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Journal of Virology, February 2008, p. 1294-1304, Vol. 82, No. 3
0022-538X/08/$08.00+0     doi:10.1128/JVI.01815-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Bovine Viral Diarrhea Virus Core Is an Intrinsically Disordered Protein That Binds RNA{triangledown}

Catherine L. Murray, Joseph Marcotrigiano,{dagger} and Charles M. Rice*

Laboratory of Virology and Infectious Disease, Center for the Study of Hepatitis C, The Rockefeller University, New York, New York 10065

Received 17 August 2007/ Accepted 6 November 2007


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ABSTRACT
 
Pestiviruses, including bovine viral diarrhea virus (BVDV), are important animal pathogens and close relatives of hepatitis C virus. Pestivirus particles are composed of an RNA genome, a host-derived lipid envelope, and four virion-encoded structural proteins, core (C), Erns, E1, and E2. Core is a small, highly basic polypeptide that is processed by three enzymatic cleavages before its incorporation into virions. Little is known about its biological properties or its role in virion assembly and structure. We have purified BVDV core protein and characterized it biochemically. We have determined that the processed form of core lacks significant secondary structure and is instead intrinsically disordered. Consistent with its highly basic sequence, we observed that core binds to RNA, although with low affinity and little discernible specificity. We found that BVDV core protein was able to functionally replace the nonspecific RNA binding and condensing region of an unrelated viral capsid protein. Together these results suggest that the in vitro properties of core may reflect its mechanism of action in RNA packaging and virion morphogenesis.


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INTRODUCTION
 
Bovine viral diarrhea virus (BVDV) is a member of the genus Pestivirus within the family Flaviviridae. Pestiviruses infect wild and agriculturally important animals worldwide and have a significant financial impact on the livestock industry (32). The family Flaviviridae also includes numerous important human pathogens, encompassed within the genera Flavivirus and Hepacivirus. Among these is hepatitis C virus (HCV), the prototype member of the Hepacivirus genus, which currently afflicts approximately 120 million chronic carriers.

The pestivirus genome is a single-stranded, positive-sense RNA molecule of approximately 12.3 kilobases (kb) in length. On introduction into the cell, an internal ribosome entry site within the 5' end of the genome directs the synthesis of a single polyprotein (reviewed in reference 28). The polyprotein is processed co- and posttranslationally by viral and cellular proteases to release the individual viral proteins, sequentially termed Npro, C, Erns, E1, E2, p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B (Fig. 1A). Core (C) and the envelope glycoproteins (Erns, E1, and E2) are structural proteins, which combine with the genomic RNA and lipid bilayer to form the physical virion. Production of the infectious particle requires as-yet-undefined actions of the nonstructural (NS) proteins p7 and NS2, with the latter functional only in the form of the uncleaved NS2-3 precursor (1, 14). Both NS2 and NS3 possess protease activities; NS2 is responsible for a single cleavage at its own carboxy terminus (26), while NS3, along with its cofactor NS4A, performs the remainder of the NS protein processing. NS3 also possesses helicase and nucleoside triphosphatase activities essential for RNA replication. NS5B is the RNA-dependent RNA polymerase (28).


Figure 1
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  		FIG. 1. Purification of recombinant BVDV core protein. (A) BVDV genome organization; the core gene is shown in black. (B) Schematics of BVDV core protein and GST fusion proteins. White, GST; black, BVDV core. (C) Nondenaturing and denaturing purification schemes for C87 and C90. Shading indicates use of column.

The mature, virion-associated form of the BVDV core protein is 90 amino acids in length and is excised from the polyprotein by three enzymatic cleavages (15). Npro generates the core protein amino terminus through a cotranslational autoproteolytic processing event (48). A signal sequence in the carboxy terminus of core then targets the nascent protein to the endoplasmic reticulum (ER) membrane, initiating dual processing events by host enzymes. Signal peptidase cleaves the protein from Erns in the ER lumen (45), and signal peptide peptidase performs an additional processing event within the core membrane anchor (15). The liberated core protein is highly basic and is presumed to bind RNA. Through mechanisms that are not well understood, the core protein and RNA genome become enveloped by an ER-derived membrane studded with glycoproteins Erns, E1, and E2 (49). The virions are then trafficked from the ER lumen, via the cellular secretory pathway, to the extracellular milieu.

Little is known about the roles that the pestivirus core protein plays in the formation or structure of the infectious virion. Here we expressed and purified recombinant BVDV core protein from bacteria and found it to be natively unfolded in solution. We determined that core does indeed bind RNA, although with low affinity and low specificity. We found that BVDV core was able to functionally replace the RNA-neutralizing and -condensing domain of an unrelated viral capsid in vivo, suggesting that the disordered and basic nature of the core protein is central to its biological function.


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MATERIALS AND METHODS
 
Cell culture. BHK 21 (baby hamster kidney) cells were propagated in modified Eagle's medium (Invitrogen, Carlsbad, CA) supplemented with 7.5% fetal bovine serum (Invitrogen). Cells were grown at 37°C in 5% CO2.

Plasmid constructs. Plasmids were constructed by standard methods. Constructs were verified by restriction enzyme digestion and sequencing of PCR-amplified segments. Plasmid and primer sequences are available upon request.

(i) pGEX-6P-1/GST-BVDV core expression constructs. Core sequences from BVDV strain NADL were PCR amplified from pBeloBac11/NADL (1). For pGEX-6P-1/GST-84, nucleotides 890 to 1141 were amplified using primers RU-2549 and RU-2550; for pGEX-6P-1/GST-C87, nucleotides 890 to 1150 were amplified using RU-2549 and RU-4453; and for pGEX-6P-1/GST-C90, nucleotides 890 to 1159 were amplified using primers RU-2549 and RU-4658. RU-2549 engineered a BamHI site upstream of core, keeping it in frame with glutathione S-transferase (GST); downstream primers contained a stop codon and an XhoI site. PCR products were digested with BamHI/XhoI and ligated to the 4,757-base-pair (bp) fragment of pGEX-6P-1 (GE Healthcare, Piscataway, NJ) digested with the same enzymes.

(ii) Sindbis virus capsid deletion and chimeric genomes. The Sindbis/luc (pToto1101/luc) backbone encodes a wild-type Sindbis virus cDNA with firefly luciferase inserted within nsP3 (3). Deletions and chimeras were constructed by assembly PCR, and final PCR products were digested with HpaI/AatII and ligated to the 14,211-bp fragment of Sindbis/luc digested with the same enzymes. Detailed descriptions of the cloning strategies are available upon request.

