknown function that is also found in NSP3 from alphaviruses and belongs to the Appr-1-processing enzyme family. The protease contains at least one Zn2+-binding site as well as a putative EF-hand Ca2+-binding motif (box with diagonal pattern). It has been proposed that the residues C1152 and H1273 constitute the catalytic dyad (stars). The minimal metal-binding domain RUBCa (aa 1143 to 1252) is bacterially expressed and used in this study. (B) Sequence alignment results of putative EF-hand Ca2+-binding motif RUBCa in the protease domain with calmodulin and calcyphosine. The motif remained conserved in both clades I (genotypes RV1a, 1B, 1C, 1D, and 1E) and II (genotypes RV2A, 2B, and 2c) of RUB. Boldface residues represent the potential Ca2+-coordinating residues. NCBI or GenBank accession numbers are the following: CaM_EF1 and CaM_EF2 (calmodulin, P62158); CAYP_hum and CAYP_rab (calcyphosine from human, Q13938; rabbit, P41150). The genomic sequence of the eight different genotypes of RUB was sequenced in our laboratory (unpublished data). (C) Homology modeling of RUB NS protease. The leader protease of FMDV (pdb code 1qmy, chain A), a papain-like cysteine protease with a high-resolution structure available, is chosen as the template for homology modeling. The active site consists of C1152 and H1273 (shown as sticks). The predicted EF-hand Ca2+-binding motif, located at the opposite side, is highlighted in blue with the calcium ion shown as a cyan sphere. The cysteine (green) and tryptophan (magenta) residues in the protease domain are shown as sticks. The cleavage site G1301-G1302 is highlighted in black.

Since the three-dimensional (3D) structure of a viral papain-like cysteine protease, the leader protease of FMDV, has been determined (25, 53), it provides an excellent template for homology modeling of the NS protease of RUB. Pairwise sequence alignment of the leader protease of FMDV (pdb code 1qmy) with the RUB NS protease revealed 12.1% sequence identity and highly similar secondary-structure arrangement. Based on the sequence alignment results, a homology model of the RUB NS protease has been generated using the program SWISS-MODEL (Fig. 1C). With the program GG (11), we further detected a potential Ca²⁺-binding pocket around the predicted EF-hand Ca²⁺-binding motif in this model structure, indicating perfect agreement between the prediction made on the basis of primary sequence and the 3D structure of RUB NS protease.

Grafting the EF-hand Ca²⁺-binding loop into CD2 domain 1 (CD2.D1). Although the RUB NS protease was previously expressed in active form as a maltose-binding protein (MBP) fusion (37), it was never purified free from the MBP domain despite numerous attempts. We have likewise expressed it as a GST fusion protein but were unable to purify it following cleavage from the GST domain, because it was insoluble (likely because it is highly hydrophobic in nature). Therefore, to study the Ca²⁺-binding capability of the putative Ca²⁺-binding motif, we grafted the 12-residue Ca²⁺-binding loop (aa 1206 to 1217) into a non-Ca²⁺-binding scaffold protein, CD2.D1. As seen in Fig. 2A, CD2 is a cellular adhesion molecule composed of nine ß-strands with an immunoglobulin-like fold, and the putative Ca²⁺-binding loop from the RUB NS protease was inserted between Gly53 and Ala54, which lie within the loop between strands C'' and D of CD2; the grafted construct was termed CD2.RUBCa. Additionally, three glycines were placed on each side of the Ca²⁺-binding loop to serve as linkers to provide sufficient conformational freedom for the grafted Ca²⁺-binding loop and to minimize perturbation on the host protein while allowing the inserted loop to retain its capability of chelating Ca²⁺ (31, 69-71). The spectroscopic silence of Ca²⁺ makes it extremely challenging to investigate Ca²⁺-binding properties directly. Therefore, lanthanides are commonly used to probe Ca²⁺-binding sites due to their similar ionic radii (Ca²⁺, 1.00 Å; Tb³⁺, 0.92 Å; La³⁺, 1.03 Å) and metal coordination chemistry. In CD2.RUBCa, four aromatic residues in the host protein spaced closely (<15 Å) to the grafted loop made it possible to monitor the binding of Tb³⁺ using FRET (Fig. 2A).