Protein expression and purification. (i) Expression and GST fusion protein purification. Amino-terminal GST fusion proteins were expressed in Escherichia coli K-12 UT5600 (NEB, Ipswich, MA) or Rosetta cells (Novagen, San Diego, CA). Expression and solubility were equivalent in either cell type. Expression was induced at an optical density at 600 nm of 0.5 by addition of 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG) (Inalco, Milan, Italy). Following induction, cells were grown at 18°C with agitation overnight. Cells pellets resuspended in 0.5 M KCl, 20 mM HEPES (pH 7.4), and 10% (vol/vol) glycerol were lysed by three passages through a cold EmulsiFlex C5 homogenizer (Avestin, Ottawa, Canada) at >10,000 lb/in2. Unlysed and insoluble material was removed by centrifugation, and soluble material was loaded on a GSTrap fast protein liquid chromatography (FPLC) column (GE Healthcare; 5-ml bed volume) equilibrated with buffer A1 (0.5 M KCl, 20 mM HEPES [pH 7.4]). After washing with buffer B1 (1 M KCl, 20 mM HEPES [pH 7.4]), fusion protein was eluted with 150 mM KCl, 100 mM Tris (pH 8.0), and 15 mM glutathione. GST tag was removed by overnight treatment with Prescission protease (GE Healthcare; 10 µg per 1 mg protein) during dialysis against buffer A2 (100 mM KCl, 20 mM HEPES [pH 7.4]).

(ii) Nondenaturing purification. After removal of GST, BVDV core was further purified over SP Sepharose or heparin columns (GE Healthcare) equilibrated with buffer A2. A linear salt gradient made from buffers A2 and B1 was used to elute the protein, with C87-containing fractions eluting at approximately 470 mM KCl and C90 at approximately 520 mM KCl.

(iii) Denaturing purification. After purification of the GST fusion proteins and cleavage of the GST as described above, final concentrations of 1.0% acetonitrile and 0.0325% trifluoroacetic acid (TFA) were added to denature the samples. Denatured protein was loaded on a Source 5RPC ST 4.4/150 reverse-phase high-pressure liquid chromatography column (GE Healthcare; 2.5-ml bed volume) equilibrated with buffer A3 (2.0% acteonitrile, 0.0625% TFA). A linear acetonitrile gradient made from buffer A3 and buffer B2 (80% acetonitrile, 0.05% TFA) was used to elute the protein, with core-containing fractions eluting at approximately 20% (C87) to 24% (C90) acetonitrile. Eluted fractions were lyophilized, resuspended in 6 M guanidine-HCl and 50 mM HEPES (pH 7.5), and refolded by dialysis into buffer A2.

Protein was quantified by absorbance at 280 nm using extinction coefficients of 2,560 M–1 cm–1 (C87) and 8,250 M–1 cm–1 (C90). Protein was concentrated or buffer exchanged using Centricon centifugal concentrators (Millipore, Billerica, MD) and stored in 100 to 500 mM KCl, 20 mM HEPES (pH 7.4), and 10% (vol/vol) glycerol or in 100 mM KCl and 20 mM HEPES (pH 7.4) without glycerol for circular dichroism (CD) applications. Amino-terminal sequencing and mass spectrometry were conducted by the University of Texas Medical Branch (Galveston, TX) and the Rockefeller University proteomics resource center, respectively.

Limited proteolysis assay. A 0.2-µg amount of endoprotease GluC (Roche, Mannheim, Germany) was used to digest 2 µg protein in 100 mM KCl and 20 mM HEPES (pH 7.4) at room temperature (RT). After incubation, reactions were stopped by the addition of an equal volume of 2x sodium dodecyl sulfate (SDS) loading buffer with β-mercaptoethanol and heating to 100°C for 5 min. Samples were resolved by SDS-12% polyacrylamide gel electrophoresis and visualized by silver staining.

Intrinsic fluorescence spectroscopy. C90 protein (purified under nondenaturing conditions) at 0.03 mg/ml in 100 mM KCl and 20 mM HEPES (pH 7.4) was analyzed using an Olis RSM 1000F spectrofluorimeter. For tyrosine spectra, the sample was excited at 280 nm and scanned from 300 to 430 nm in 1-nm increments.

CD spectroscopy. Protein (purified under nondenaturing conditions) at 0.8 to 1.0 mg/ml (C87) or 0.3 mg/ml (C90) in 100 mM KCl and 20 mM HEPES (pH 7.4) was analyzed using an Aviv 202 CD spectrometer. Spectra were read from 250 to 190 nm at 1-nm intervals. CD readings were converted to molar ellipticity ([{theta}]) using the equation [{theta}] = ({theta}obs x 100 x MW)/(c x l x n), where [{theta}] is in units of degrees cm2/dmol, {theta}obs is the CD reading in degrees, MW is molar mass of the protein in g/mol, c is concentration of the protein in g/liter, l is the path length in cm, and n is the number of residues in the protein.

RNA homopolymer labeling and filter-binding assay. Twenty picomoles of each RNA homopolymer (Dharmacon, Lafayette, CO) was end labeled in a 20-µl reaction volume containing 60 µCi [{gamma}-32P]ATP, 10 U T4 polynucleotide kinase (NEB), and a final concentration of 1x polynucleotide kinase buffer (NEB) for 30 min at 37°C. RNA was purified over a P30 spin column (Bio-Rad, Hercules, CA) and resuspended to 1,000 cpm/µl in 1x BB [200 mM potassium acetate (KOAc), 50 mM TrisOAc (pH 7.7), 5 mM Mg(OAc)2]. RNA was heated to 60°C for 5 min and slowly cooled to RT immediately before use.

For filter-binding assays (17), 0.05 µM to 5 µM dilutions of protein in 1x BB were incubated with 10,000 cpm of labeled homopolymer for 10 min at RT. Each reaction mixture was spotted on a 0.45-µm nitrocellulose filter (Millipore) with a vacuum source applied and immediately washed with 1x BB. RNA alone, spotted without vacuum or washing, was used as a measure of total counts. Filters were dried, and radioactivity was counted in ReadySafe scintillation cocktail (Beckman Coulter, Fullerton, CA). Curves are fit using nonlinear regression for sigmoidal dose-response (variable slope; Prism Graph pad). Dissociation constants (Kd) represent the concentration of protein resulting in 50% maximal binding.

For competition filter-binding assays, 50 nM to 3 mM dilutions of U oligomer in 1x BB were incubated with 10,000 cpm of labeled U30 and 0.75 µM C90. Reaction mixtures were incubated for 10 min at RT, filtered, washed, and dried, and radioactivity was counted as described above. Curves were fit using one-site competition (Prism Graph pad).

Systematic evolution of ligands by exponential enrichment. A DNA template for a 25-mer random RNA library was created by annealing oligonucleotide primers (5 nmol each) RU-4447 (5'-TCCCGCTCGTCGTCT[25N]CCGCATCGTCCTCCCT-3') and RU-4448 (5'-GAAATTAATACGACTCACTATAGGGAGGACGATGCGG-3') (17). The mixed oligonucleotides were filled in with 25 U DNA polymerase I (Klenow fragment; NEB) and 1 mM deoxynucleoside triphosphates (dNTPs) in 1x EcoPol buffer (NEB) for 30 min at 37°C. The template was gel purified and quantified by absorbance at 260 nm. For RNA synthesis, 500 pmol of template was transcribed in a 1-ml reaction mixture containing final concentrations of 1x T7 transcription buffer (Promega), 2 mM NTPs, 10 mM dithiothreitol, 2,000 U T7 RNA polymerase (Ambion, Austin, TX), 800 U RNasin (Promega, Madison, WI), and 200 µCi [{alpha}-32P]UTP. Reaction mixtures were incubated at 37°C for 4 to 6 h and for an additional 15 min with 60 U DNase I (Invitrogen) before RNA was gel purified. The affinity of C87 for the RNA pool was measured by filter-binding assay as described above.

For selection, protein at a concentration found to bind 5% of the library was incubated with a 10- to 50-fold molar excess of RNA for 10 min at RT. Unbound RNA was then removed by filtration, and bound RNA was extracted from the filter, precipitated, and used for reverse transcription and PCR. For reverse transcription-PCR, extracted RNA was incubated with a final concentration of 0.5 mM dNTPs and 50 pmol RU-4440 (5'-TCCCGCTCGTCGTCTG-3') for 3 min at 70°C before addition of 1x first-strand buffer (Invitrogen), 10 mM dithiothreitol, 20 U RNasin (Promega), and 100 U SuperScript II reverse transcriptase (Invitrogen) and incubation at 42°C for 1.5 h, followed by 72°C for 7 min. Ten units RNase H (Epicenter, Madison, WI) and 1,000 U RNase T1 (Ambion) were added and incubated at 37°C for 20 min. The reverse transcription reaction product was then PCR amplified using RU-4440 and RU-4448, purified, and used for the next round of transcription.

Creation of polyclonal antibodies. Anti-BVDV core polyclonal antibodies were produced in rabbits (Cocalico, Reamstown, PA) using C84 as an immunogen.

RNA transcription. Plasmids were linearized by digestion with XhoI for 3 h at 37°C, and templates were purified over a Minelute column (Qiagen, Valencia, CA). A 0.6-µg amount was transcribed in a 10-µl reaction mixture using the SP6 mMessage mMachine kit containing cap analog (Ambion). Reaction mixtures were incubated for 3 h at 37°C, with subsequent treatment with 3 U DNase I (Ambion) for 15 min at 37°C. RNA was purified with an RNeasy column (Qiagen) and quantified by absorbance at 260 nm, and its integrity was verified by 0.8% agarose gel electrophoresis.

RNA transfection and viral replication assay. For each genome, 3 µg of RNA was electroporated as described previously (3), and cells were plated into 24-well and P100 tissue culture dishes. At 6 h postelectroporation, cells in 24-well plates were washed and lysed with cell culture lysis buffer (Promega) for assay of firefly luciferase activity. Infectivity was assayed by inoculation of naive cells with clarified supernatant harvested at 24 h postelectroporation. After 1.5 h, the medium was changed, and after a total of 4 h, cells were lysed with cell culture lysis buffer (Promega) and analyzed for luciferase activity. Each infection was done in triplicate.

Western blotting. Cells were lysed at 6 h postelectroporation with cell culture lysis buffer (Promega) and lysates separated by SDS-12% polyacrylamide gel electrophoresis. Blots were blocked for 1 h with 5% milk-TBST (0.02 M Tris-Cl [pH 7.4], 0.2 M NaCl, 0.1% [vol/vol] Tween 20). For Sindbis virus capsid detection, anticapsid polyclonal antibody (40) was diluted 1:5,000 in TBST; for BVDV core detection, polyclonal anticore antiserum from rabbit 886, bleed date, 10 October 2002, was diluted 1:5,000 in 5% milk-TBST. After overnight incubation at 4°C and extensive washing, goat anti-rabbit immunoglobulin-horseradish peroxidase secondary antibody (Pierce) was added at a 1:5,000 dilution in 5% milk-TBST and left for 30 min at RT. After additional washing, blots were developed with SuperSignal West Femto chemiluminescent substrate (Pierce).


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RESULTS
 
Expression and purification of recombinant BVDV core protein. We cloned and expressed two versions of BVDV core protein encompassing the amino-terminal 87 (C87) or 90 (C90) residues fused to GST (Fig. 1B). Deletion of the majority of the Erns signal sequence was expected to facilitate purification and result in proteins related (C87) or identical (C90) to the mature, virion-associated core (15). C87 and C90 were initially purified under nondenaturing conditions using a two-step scheme (Fig. 1C). GST-core fusion proteins were bound to a GSTrap column, and after washing with high salt to remove nonspecifically bound proteins and nucleic acid, the fusion proteins were eluted with glutathione. The GST moiety was removed by Prescission protease treatment during overnight dialysis. C87 was further purified over an SP Sepharose column and eluted within a salt gradient at approximately 470 mM KCl. C90 was further purified by binding to a heparin column, eluting at approximately 520 mM KCl. C87 yields were approximately 3 mg/liter bacterial culture, whereas C90 yields were between 0.05 and 0.1 mg/liter.

Since C90 could not be purified to satisfactory yields and purity under nondenaturing conditions, a denaturing purification scheme was devised (Fig. 1C). After purification of GST-C87 or GST-C90 fusion proteins and removal of GST as described above, a final concentration of 1% acetonitrile-0.0325% TFA was added. Denatured protein was loaded on a Source 5RPC ST 4.4/150 reverse-phase column and eluted with an acetonitrile gradient. Eluted proteins were lyophilized, resuspended in 6 M guanidine-HCl and 50 mM HEPES (pH 7.5), and refolded by dialysis into 100 mM KCl and 20 mM HEPES (pH 7.4). Although C90 yields were again 0.1 mg/liter, the denaturing protocol had the advantage of destroying a copurifying phosphatase activity (data not shown), allowing the core protein to be assayed for binding to end-labeled nucleic acids without additional purification steps. The identities of recombinant proteins produced by denaturing and nondenaturing methods were verified by mass spectrometry and amino-terminal sequencing.

BVDV core is an intrinsically disordered protein. As an initial analysis of the BVDV core protein, we used secondary structure prediction algorithms to estimate the helical content of the unprocessed sequence (residues 1 to 102). Helix formation was predicted with high confidence only for the carboxy-terminal region, consistent with the function of these amino acids as a signal sequence for Erns (Fig. 2A) (43). The majority of the core protein was not predicted to possess secondary structure and was suggested by a predictor of natively disordered regions (PONDR) algorithm to have a high propensity for intrinsic flexibility (27, 41, 42). These predictions suggested that BVDV core consists of a disordered amino-terminal 75 to 84 residues followed by a helical transmembrane domain.


Figure 2
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  		FIG. 2. BVDV core is intrinsically disordered. (A) BVDV core protein sequence and secondary structure predictions. Predicted disordered regions are in bold, and proposed {alpha}-helices are underlined. The signal peptide peptidase cleavage site is designated by an arrow. (B) Limited proteolysis of C87 (left) and C90 (right) with endoprotease GluC. Incubation times at RT are indicated (min). (C) Tyrosine intrinsic fluorescence spectrum of C90; data are representative of three independent experiments. The dotted line indicates 340 nm. (D) CD spectrum of C87. Means and standard errors of the means from duplicate scans are shown. Data are representative of multiple independent experiments.

In order to test the hypothesis that BVDV core is natively unfolded, C87 and C90 purified under nondenaturing conditions were assayed for characteristics of intrinsically disordered proteins. Natively unstructured proteins have a large proportion of solvent-exposed residues and are therefore highly susceptible to trace amounts of protease. Treatment of C87 or C90 with endoprotease GluC resulted in almost complete digestion within 1 minute when the core protein was present in a 10-fold excess (Fig. 2B). Similar results were seen with trypsin and chymotrypsin, and, for all three enzymes, proteolysis of C87 was complete within 1 hour even when core protein was in a 300-fold excess (data not shown). These results suggested that the majority of core residues are accessible to proteases.