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FIG. 2. Grafting the predicted EF-hand Ca2+-binding loop into CD2.D1 and formation of metal-protein complex. (A) Model structure of the engineered protein CD2.RUBCa. The Ca2+-binding loop (black) from the RUB NS protease (aa 1206 to 1217), with two glycines on the left and three glycines on the right, rendering flexibility, is grafted to the loop which connects strands C'' and D. The model structure was built using the automated comparative protein-modeling server SWISS-MODEL. Aromatic residues Trp (black) are shown as sticks. The Ca2+ ion is shown as a sphere. Intrinsic Trp fluorescence emission spectra (B) and far-UV CD spectra (C) of CD2 (open circle) and engineered protein CD2.RUBCa (closed circle) are compared to examine the perturbation of insertion on the scaffold protein CD2.D1. Buffers consists of 10 mM Tris, 10 mM KCl, pH 7.4. (D) Electrospray mass spectra of CD2 with a grafted EF-loop from RUB NS protease (P) in the presence of a fivefold molar excess of TbCl3 (+156).

A variety of spectroscopic methods confirmed that the structure of the CD2.D1 had not been perturbed by the inserted loop either in the absence or presence of metal ions. The Trp intrinsic fluorescence spectrum of CD2 overlapped with that of CD2.RUBCa, both of which exhibited the emission maximum at 325 nm and a shoulder peak at 313 nm (Fig. 2B). The far-UV CD spectra of CD2.RUBCa remained similar to that of CD2.D1 alone with a deep trough at 216 nm, characteristic of typical ß-sheet elements (Fig. 2C). The differences observed could be primarily from the inserted 12-residue loop and flanking triple-glycine linkers. The 2D ¹H NMR further showed that the majority of the dispersed signals in the fingerprint regions of the total correlated spectroscopy and nuclear overhauser enhancement spectroscopy spectra of CD2.RUBCa were located at the same positions as that of CD2, suggesting that the integrity and packing of the host protein frame are maintained after the insertion of the Ca²⁺-binding loop from RUB NS protease (unpublished data). In general, the insertion of the foreign acidic sequence did not cause major conformational changes of the host protein, and the overall fold of CD2.RUBCa remained similar to that of CD2.D1. The ability of the grafted protein (calculated molecular mass, 12,712.9 Da) to form 1:1 metal/protein complexes was confirmed by ESI-MS. As shown in Fig. 2D, the molecular mass of the engineered protein CD2.RUBCa agreed well with theoretical mass. The presence of a fivefold excess of Tb³⁺ led to the emergence of a new peak and additional mass of 156 Da. Similar results were also obtained for La³⁺. This provides the foundation for measuring the intrinsic metal-binding affinity with a minimized contribution from protein conformational change and minimal influence of the host protein environment on the grafted EF-hand loop (69).

Obtaining the metal-binding affinities of grafted Ca²⁺-binding loop by FRET and NMR. As shown in Fig. 3A, the addition of increasing amounts of CD2.RUBCa to 10 µM Tb³⁺ resulted in an increase of Tb³⁺ emission fluorescence intensity at 545 nm when excited at 282 nm, suggesting the formation of a metal-protein complex. However, the addition of both CD2.D1 and CD2.RUBCa.AA, a mutated construct with glutamic acid-to-alanine changes at two of the ligands within the grafted loop (D5A/D12A), had substantially weaker enhancement than that of CD2.RUBCa, suggesting that the mutant lost its specific Tb³⁺-binding capability and that Tb³⁺ bound to the grafted loop through the ligands D5 and D12. By monitoring the changes of Tb³⁺ emission enhancement of a fixed amount of proteins as a function of Tb³⁺ concentration (Fig. 3B) and by assuming a 1:1 (Tb³⁺ to protein) binding ratio, a dissociation constant (K_d) of 47 ± 4 µM was obtained for CD2.RUBCa. In addition, the dissociation constant of Ca²⁺ was indirectly estimated by a metal ion competition assay in which 1.5 µM of CD2.RUBCa with 40 µM Tb³⁺ was titrated with increasing amounts of Ca²⁺ in excess. As shown in the inset of Fig. 3B, the Tb³⁺ fluorescence emission maximum at 545 nm decreased due to the competition between Tb³⁺ and Ca²⁺ for the grafted metal-binding site, yielding an apparent dissociation constant of 395 ± 135 µM and a real dissociation constant of 214 ± 73 µM for Ca²⁺ according to equation 2.