We next used intrinsic fluorescence spectroscopy to determine the environment of tyrosine residues within the core protein. The peak emission of C90 was observed at approximately 360 nm. Since an emission maximum at a wavelength greater than 340 nm indicates a high degree of solvent exposure, this again suggested that the core protein lacked significant globular structure (Fig. 2C). The core protein was further analyzed for the presence of secondary structural elements by far-UV CD (reviewed in reference 19). The CD spectrum of C87 indicated a minimum at approximately 195 nm, consistent with a protein that lacks secondary structure (Fig. 2D). A minimum at 208 nm or 222 nm, indicative of {alpha}-helical or β-sheet secondary structures, was not observed. C90 produced similar results, although the lower protein concentration made the data less reliable (data not shown). Taken together, these results suggest that the BVDV core protein lacks significant secondary structure and is likely to be disordered in its native state.

BVDV core binds RNA with low affinity and with little discernible specificity. The extremely basic nature of the BVDV core protein, as well as its suspected close proximity to the viral genome within the infectious particle, led us to hypothesize that core may bind to RNA. In order to test C87 and C90 for RNA-binding activity, we used a quantitative filter-binding assay (17). Radiolabeled RNA homo-oligomers (30 nucleotides in length) of adenosine (A), cytidine (C), guanosine (G), and uridine (U) were added at constant concentration to a dilution series of protein. Protein purified under denaturing conditions was used in this assay in order to eliminate a bacterial phosphatase that copurified under native conditions (data not shown). After binding of protein and labeled RNA in solution, reaction mixtures were applied to nitrocellulose filters under vacuum and washed, and RNA retained on the filter was quantified. C87 and C90 were found to bind RNA equivalently and with approximately 1 µM affinity; a slight preference was seen for G and U homo-oligomers (Fig. 3 and Table 1). The low affinity and low specificity of core binding to RNA in this assay suggested a nonspecific, charge-charge interaction of the highly basic protein and negatively charged phosphate backbone of the nucleic acid.


Figure 3
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  		FIG. 3. BVDV core protein binds RNA. Binding of C87 (A) and C90 (B) to RNA homo-oligomers is shown. Open circles, A30; open squares, C30; black circles, G30 black squares, U30. Means and standard errors of the means from at least duplicate binding assays are shown. Curves are fit using a sigmoidal variable-slope model.


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  		TABLE 1. Kds of core binding to RNA homo-oligomers

To further investigate the involvement of specificity in core-RNA interactions, systematic enhancement of ligand binding by exponential enrichment was employed. In this approach, an RNA library is enriched for sequences with high affinity for the target protein. A random library of 25 nucleotides, with a T7 promoter and flanking primer-binding sites, was created from synthesized oligonucleotide primers. RNA transcribed from this library was selected for sequences better able to bind C87, as described in Materials and Methods. After eight rounds of selection, the reverse transcription-PCR-amplified RNA pool was cloned and multiple individual colonies were sequenced. Although the affinity of the pool had increased less than twofold during selection, a consensus sequence emerged in all of the 13 clones sequenced (Fig. 4). This sequence was not detected in six clones sequenced from the original library. The consensus sequence, GUGGGA(A/U)NNA, is G rich, in keeping with the slightly higher affinity for G observed in the homo-oligomer studies. The minimal increase in affinity of the pool is also consistent with the similar affinities of the homo-oligomers for core and again suggests that core binds RNA with little specificity. We cannot, however, rule out the possibility that other selective factors, such as nuclease resistance, could have also contributed to the emergence of a consensus sequence. Taken together, these results indicate that BVDV core binds RNA, but with low affinity and low specificity under the conditions tested. The slight preference for oligo(G) could indicate a propensity to bind structured or double-stranded RNA molecules.


Figure 4
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  		FIG. 4. Selection of a consensus RNA for C87 binding. (A) Binding of round 1 (white) and round 8 (black) RNA pools to C87. For round 1 binding, means and standard errors of the means for triplicate measurements are shown. (B) Sequences of the randomized region of cloned cDNAs from round 8 of selection. The consensus is also shown; W, A or U; N, any nucleotide.

C90 binds to approximately 14 nucleotides of oligo(U) RNA. To further investigate the nature of the core-RNA interaction, we determined the minimal RNA size that could act as a core-binding site. We used the filter-binding assay to quantify the abilities of oligo(U) sequences of different lengths to compete for U30 binding to C90; oligo(G) was not chosen because of the possible complication of secondary structures. In this assay the C90 concentration was held constant at approximately the Kd (0.75 µM), the radiolabeled U30 concentration was also constant at approximately 100 pM, and unlabeled competitor was diluted from 3 mM to 50 nM. While U6 was not able to compete for U30 binding, as the length was increased competition became more efficient (Fig. 5). From the competition assay, the binding site size for binding of C90 to oligo(U) RNA was estimated at 12 to 14 nucleotides.


Figure 5
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  		FIG. 5. C90 minimal RNA binding site size is 12 to 14 nucleotides. Competition of C90 binding to U30 was determined. Residual U30 binding in the presence of the unlabeled indicated oligomers is shown. Means and standard errors of the means from duplicate binding assays are plotted. Curves are fit using a one-site competition model.

To verify the binding site size, we conducted a direct filter-binding assay of labeled U14 or U30 to C90. As observed previously, the binding affinity of C90 to U30 was approximately 1 µM. The affinity of C90 binding to U14 was lower, but binding was detectable (Kd = 1.8 µM) (Fig. 6A). The low retention efficiency is consistent with previous reports of an RNA-binding protein associating with its minimal substrate (2). To investigate cooperativity, binding data were expressed as a Hill plot. The slope of the graph indicates the number of core molecules binding cooperatively and suggested that two molecules of C90 bind to U30, whereas a single molecule binds to U14 (Fig. 6B). This analysis is consistent with a minimal C90 binding site size of 14 nucleotides.


Figure 6
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  		FIG. 6. C90 binding to U14 and U30. (A) Direct binding of C90 to radiolabeled U14 (black) or U30 (white). Means and standard errors of the means of at least quadruplicate measurements are shown. Curves are fit using a sigmoidal variable-slope model. (B) Hill plots of C90 binding to U14 (black) or U30 (white). Slopes of linear regression are 1.04 ± 0.428 (r2 = 0.5985) (U14) and 1.99 ± 0.276 (r2 = 0.8124) (U30).

BVDV core acts as a nonspecific RNA binding protein in vivo. Our in vitro analyses of BVDV core protein suggested a nonspecific, low-affinity association with RNA. We hypothesized that core might act as a nonspecific RNA-binding protein in vivo and that this type of interaction might allow it to condense and neutralize viral genomic RNA. To test this hypothesis, we investigated whether BVDV core protein could functionally replace a known nonspecific RNA-condensing sequence. The well-documented RNA-packaging activity of an alphavirus capsid protein provided an ideal system for this investigation. Sindbis virus, the prototype alphavirus, encodes a 264-amino-acid capsid protein, the carboxy-terminal 150 residues of which form a chymotrypsin-like serine autoprotease (Fig. 7A). The amino-terminal region of Sindbis virus capsid is rich in lysine, arginine, and proline residues; is natively disordered; and forms specific and nonspecific associations with RNA (13, 29). Cryo-electron microscopy image reconstructions, combined with fitting of individual protein crystal structures, have provided a view of capsid protein architecture within the intact alphavirus particle (35, 53). Beneath the virion envelope, the nucleocapsid is a well-ordered icosahedral shell, with the capsid protease domains forming pentameric and hexameric protrusions from the RNA-containing interior. The flexible amino-terminal residues of the capsid protein extend from the ordered protease domains into the nucleocapsid core, forming a mixed protein-RNA region that does not have a well-resolved structure (5, 53). These studies demonstrated that the disordered amino terminus of Sindbis virus capsid protein is involved in nonspecifically binding and condensing the genomic RNA.


Figure 7
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  		FIG. 7. Sindbis capsid and BVDV core proteins. (A) Schematics of Sindbis capsid (top) and BVDV core (bottom) functional regions. (B) Deletions in Sindbis virus capsid protein created in the context of Sindbis/luc. (C) Chimeras of Sindbis virus (gray) and BVDV (black) capsid protein sequences in the Sindbis/luc genome.

In order to assess the ability of BVDV core to functionally replace the charge neutralization activity of Sindbis virus capsid, Sindbis virus genomes with in-frame deletions engineered into the amino-terminal domain of the capsid protein were created (Fig. 7B). The native sequence from amino acid 97 to 264 was maintained in all constructs, preserving the specific RNA interaction sequence and protease activity (6, 36). In the largest deletion, the methionine start codon was fused directly to amino acid 97. This genome was predicted to be severely impaired in RNA packaging and therefore in infectious virus production (11). To test the ability of the BVDV core protein to rescue the deletion mutants, BVDV sequences were inserted in the deleted region, while preserving the approximate native length of the Sindbis virus capsid amino-terminal domain (Fig. 7C). The parental sequence was a fully infectious Sindbis virus genome encoding firefly luciferase as a fusion with the nsP3 protein (Sindbis/luc) (3), facilitating sensitive detection of infectious virus production by reporter gene activity.

In vitro-generated RNA transcripts of each genome were electroporated into BHK 21 cells. Wild-type Sindbis/luc and Sindbis/luc({Delta}KpnI), a replication-defective genome with a deletion in nsP4 (3), were electroporated in parallel. Replication was assayed at 6 h postelectroporation by quantification of intracellular luciferase activity. Consistent with the nonessential role of Sindbis virus capsid in RNA replication, luciferase activities were similar for each of the mutant genomes; Sindbis/luc({Delta}KpnI) did not replicate (Fig. 8A). Intracellular capsid protein expression was also measured at 6 h postelectroporation. Western blotting with anti-Sindbis virus capsid (39) and anti-BVDV core polyclonal antibodies (see Materials and Methods) confirmed the expression of the appropriate sequences (Fig. 8B). The reduced expression of Sindbis/luc(C{Delta}2-96) and related chimeras is likely due to the deletion of a translational enhancer present in the capsid-coding sequence (12).


Figure 8
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  		FIG. 8. BVDV core can functionally replace the Sindbis virus capsid amino-terminal domain. (A) Replication of capsid deletion and chimeric genomes at 6 h postelectroporation as measured by luciferase assay. Means and standard errors of the means from duplicate experiments are shown. (B) Protein expression at 6 h postelectroporation. BVDV core (top panel) and Sindbis virus capsid (middle panel) were detected using polyclonal antibodies. (C) Infectious virus production at 24 h postelectroporation as measured by luciferase assay of inoculated cells. Means and standard errors of the means from duplicate experiments are shown.

At 24 h postelectroporation, cell culture supernatants were harvested, clarified by filtration, and used to infect naïve BHK 21 cells. The infected cells were incubated for 4 h before assay for luciferase activity. Sindbis/luc(C{Delta}2-96) was severely impaired in infectious virus production, while the less drastic deletions, Sindbis/luc(C{Delta}50-96) and Sindbis/luc(C{Delta}72-96), showed only moderate reductions (Fig. 8C). In each case, the inclusion of BVDV sequences led to increased signal compared to the deletion mutants, suggesting that the BVDV residues positively influenced infectious virion production. In the case of Sindbis/luc(C{Delta}2-96/BVDV1-90), in which the entire virion-associated form of BVDV core replaces the basic amino terminus of Sindbis virus capsid, infectious virus was only about 10-fold reduced from wild-type Sindbis/luc (Fig. 8C). This strongly suggested that the BVDV core protein could functionally replace the Sindbis virus capsid amino-terminal domain. Interestingly, infectious titers of Sindbis/luc(C{Delta}2-96/BVDV1-90) were markedly increased over those of Sindbis/luc(C{Delta}2-96/BVDV1-84) (Fig. 8C). The requirement for these carboxy-terminal six amino acids, which are actually not basic in nature and have predicted helical character, might point to an additional functional sequence within the BVDV core protein.

Taken together, these results suggest that the nonspecific RNA-binding properties of BVDV core observed in vitro are responsible for its ability to functionally replace a heterologous RNA condensation sequence in vivo, and they indicate that the BVDV core protein likely functions by a similar mechanism in packaging its own genomic RNA.


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DISCUSSION
 
We have expressed and purified recombinant BVDV core protein from bacteria and characterized it biochemically. Sequence analysis, limited proteolysis, and spectroscopic studies indicated that BVDV core protein does not possess significant secondary structure but is instead intrinsically disordered. Proteins that lack secondary structure in their native state are a growing class of sequences identified from an increasing variety of sources (reviewed in references 10, 50, and 51). The intrinsic flexibility of natively disordered sequences allows a large, accessible surface area for interactions and posttranslation modifications and also facilitates conformational switching and induced binding. As a result, disordered regions are often involved in macromolecular complex assembly, signaling, and nucleic acid binding (10). Unstructured regions are not uncommon in viral capsid proteins. For enveloped positive-sense RNA viruses, disordered sequences containing a high concentration of positively charged residues are ubiquitously found in the capsid protein amino-terminal domains. Among the Flaviviridae, both dengue virus and West Nile virus capsid proteins contain flexible, basic amino-terminal regions of approximately 20 amino acids (9, 30); similarly, the highly basic 117 to 124 amino-terminal residues of the HCV core protein have been shown to be unstructured (4, 25).