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FIG. 3. Obtaining metal-binding affinity using aromatic residue-sensitized Tb3+-FRET and 1D 1H NMR. (A) Tb3+ fluorescence enhancement at 545 nm due to resonance energy transfer (excited at 282 nm) as a function of protein concentrations of CD2 (open diamond), CD2.RUBCa (closed circle), and its mutant D5A/D12A (open circle). (B) Tb3+ titration of CD2.RUBCa. Normalized fluorescence intensity was plotted as a function of the Tb3+ concentration. The inset shows the Ca2+ competition titration curve of CD2.RUBCa (1.5 µM) preincubated with 40 µM Tb3+ in 20 mM PIPES, 10 mM KCl, pH 6.8. (C) La3+ titration of CD2.RUBCa monitored by 1D 1H NMR. Some resonances (arrows) at amide regions of 1D 1H NMR spectra of CD2.RUBCa (0.2 mM) shift with the increased concentrations of La3+ (from bottom to top: 0, 39.2, 113.2, 214.3, 442.2, 360.7, 605.1, 929.1, and 1,411.2 µM, respectively) in 20 mM PIPES, 10 mM KCl, at pH 7.4. (D) The chemical shift change at different resonant regions as a function of the concentration of La3+. ppm, parts per million.

1D ¹H NMR was conducted to monitor the La³⁺-induced chemical shift changes of CD2.RUBCa as a function of La³⁺ concentration. Several resonances, such as those at 7.97, 6.98, 6.79, and 2.67 ppm (Fig. 3C), gradually shifted or sharpened as the La³⁺ concentration increased, indicating a single binding process. The La³⁺-binding affinity, with an average of 14 ± 7 µM, was obtained by plotting the chemical shift of these peaks as a function of La³⁺ concentration (Fig. 3D).

Metal selectivity. Competition assays based on Tb³⁺-FRET have been used as a convenient method to probe the binding capacity of physiologically competing metal ions (e.g., 100 mM K⁺ and 10 mM Mg²⁺) to EF-hand CaBPs, such as calmodulin (64) and galactose-binding protein (14). As seen in Fig. 4A, a significant decrease in Tb³⁺-FRET elicited from Tb³⁺-loaded CD2.RUBca was only observed following the addition of 1 mM Ca²⁺ or 0.1 mM La³⁺ (another Ca²⁺ analog, as discussed above). However, the addition of 100 mM K⁺ or 10 mM Mg²⁺ led to only a slight decrease (<10%) in the intensity of Tb³⁺ fluorescence. To further clarify whether the engineered protein CD2.RUBCa exhibited selectivity for Ca²⁺, we monitored the 1D ¹H NMR spectrum with sequential additions of K⁺, Mg²⁺, and Ca²⁺. As shown in Fig. 4B, while excess K⁺ and Mg²⁺ did not result in any changes in the chemical shifts, Ca²⁺ was able to specifically induce changes of chemical shifts in the main-chain amide proton region even in the presence of 100 mM K⁺ and 10 mM Mg²⁺. These results clearly demonstrate that CD2.RUBCa is capable of binding Ca²⁺ with selectivity over Mg²⁺ or K⁺, similar to other known EF-hand proteins, such as calmodulin (44, 50).

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FIG. 4. Metal selectivity of the engineered protein CD2.RUBCa. (A) Metal competition assay. The addition of 1.5 µM CD2.RUBCa to free Tb3+ (40 µM) solution resulted in an increase of fluorescence intensity at 545 nm by over 20-fold due to the binding of Tb3+ to the protein and the resultant FRET. K+ (100 mM), 10 mM Mg2+, 1 mM Ca2+, and 0.1 mM La3+ were subsequently added to individually prepared solutions containing 40 µM Tb3+ and 1.5 µM CD2.RUBCa. (B) Amide region of 1D 1H NMR spectrum of CD2.RUBCa with sequential addition of 100 mM K+, 10 mM Mg2+, and 1 mM Ca2+. Resonances exhibiting changes are indicated by arrows. ppm, parts per million.