Unfolded regions of viral capsid proteins are often involved in nucleic acid binding (20, 29, 46). We found that BVDV core protein was able to associate with RNA in vitro and that it could functionally replace the nonspecific nucleic acid-binding region of the Sindbis virus capsid protein in vivo. The in vitro nonspecific RNA binding affinities of BVDV core and a portion of the Sindbis virus capsid amino-terminal region are similar, at approximately 0.5 to 0.6 µM (29). This relatively low binding affinity likely allows the RNA genome to be released and accessed for translation upon entry into a new host cell. The ability of BVDV core to functionally replace the role of the Sindbis virus capsid amino terminus in infectious virus production indicates a recapitulation not only of RNA binding and condensation activities but also of appropriate genome release upon uncoating. While Sindbis virus capsid protein residues 97 to 106 interact specifically with an encapsidation signal in the genomic RNA (36, 52), BVDV core appears to be far less discerning. Apart from a slight preference for guanosine, we found little specificity in core-RNA interactions. Although this could be an artifact of the in vitro experimental conditions, the ability of BVDV core to readily bind Sindbis RNA in vivo favors the hypothesis of largely ionic interactions. This putative inability to discriminate between genomic, antisense, and cellular RNAs may be overcome by local concentration effects caused by intracellular compartmentalization of viral replication and assembly. No evidence for a specific encapsidation signal has been found for any member of the Flaviviridae.

While the amino terminus of Sindbis virus capsid protein, and likely those of other viral capsid proteins, appears to be functionally homologous to BVDV core, the pestivirus sequence has the unusual lack of a structured carboxy-terminal domain. In the absence of such a domain, it is difficult to envision how the BVDV core protein might be able to form a structured shell around the viral RNA. Canonical viral nucleocapsids consist of an ordered array of capsid protein subunits, arranged symmetrically around the nucleic acid interior. These nucleocapsids, including those of Sindbis virus, can often be assembled in vitro from capsid protein and nucleic acid components and visualized and isolated from infected cells. For some viruses, cellular chaperones are employed to dictate the assembly of disordered structural protein components into an ordered array (7). Interestingly, however, nonenveloped nucleocapsids are rarely seen in flavivirus- or pestivirus-infected cells (28), and limited studies of HCV have also so far failed to detect intracellular nucleocapsids (44). This suggests that Flaviviridae core proteins may not form discrete, stable, proteinaceous structures around the viral genome. Consistent with this, purification and in vitro assembly of Flaviviridae nucleocapsids have been extremely difficult and usually result in structures with pleiomorphic shapes and heterogeneous sizes (21, 24; C. L. Murray and C. M. Rice, unpublished observations).

Further evidence for an absence of defined symmetry in the nucleoprotein interior of Flaviviridae particles comes from the cryo-electron microscopy image reconstructions of dengue virus and West Nile virus particles (23, 33, 54). In these virion structures, no discrete nucleocapsid was detected despite a defined symmetry of the outer envelope protein shell (23, 33, 54, 55). In addition, no transmembrane connections between the envelope proteins and particle inner core were seen, suggesting that a symmetry match between the two layers is likely not required (54). The flavivirus capsid protein dimers are predicted to be randomly dispersed within this disordered interior, with their overall positive charge neutralizing the genomic RNA (34). While the flavivirus capsid proteins have defined helix-rich tertiary folds (9, 30), these proteins tolerate large deletions, suggesting a functional flexibility of structure (22, 37). The pestivirus core protein has also been shown to be flexible in size, tolerating the insertion of an epitope tag (15). We propose that the disordered BVDV core protein randomly associates with RNA in a manner analogous to the flavivirus capsid, with its extended conformation displaying numerous basic residues for RNA neutralization. In this way, the pestivirus core protein would perform a function similar to that of positively charged polyamines, such as spermidine, which have been found incorporated into particles of several viruses, including encephalomyocarditis virus and turnip yellow mosaic virus (8, 47).

A simple charge neutralization mechanism, in the absence of an ordered nucleocapsid or specific genomic RNA interactions, may preclude the need for a folded core protein domain. Interestingly, however, the predicted short carboxy-terminal helix of BVDV core did appear to be required for optimal functional replacement of the Sindbis virus capsid amino-terminal domain. The region of the Sindbis virus capsid protein deleted in the chimeric genomes included not only nonspecific RNA-binding sequences but also a short, helical homotypic interaction domain (38). This helix is required for stabilization of nucleic acid-dependent dimers and imparts specificity to capsid-capsid interactions (16, 38). The possible compensation for this deletion might suggest the presence of at least one other functional region in the BVDV core protein. Indeed, a chimera in which this Sindbis virus helix was left intact [Sindbis/luc(C{Delta}72-96/BVDV54-83)] achieved close to wild-type levels of infectious virus production in the absence of the BVDV core carboxy-terminal residues. This suggested that there are separable charge neutralization and additional functions within the BVDV core protein. It remains to be investigated whether the pestivirus core protein forms dimers and whether hydrophobic resides 85 to 90 might be involved in the mediation of these interactions. Possible dimer formation would be consistent with our observed cooperativity of RNA binding and reminiscent of the functional dimers formed by other Flaviviridae capsid proteins (4, 18, 21, 31).

In conclusion, we found that BVDV core is a natively unfolded protein with a large number of basic residues involved in mediating low-affinity nonspecific interactions with RNA. We further determined that this in vitro behavior likely reflects its in vivo mechanism of action as a nonspecific RNA-condensing agent.


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ACKNOWLEDGMENTS
 
We thank Patricia Holst, Michelle Hunter, Maryline Panis, Jodie Tassello, Merna Torres, and Anesta Webson for laboratory support and technical assistance. We are grateful to Santosh Menon, Kirk Jensen, Michael Rout, Kiran Musunuru, Jennifer Darnell, and Karla Kirkegaard for helpful discussions. We thank Heinz-Jürgen Thiel and Till Rümenapf for communication of prepublication data regarding the BVDV core carboxy terminus. We thank Christopher Jones for valuable insights and for critical reading of the manuscript.

This work was supported by the Greenberg Medical Research Institute, the Starr Foundation, and grant R01 AI075099 from the National Institutes of Health. C.L.M. was supported by a grant from the Natural Sciences and Engineering Research Council of Canada (PGSA-232691).


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FOOTNOTES
 
* Corresponding author. Mailing address: Laboratory of Virology and Infectious Disease, Center for the Study of Hepatitis C, The Rockefeller University, 1230 York Ave., New York, NY 10065. Phone: (212) 327-7046. Fax: (212) 327-7048. E-mail: ricec{at}rockefeller.edu Back

{triangledown} Published ahead of print on 21 November 2007. Back

{dagger} Present address: Center for Advanced Biotechnology and Medicine, Department of Chemistry and Chemical Biology, Rutgers University, 679 Hoes Lane, Piscataway, NJ 08854. Back