Using the minimal metal-binding domain RUBCa for metal-binding and conformational studies. While our attempts to express the entire NS protease (aa 1000 to 1300) of P150 were fruitless, we successfully expressed a minimal metal-binding domain as a GST fusion protein and purified the minimal metal-binding domain (RUBCa, aa 1143 to 1252; see Fig. 1), which contains the Ca²⁺-binding loop, both catalytic residues, and the ligands thought to be involved in Zn²⁺ binding. Purified RUBCa was used to characterize the predicted EF-hand Ca²⁺-binding motif in its native protein environment. When purified RUBCa was reconstituted in the presence of Ca²⁺ and subjected to ICP-MS to measure its metal content, it was found to bind Ca²⁺ with a molar ratio (Ca²⁺/protein) of 0.7 ± 0.2 to 1 (n = 2).

RUBCa contains three Trp residues, and according to the model of the structure of the RUB NS protease (Fig. 1C), one of these aromatic residues, W1153, is in close proximity to the predicted Ca²⁺-binding loop (3.5 Å) (Fig. 1C). This feature enabled us to probe the metal-binding properties of RUBCa using Tb³⁺-FRET. As shown in Fig. 5A, a significant enhancement of Tb³⁺ fluorescence intensity was observed when Tb³⁺ was added to RUBCa, indicating Tb³⁺ binding to the predicted loop. A K_d for Tb³⁺ of 3 ± 1 µM was obtained (Fig. 5A, inset).

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FIG. 5. Metal ion titration of the minimal metal-binding domain RUBCa monitored by aromatic residue-sensitized Tb3+ fluorescence (A) and intrinsic Trp fluorescence (B). (A) Normalized Tb3+ fluorescence spectra of RUBCa with increasing concentrations of Tb3+ (from bottom to top: 0, 1.0, 4.0, 9.9, 14.8, 19.6, and 14.4 µM, respectively). The inset shows the Tb3+ fluorescence enhancement at 545 nm due to energy transfer as a function of the concentration of Tb3+. (B) Intrinsic Trp fluorescence emission spectra of RUBCa (2.5 µM) with increasing concentrations of Ca2+ (from top to bottom: 0, 49.8, 291.3, 566.0, 740.7, and 909.1 µM, respectively). The inset shows the intrinsic Trp fluorescence intensity plotted as a function of the concentration of Ca2+. An average dissociation constant of 316 µM was obtained by assuming a 1:1 binding model. The excitation wavelength was set at 282 nm. All the buffers used in metal titration consist of 20 mM PIPES, 10 mM KCl, pH 6.8.

In comparison with the 355 nm for free Trp, the emission maximum of the purified RUBCa blue-shifted to 339 nm, suggesting that at least some Trp in RUBCa was partly shielded from the solvent, though not fully buried inside the hydrophobic core (Fig. 5B). The shoulder at 355 nm suggests that some Trp residues were exposed to the solvent. No Ca²⁺-induced emission peak position change was observed. However, the addition of Ca²⁺ led to the decrease of the emission intensity, indicating local changes of the chemical environment around aromatic residues (Fig. 5B). By monitoring this intensity change, a dissociation constant of 316 ± 4 µM was obtained (Fig. 5B, inset), which was in agreement with the Ca²⁺-binding affinity obtained using the grafted protein CD2.RUBCa (Table 1).

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TABLE 1. The metal-binding affinities of the engineered protein CD2.RUBCa and the minimal metal-binding domain RUBCa