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REFERENCES
 
    1
  1. Agapov, E. V., C. L. Murray, I. Frolov, L. Qu, T. M. Myers, and C. M. Rice. 2004. Uncleaved NS2-3 is required for production of infectious bovine viral diarrhea virus. J. Virol. 78:2414-2425.[Abstract/Free Full Text]
    2. 2
  2. Beckman, M. T., and K. Kirkegaard. 1998. Site size of cooperative single-stranded RNA binding by poliovirus RNA-dependent RNA polymerase. J. Biol. Chem. 273:6724-6730.[Abstract/Free Full Text]
    3. 3
  3. Bick, M. J., G. Gao, S. P. Goff, C. M. Rice, and M. R. MacDonald. 2003. Expression of the zinc-finger antiviral protein inhibits alphavirus replication. J. Virol. 77:11555-11562.[Abstract/Free Full Text]
    4. 4
  4. Boulant, S., C. Vanbelle, C. Ebel, F. Penin, and J. P. Lavergne. 2005. Hepatitis C virus core protein is a dimeric alpha-helical protein exhibiting membrane protein features. J. Virol. 79:11353-11365.[Abstract/Free Full Text]
    5. 5
  5. Cheng, R. H., R. J. Kuhn, N. H. Olson, M. G. Rossmann, H. K. Choi, T. J. Smith, and T. S. Baker. 1995. Nucleocapsid and glycoprotein organization in an enveloped virus. Cell 80:621-630.[CrossRef][Medline]
    6. 6
  6. Choi, H.-K., L. Tong, W. Minor, P. Dumas, U. Boege, M. G. Rossman, and G. Wengler. 1991. Structure of Sindbis virus core protein reveals a chymotrypsin-like serine proteinase and the organization of the virion. Nature 354:37-43.[CrossRef][Medline]
    7. 7
  7. Chromy, L. R., J. M. Pipas, and R. L. Garcea. 2003. Chaperone-mediated in vitro assembly of Polyomavirus capsids. Proc. Natl. Acad. Sci. USA 100:10477-10482.[Abstract/Free Full Text]
    8. 8
  8. Cohen, S. S., and M. L. Greenberg. 1981. Spermidine, an intrinsic component of turnip yellow mosaic virus. Proc. Natl. Acad. Sci. USA 78:5470-5474.[Abstract/Free Full Text]
    9. 9
  9. Dokland, T., M. Walsh, J. M. Mackenzie, A. A. Khromykh, K. H. Ee, and S. Wang. 2004. West Nile virus core protein; tetramer structure and ribbon formation. Structure 12:1157-1163.[Medline]
    10. 10
  10. Dunker, A. K., C. J. Brown, J. D. Lawson, L. M. Iakoucheva, and Z. Obradovic. 2002. Intrinsic disorder and protein function. Biochemistry 41:6573-6582.[CrossRef][Medline]
    11. 11
  11. Forsell, K., M. Suomalainen, and H. Garoff. 1995. Structure-function relation of the NH2-terminal domain of the Semliki Forest virus capsid protein. J. Virol. 69:1556-1563.[Abstract]
    12. 12
  12. Frolov, I., and S. Schlesinger. 1994. Translation of Sindbis virus mRNA: effects of sequences downstream of the initiating codon. J. Virol. 68:8111-8117.[Abstract/Free Full Text]
    13. 13
  13. Geigenmuller-Gnirke, U., H. Nitschko, and S. Schlesinger. 1993. Deletion analysis of the capsid protein of Sindbis virus: identification of the RNA binding region. J. Virol. 67:1620-1626.[Abstract/Free Full Text]
    14. 14
  14. Harada, T., N. Tautz, and H. J. Thiel. 2000. E2-p7 region of the bovine viral diarrhea virus polyprotein: processing and functional studies. J. Virol. 74:9498-9506.[Abstract/Free Full Text]
    15. 15
  15. Heimann, M., G. Roman-Sosa, B. Martoglio, H. J. Thiel, and T. Rumenapf. 2006. Core protein of pestiviruses is processed at the C terminus by signal peptide peptidase. J. Virol. 80:1915-1921.[Abstract/Free Full Text]
    16. 16
  16. Hong, E. M., R. Perera, and R. J. Kuhn. 2006. Alphavirus capsid protein helix I controls a checkpoint in nucleocapsid core assembly. J. Virol. 80:8848-8855.[Abstract/Free Full Text]
    17. 17
  17. Jensen, K. B., K. Musunuru, H. A. Lewis, S. K. Burley, and R. B. Darnell. 2000. The tetranucleotide UCAY directs the specific recognition of RNA by the Nova K-homology 3 domain. Proc. Natl. Acad. Sci. USA 97:5740-5745.[Abstract/Free Full Text]
    18. 18
  18. Jones, C. T., L. Ma, J. W. Burgner, T. D. Groesch, C. B. Post, and R. J. Kuhn. 2003. Flavivirus capsid is a dimeric alpha-helical protein. J. Virol. 77:7143-7149.[Abstract/Free Full Text]
    19. 19
  19. Kelly, S. M., T. J. Jess, and N. C. Price. 2005. How to study proteins by circular dichroism. Biochim. Biophys. Acta 1751:119-139.[Medline]
    20. 20
  20. Khromykh, A. A., and E. G. Westaway. 1996. RNA binding properties of core protein of the flavivirus Kunjin. Arch. Virol. 141:685-699.[CrossRef][Medline]
    21. 21
  21. Kiermayr, S., R. M. Kofler, C. W. Mandl, P. Messner, and F. X. Heinz. 2004. Isolation of capsid protein dimers from the tick-borne encephalitis flavivirus and in vitro assembly of capsid-like particles. J. Virol. 78:8078-8084.[Abstract/Free Full Text]
    22. 22
  22. Kofler, R. M., F. X. Heinz, and C. W. Mandl. 2002. Capsid protein C of tick-borne encephalitis virus tolerates large internal deletions and is a favorable target for attenuation of virulence. J. Virol. 76:3534-3543.[Abstract/Free Full Text]
    23. 23
  23. Kuhn, R. J., W. Zhang, M. G. Rossmann, S. V. Pletnev, J. Corver, E. Lenches, C. T. Jones, S. Mukhopadhyay, P. R. Chipman, E. G. Strauss, T. S. Baker, and J. H. Strauss. 2002. Structure of dengue virus: implications for flavivirus organization, maturation, and fusion. Cell 108:717-725.[CrossRef][Medline]
    24. 24
  24. Kunkel, M., M. Lorinczi, R. Rijnbrand, S. M. Lemon, and S. J. Watowich. 2001. Self-assembly of nucleocapsid-like particles from recombinant hepatitis C virus core protein. J. Virol. 75:2119-2129.[Abstract/Free Full Text]
    25. 25
  25. Kunkel, M., and S. J. Watowich. 2004. Biophysical characterization of hepatitis C virus core protein: implications for interactions within the virus and host. FEBS Lett. 557:174-180.[CrossRef][Medline]
    26. 26
  26. Lackner, T., A. Muller, A. Pankraz, P. Becher, H. J. Thiel, A. E. Gorbalenya, and N. Tautz. 2004. Temporal modulation of an autoprotease is crucial for replication and pathogenicity of an RNA virus. J. Virol. 78:10765-10775.[Abstract/Free Full Text]
    27. 27
  27. Li, X., P. Romero, M. Rani, A. K. Dunker, and Z. Obradovic. 1999. Predicting protein disorder for N-, C-, and internal regions. Genome Inform. Ser. Workshop Genome Inform. 10:30-40.[Medline]
    28. 28