Far-UV CD was performed to reveal any possible changes of secondary structure of RUBCa induced by Ca²⁺. As seen in Fig. 6A, the spectra of both EGTA-treated and Ca²⁺-loaded RUBCa had two troughs at 222 and 206 nm, indicating the existence of -helical secondary structures. Using the program DICHROWEB (67), the best fit of the CD spectrum of RUBCa indicated that 17.8% was -helix and 19.5% was ß-sheet, whereas the remainder was random coil. In excess Ca²⁺, the CD signal of RUBCa at 208 nm and 222 nm was 5% more negative than that of RUBCa in 1 mM EGTA, and the DICHROWEB-predicted -helix and ß-sheet contents were 23.8% and 14.9%, respectively. Thus, the observed gain in negative ellipticity could be attributed to the formation of a higher degree of -helical content induced by Ca²⁺ binding. The anionic amphiphile ANS was further used as a hydrophobic probe to examine the conformational properties of RUBCa. As shown in Fig. 6B, upon addition of RUBCa the emission peak of ANS fluorescence blue-shifted from 510 nm to 490 nm and the maximal emission intensity increased by 30%, suggesting that part of the hydrophobic regions of the purified RUBCa were exposed to the solvent and thus were accessible to ANS. The addition of excess Ca²⁺ did not cause significant conformational changes in these hydrophobic regions, considering the overlapping emission fluorescence spectra of RUBCa in the presence of 1 mM EGTA or 1 mM Ca²⁺. Taken together, these data indicated that the binding of Ca²⁺ induced a local conformational change, whereas the secondary structure and hydrophobic surface were not significantly altered.

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FIG. 6. Ca2+-induced conformational changes and thermal unfolding of the putative Ca2+-binding domain RUBCa. (A) Far-UV CD spectra of RUBCa with 1 mM EGTA (open circle) or 1 mM Ca2+ (closed circle) in 10 mM Tris-HCl, 100 mM KCl. The inset shows normalized CD signal at 222 nm plotted as a function of increasing temperature (5 to 90°C) in the presence of 1 mM EGTA (open circle) or 1 mM Ca2+ (closed circle). (B) Fluorescence emission spectra of 40 µM ANS (open square) and ANS-RUBCa complex with 1 mM EGTA (open circle) or 1 mM Ca2+ (closed circle). The excitation wavelength was set at 390 nm. The buffer consisted of 10 mM Tris-HCl, 10 mM KCl (pH 7.4).

In order to gain more insight into the possible role of Ca²⁺ binding, thermal unfolding was carried out by monitoring the CD signal change at 222 nm as a function of temperature under Ca²⁺-depleted or Ca²⁺-loaded conditions. With increasing temperature from 5 to 90°C, RUBCa gradually underwent thermal denaturation, leading to the decrease of CD signals. Compared with 1 mM EGTA, the melting temperature (T_m) of RUBCa with Ca²⁺ increased from 37.7°C ± 0.8°C to 41.8°C ± 0.4°C (Fig. 6A). The observed increase of melting temperature suggested that the binding of Ca²⁺ stabilized the overall structure of RUBCa under physiological conditions (100 mM KCl).

Mutating the potential Ca²⁺-binding ligands in infectious cDNA clones and replicons. To determine if the sequence that could potentially bind Ca²⁺ is of physiological significance, the D5A/D12A double mutant shown to abrogate binding in CD2.RUBCa was introduced into the Robo502 infectious cDNA clone of RUB (the mutant was termed Robo502AA). Following transfection, the virus titer obtained from Robo502AA was 20-fold lower than that from the Robo502 parent (Fig. 7A), indicating that these residues with their potential Ca²⁺ binding were necessary for optimal RUB replication. Following two subsequent passages of the transfected culture fluids, the Robo502AA titer rose to within threefold of that of Robo502 (Fig. 7A and B). When four individual plaques from the first passage of Robo502AA were amplified and the sequence of the NS protease determined, three were found to have reverted to the wild-type (wt) sequence at both sites, while the fourth had reverted to the wt sequence at one site (Fig. 7C). Thus, the increase in titer was due to reversion of the mutant sequences.

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FIG. 7. Effects of mutations of the potential Ca2+ coordination ligands on RUB replication. Transcripts from the wt infectious cDNA clone, Robo502, or Robo502AA containing the D1210A and D1217A mutations in the Ca2+-binding loop were used to transfect Vero cells. Culture fluid from the transfection plate (P0) was harvested on day 7 posttransfection and passaged twice in Vero cells (P1 and P2). The virus titer in the P0, P1, and P2 culture fluids was determined by plaque assay in triplicate (A). Open bar, Robo502; black bar, Robo502AA. (B) Representative plaques at each passage. (C) To check for the generation of revertants in the Robo502AA population, four plaques were picked from terminal plaque assay dilution plates from P1 (left) culture fluid, and after one round of amplification in Vero cells, the sequence of the metal-binding domain in the NSP was determined, as shown in comparison to the wt sequence.

To prevent the accumulation of revertants, the AA mutation was also introduced into the replicon, RUBrep/GFP, in which the SP-ORF is replaced with the GFP reporter gene. Following transfection, RUBrep/GFP replicates and expresses GFP but does not spread from cell to cell. As shown in Fig. 8A, RNA synthesis by RUBrep-AA/GFP was delayed by 1 day postinfection in comparison with the wt replicon. Concomitantly, GFP expression was similarly delayed (data not shown). We also assayed P200 NSP precursor cleavage in cells transfected with a RUBrep-HA/GFP derivative, replicons expressing a P150 tagged with the HA epitope (63). These experiments were done at 6 h posttransfection, when translation from the input transcripts is detectable but replication has not yet started. As shown in Fig. 8B, at 35°C P200 to P150 cleavage was efficient in wt RUBrep-HA-transfected cells but only 50% efficient in RUBrepAA-HA/GFP-transfected cells. RUBrep-NS*-HA/GFP, a construct with a C1152S substitution at the catalytic site unable to undertake cleavage, served as an uncleaved control. Given our biophysical measurements on RUBCa that indicate that Ca²⁺ binding increased the T_m of RUBCa from 37.7°C to 41.8°C (Fig. 6A), we also did the experiment in cells transfected at 39°C. As shown in Fig. 8B, wt replicon cleavage of the P200 precursor was more efficient than that at 35°C; however, cleavage in AA mutant replicon-transfected cells could not be detected, indicating that the mutant protease is temperature sensitive. Thus, not only is Ca²⁺ binding important for NS protease activity, in the absence of the Ca²⁺ binding the protease is rendered temperature sensitive.

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FIG. 8. Replicon RNA synthesis and P200 cleavage in transfected Vero cells. Vero cells were transfected with transcripts from RUBrep/GFP or RUBrepAA/GFP containing mutations D5A and D12A (A) or from RUBrep-HA/GFP or RUBrepAA-HA/GFP, which express an HA-epitope-tagged P150 (B). (A) Total cell RNA was extracted 1 to 4 days posttransfection, and replicon plus-strand RNA species (G, genomic; SG, subgenomic) were resolved by Northern blotting following agarose gel electrophoresis. (B) Transfections were performed at 35°C or 39°C. Six hours posttransfection, cells were lysed and the P200 precursor and P150 product were resolved by Western blotting probed with anti-HA antibodies following SDS-PAGE (the other product, P90, does not appear because it does not contain the HA epitope). Cells transfected with a replicon containing a C1152S catalytic site mutation (RUBrep-NS*-HA/GFP) that cannot mediate P200 cleavage served as an uncleaved control.

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Identification of an EF-hand Ca²⁺-binding motif in a viral NSP. In this report, we have predicted and further characterized a Ca²⁺-binding motif in a virus NSP. Most other viral Ca²⁺-binding motifs occur in virion structural proteins, and of these, only the rotavirus VP7 outer capsid protein (DITADPTTAPQTE) (19), the human immunodeficiency virus type 1 transmembrane protein gp41 (16), and the polyomavirus VP1 (DENGVGPLCKGE) (26) contain EF-hand Ca²⁺-binding motifs. In a canonical EF-hand Ca²⁺-binding motif, the coordination geometry in the loop is formed by oxygen atoms from the side chain carboxyl or hydroxyl groups (loop positions 1, 3, 5, and 12), the main chain carbonyls (position 7), and a bridged water (position 9), and the flanking sequences around the loop preferentially form a helical secondary structure. The negatively charged residue pairs at the Z axis (loop positions 5 and 12) are essential for the binding of cations (50). Examination of the primary sequence of the motifs in the above-mentioned virion structural proteins reveals that none of these are genuine canonical EF-hand Ca²⁺-binding moieties due to deviations in flanking sequence structural requirements (HIV gp41), the length of the loop (13 residues instead of 12 in rotavirus VP7), and the coordinating residues (V instead of D/E/N/Q/T/S at loop position 5 in polyomavirus VP1). Thus, the Ca²⁺-binding motif in the RUB NS protease is the first to be identified with a canonical EF-hand structure. Interestingly, using our prediction method we also detected a putative EF-hand Ca²⁺-binding motif close to the methyltransferase domain in nonstructural protein nsP1 that is conserved among the alphaviruses, the other Togavirus genus (unpublished data).