  28. Lindenbach, B. D., H. J. Thiel, and C. M. Rice. 2007. Flaviviridae: the viruses and their replication, p. 1101-1152. In D. M. Knipe and P. M. Howley (ed.), Fields virology, 5th ed., vol. 1. Lippincott-Raven Publishers, Philadelphia, PA.
    29. 29
  29. Linger, B. R., L. Kunovska, R. J. Kuhn, and B. L. Golden. 2004. Sindbis virus nucleocapsid assembly: RNA folding promotes capsid protein dimerization. RNA 10:128-138.[Abstract/Free Full Text]
    30. 30
  30. Ma, L., C. T. Jones, T. D. Groesch, R. J. Kuhn, and C. B. Post. 2004. Solution structure of dengue virus capsid protein reveals another fold. Proc. Natl. Acad. Sci. USA 101:3414-3419.[Abstract/Free Full Text]
    31. 31
  31. Matsumoto, M., S. B. Hwang, K.-S. Jeng, N. Zhu, and M. M. C. Lai. 1996. Homotypic interaction and multimerization of hepatitis C virus core protein. Virology 218:43-51.[CrossRef][Medline]
    32. 32
  32. Moennig, V., and P. G. Plagemann. 1992. The pestiviruses. Adv. Virus Res. 41:53-98.[Medline]
    33. 33
  33. Mukhopadhyay, S., B. S. Kim, P. R. Chipman, M. G. Rossmann, and R. J. Kuhn. 2003. Structure of West Nile virus. Science 302:248.[Free Full Text]
    34. 34
  34. Mukhopadhyay, S., R. J. Kuhn, and M. G. Rossmann. 2005. A structural perspective of the flavivirus life cycle. Nat. Rev. Microbiol. 3:13-22.[CrossRef][Medline]
    35. 35
  35. Mukhopadhyay, S., W. Zhang, S. Gabler, P. R. Chipman, E. G. Strauss, J. H. Strauss, T. S. Baker, R. J. Kuhn, and M. G. Rossmann. 2006. Mapping the structure and function of the E1 and E2 glycoproteins in alphaviruses. Structure 14:63-73.[Medline]
    36. 36
  36. Owen, K. E., and R. J. Kuhn. 1996. Identification of a region in the Sindbis virus nucleocapsid protein that is involved in specificity of RNA encapsidation. J. Virol. 70:2757-2763.[Abstract]
    37. 37
  37. Patkar, C. G., C. T. Jones, Y. H. Chang, R. Warrier, and R. J. Kuhn. 2007. Functional requirements of the yellow fever virus capsid protein. J. Virol. 81:6471-6481.[Abstract/Free Full Text]
    38. 38
  38. Perera, R., K. E. Owen, T. L. Tellinghuisen, A. E. Gorbalenya, and R. J. Kuhn. 2001. Alphavirus nucleocapsid protein contains a putative coiled coil alpha-helix important for core assembly. J. Virol. 75:1-10.[Abstract/Free Full Text]
    39. 39
  39. Rice, C. M., J. R. Bell, M. W. Hunkapiller, E. G. Strauss, and J. H. Strauss. 1982. Isolation and characterization of the hydrophobic COOH-terminal domains of the Sindbis virion glycoproteins. J. Mol. Biol. 154:355-378.[CrossRef][Medline]
    40. 40
  40. Rice, C. M., and J. H. Strauss. 1982. Association of Sindbis virion glycoproteins and their precursors. J. Mol. Biol. 154:325-348.[CrossRef][Medline]
    41. 41
  41. Romero, P., Z. Obradovic, and K. Dunker. 1997. Sequence data analysis for long disordered region prediction in the calcineurin family. Genome Inform. Ser. Workshop Genome Inform. 8:110-124.[Medline]
    42. 42
  42. Romero, P., Z. Obradovic, X. Li, E. C. Garner, C. J. Brown, and A. K. Dunker. 2001. Sequence complexity of disordered protein. Proteins 42:38-48.[CrossRef][Medline]
    43. 43
  43. Rost, B., and C. Sander. 1993. Prediction of protein secondary structure at better than 70% accuracy. J. Mol. Biol. 232:584-599.[CrossRef][Medline]
    44. 44
  44. Rouille, Y., F. Helle, D. Delgrange, P. Roingeard, C. Voisset, E. Blanchard, S. Belouzard, J. McKeating, A. H. Patel, G. Maertens, T. Wakita, C. Wychowski, and J. Dubuisson. 2006. Subcellular localization of hepatitis C virus structural proteins in a cell culture system that efficiently replicates the virus. J. Virol. 80:2832-2841.[Abstract/Free Full Text]
    45. 45
  45. Rümenapf, T., G. Unger, J. H. Strauss, and H.-J. Thiel. 1993. Processing of the envelope glycoproteins of pestiviruses. J. Virol. 67:3288-3294.[Abstract/Free Full Text]
    46. 46
  46. Santolini, E., G. Migliaccio, and N. La Monica. 1994. Biosynthesis and biochemical properties of the hepatitis C virus core protein. J. Virol. 68:3631-3641.[Abstract/Free Full Text]
    47. 47
  47. Sheppard, S. L., A. T. Burness, and S. M. Boyle. 1980. Polyamines in encephalomyocarditis virus. J. Virol. 34:266-267.[Abstract/Free Full Text]
    48. 48
  48. Stark, R., G. Meyers, T. Rümenapf, and H.-J. Thiel. 1993. Processing of pestivirus polyprotein: cleavage site between autoprotease and nucleocapsid protein of classical swine fever virus. J. Virol. 67:7088-7095.[Abstract/Free Full Text]
    49. 49
  49. Thiel, H.-J., R. Stark, E. Weiland, T. Rümenapf, and G. Meyers. 1991. Hog cholera virus: molecular composition of virions from a pestivirus. J. Virol. 65:4705-4712.[Abstract/Free Full Text]
    50. 50
  50. Tompa, P. 2005. The interplay between structure and function in intrinsically unstructured proteins. FEBS Lett. 579:3346-3354.[CrossRef][Medline]
    51. 51
  51. Uversky, V. N. 2002. What does it mean to be natively unfolded? Eur. J. Biochem. 269:2-12.[Medline]
    52. 52
  52. Weiss, B., U. Geigenmuller-Gnirke, and S. Schlesinger. 1994. Interactions between Sindbis virus RNAs and a 68 amino acid derivative of the viral capsid protein further defines the capsid binding site. Nucleic Acids Res. 22:780-786.[Abstract/Free Full Text]
    53. 53
  53. Zhang, W., S. Mukhopadhyay, S. V. Pletnev, T. S. Baker, R. J. Kuhn, and M. G. Rossmann. 2002. Placement of the structural proteins in Sindbis virus. J. Virol. 76:11645-11658.[Abstract/Free Full Text]
    54. 54
  54. Zhang, Y., J. Corver, P. R. Chipman, W. Zhang, S. V. Pletnev, D. Sedlak, T. S. Baker, J. H. Strauss, R. J. Kuhn, and M. G. Rossmann. 2003. Structures of immature flavivirus particles. EMBO J. 22:2604-2613.[CrossRef][Medline]
    55. 55
  55. Zhang, Y., V. A. Kostyuchenko, and M. G. Rossmann. 2007. Structural analysis of viral nucleocapsids by subtraction of partial projections. J. Struct. Biol. 157:356-364.[CrossRef][Medline]

Journal of Virology, February 2008, p. 1294-1304, Vol. 82, No. 3
0022-538X/08/$08.00+0     doi:10.1128/JVI.01815-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



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