Because of difficulty in expressing the entire NS protease, the metal-binding properties of this Ca²⁺-binding motif were probed by grafting the loop into the scaffold protein CD2.D1 and by expressing the entire motif within the native minimal metal-binding domain of the NS protease. Table 1 provides a summary of the metal-binding affinities of the Ca²⁺-binding motif in both the engineered CD2.RUBCa and the minimal metal-binding domain, RUBCa. It is worth noting that numerous studies of EF-hand calcium-binding motifs have been made at the peptide or subdomain level (17, 58, 68). The Ca²⁺-binding affinity of the RUBCa loop grafted into CD2 (214 µM) is weaker than the grafted EF-loop I from calmodulin (34 µM) (69), likely due to the presence of an Asp at the bidentate position 12 of the loop rather than a Glu, which ensures a high Ca²⁺-binding affinity due to its larger side chain and stronger interaction with Ca²⁺. Replacement of Glu at position 12 in CaM with Lys or Gln reduces Ca²⁺-binding affinity by 10- to 100-fold (2, 40, 68, 74), while the replacement of Asp with Glu at position 12 in the regulatory light chain of myosin leads to a 15-fold increase in binding affinity (3). The Ca²⁺-binding motif in the RUB NS protease also exhibits selectivity for Ca²⁺ and La³⁺ in the presence of excess Mg²⁺ and K⁺, similar to paired EF-hand Ca²⁺ motifs in CaBPs such as calmodulin and troponin C (35).

Ca²⁺-induced local conformational change in the RUB NS protease and its potential stabilizing role. With the inability to express and purify the entire NS protease, conformational studies were done using the RUBCa subdomain which contained both catalytic residues, the putative Zn²⁺-binding ligands, and the EF-hand Ca²⁺-binding motif. Boundaries of the RUBCa subdomain were chosen near unstructured or loop regions according to our model (Fig. 1C), and within this model the RUBCa subdomain had a well-folded, globular structure. We found that the RUBCa subdomain bound stoichiometric amounts of Ca²⁺ and underwent metal-induced local changes in conformation (Fig. 5 to 6). Ca²⁺ binding to RUBCa resulted in a modest decrease in its intrinsic Trp fluorescence intensity without emission peak position shift. However, the addition of Ca²⁺ did not significantly alter the secondary structure of RUBCa as revealed by far-UV CD. In addition, ANS binding suggested that no hydrophobic surface was perturbed upon Ca²⁺ binding. These results indicate that Ca²⁺ binding does not lead to a global conformational change of RUBCa. In the RUB NS protease model structure, the predicted helix-loop-helix EF-hand Ca²⁺-binding site is located at the exposed surface on the opposite side of the active-site C1152 and H1273 (Fig. 1C), consistent with our finding that Ca²⁺ binding to RUBCa did not induce global conformational changes. In the model, the W1153 residue is 3.5 Å, on average, away from the predicted EF-hand motif (magenta in Fig. 1C). This proximity could lead to perturbation in the chemical environment around W1153 upon metal binding and thus the model is also consistent with our observation of significant enhancement of Tb³⁺ fluorescence intensity in RUBCa due to Tb³⁺-FRET (Fig. 5A). Finally, we found that the binding of Ca²⁺ increased the T_m of RUBCA from 37.7 to 41.8°C, indicating that Ca²⁺ binding serves to stabilize the NS protease. Significantly, we found that a replicon with two mutations in the EF-hand Ca²⁺-binding loop that abrogate metal binding by the CD2.RUBCa chimera expressed a protease that was temperature sensitive at 39°C, confirming this prediction. Such a stabilizing role of Ca²⁺ has been observed in nonviral protease, such as subtilisin (59) and thermolysin (60). It will be of interest to determine if the putative EF-hand Ca²⁺-binding motif in the MT domain of the alphaviruses plays a similar stabilizing role. It should be pointed out that the effect of Ca²⁺ on the RUB NS protease cannot be studied in an in vitro translation system, because such systems (i) include EGTA to chelate Ca²⁺ to inactivate the micrococcal nuclease employed to remove endogenous mRNA and (ii) are incubated at 30°C, below the 39°C required to observe an effect in vivo.

Role of Ca²⁺ in the replication cycle of RUB. When the double Ca²⁺-binding mutant was introduced into the Robo502 RUB infectious cDNA clone, titers produced were 20-fold lower than those produced by wt Robo502; however, during subsequent passaging, the mutant virus titer rose by over 10-fold, and sequencing of isolated virus plaques indicated that this was due to reversion of the mutants to the wt sequence. In a replicon incapable of cell-to-cell spread, the double Ca²⁺-binding mutant exhibited delayed RNA synthesis. Using the replicon constructs, we finally showed that cleavage of the P200 nonstructural precursor was impaired at 35°C and temperature sensitive at 39°C. While minus-strand RNA synthesis is catalyzed by the uncleaved precursor, the precursor must be cleaved before plus-strand RNA synthesis can occur (33). Thus, an impaired cleavage is consistent with delayed plus-strand RNA synthesis, as observed with the replicon, and lower titers, as observed with the infectious cDNA clone. However, ongoing (though delayed) RNA synthesis would lead to the generation of revertants, as observed with the infectious cDNA clone.

A submillimolar K_d for Ca²⁺ binding (200 to 300 µM) was derived from both CD2.RUBCa and RUBCa. In the cell, cytosolic Ca²⁺ concentrations range from 0.1 to 10 µM, while the Ca²⁺ concentration in endosomes or lysosomes is as high as 400 to 600 µM and is maintained in part by the proton gradient across lysosomal membranes (8, 21). Presumably, in the replication complex the protease resides on the cytosolic side of the lysosomal membrane; however, release of Ca²⁺ from the lysosome could result in local Ca²⁺ concentrations high enough for binding. For example, in acidifying endosomes the uptake of extracellular Ca²⁺ (in millimolars) is immediately accompanied by rapid release of Ca²⁺ to the cytoplasm, and with an external [Ca²⁺] of 2 mM the endocytosed Ca²⁺ may be sufficient to increase the total cellular Ca²⁺ concentration by 2 µM per min (21). It is also possible that association of the viral replicase proteins with late endosomal or lysosomal membranes alters the Ca²⁺ gradient, leading to a localized increase in Ca²⁺ concentration. Nevertheless, given the weak Ca²⁺-binding affinity of the Ca²⁺-binding loop, the protease is possibly inactive in the cytosol and must be associated with the late endosomal or lysosomal membrane before encountering Ca²⁺ concentrations sufficient for binding. Thus, Ca²⁺ may play a role in regulating the virus replication cycle by delaying the nonstructural precursor cleavage until it is associated with late endosomal or lysosomal membranes and thus modulating the synthesis of plus strand RNA. A similar situation has been shown to be the case with rotaviruses whose NSP4 glycoprotein mediates an increase in intracellular Ca²⁺ concentration, which modulates transcriptase activity (4, 13, 45, 55).

 
We appreciate the critical review by Dan Adams and other members in J.J.Y.'s group. We are thankful to Shunyi Li and Siming Wang for help in ESI-MS, to Yun Huang and Yanyi Chen for protein expression, and to Suganthi Suppiah for helpful discussion.

This work was supported in part by grant R01 AI21389 from the NIAID to T.K.F. and J.J.Y. and in part by the MCB-0092486 (NSF) and GM62999 and GM070555 (NIH) grants for J.J.Y. Y.Z. is a fellow of the Molecular Basis of Disease Area of Focus at Georgia State University.

 
* Corresponding authors. Mailing address for Jenny J. Yang: Department of Chemistry, Georgia State University, 50 Decatur St., Atlanta, GA 30303. Phone: (404) 651-4620. Fax: (404) 651-2751. E-mail: chejjy{at}langate.gsu.edu. Mailing address for Teryl K. Frey: Department of Biology, Georgia State University, 50 Decatur St., Atlanta, GA 30303. Phone and fax: (404) 651-3105. E-mail: tfrey{at}gsu.edu

Published ahead of print on 2 May 2007.

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Journal of Virology, July 2007, p. 7517-7528, Vol. 81, No. 14
0022-538X/07/$08.00+0     doi:10.1128/JVI.00605-07
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