JOURNAL OF VIROLOGY, Dec. 2010, p. 12480–12491 0022-538X/10/$12.00 doi:10.1128/JVI.01319-10 Copyright © 2010, American Society for Microbiology. All Rights Reserved.
Vol. 84, No. 24
Hepatitis C Virus Nonstructural Protein 5A: Biochemical Characterization of a Novel Structural Class of RNA-Binding Proteins䌤 Jungwook Hwang,1† Luyun Huang,1† Daniel G. Cordek,1 Robert Vaughan,2 Shelley L. Reynolds,1 George Kihara,1 Kevin D. Raney,3 C. Cheng Kao,2 and Craig E. Cameron1* Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania 168021; Department of Molecular and Cellular Biochemistry, Indiana University, Bloomington, Indiana 474052; and Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 722053 Received 21 June 2010/Accepted 23 September 2010
Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) exhibits a preference for G/U-rich RNA in vitro. Biological analysis of the NS5A RNA-binding activity and its target sites in the genome will be facilitated by a description of the NS5A-RNA complex. We demonstrate that the C-4 carbonyl of the uracil base and, by inference, the C-6 carbonyl of the guanine base interact with NS5A. U-rich RNA of 5 to 6 nucleotides (nt) is sufficient for high-affinity binding to NS5A. The minimal RNA-binding domain of NS5A consists of residues 2005 to 2221 (referred to as domain I-plus). This region of the protein includes the amino-terminal domain I as well as the subsequent linker that separates domains I and II. This linker region is the site of adaptive mutations. U-rich RNA-binding activity is not observed for an NS5A derivative containing only residues 2194 to 2419 (domains II and III). Mass spectrometric analysis of an NS5A-poly(rU) complex identified domains I and II as sites for interaction with RNA. Dimerization of NS5A was demonstrated by glutaraldehyde crosslinking. This dimerization is likely mediated by domain I-plus, as dimers of this protein are trapped by cross-linking. Dimers of the domain II-III protein are not observed. The monomer-dimer equilibrium of NS5A shifts in favor of dimer when U-rich RNA is present but not when A-rich RNA is present, consistent with an NS5A dimer being the RNA-binding-competent form of the protein. These data provide a molecular perspective of the NS5A-RNA complex and suggest possible mechanisms for regulation of HCV and cellular gene expression.
Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) is essential for replication (16). The protein has a calculated molecular mass of 49 kDa and is organized into three domains: I, II, and III (Fig. 1A) (26). The domains are separated by low-complexity sequences (linkers) that likely lack defined structure (26). NS5A migrates as 56- and 58-kDa species in SDS-polyacrylamide gels (12). This greater-than-expected molecular mass was thought to be the result of phosphorylation (25). The 56-kDa form (p56) was denoted the basal phosphorylation state; p58 was denoted the hyperphosphorylated state. Three conserved clusters of serines (PI to PIII in Fig. 1A) are thought to serve as sites of phosphorylation (1), with cluster I being responsible for the hyperphosphorylated state (20). Interestingly, loss or reduction of p58 occurs during adaptation of HCV subgenomic replicons to cell culture. Among the nonstructural proteins of HCV, NS5A has the most interactions with the host and by far the fewest functions in the genome replication process. There is evidence for a role of NS5A in antagonizing innate immune responses of the host, interacting with numerous signal transduction pathways, HCV
* Corresponding author. Mailing address: Department of Biochemistry and Molecular Biology, The Pennsylvania State University, 201 Althouse Laboratory, University Park, PA 16802. Phone: (814) 8638705. Fax: (814) 865-7927. E-mail:
[email protected]. † These authors contributed equally to this study. 䌤 Published ahead of print on 6 October 2010.
replication and assembly, and more (18). The ability of NS5A to exhibit so many different activities may reflect the ability of domains II and III to adopt different conformations as a result of the natively unfolded nature of these domains (8, 9). In addition, the wide range of combinatorial patterns of phosphorylation could also contribute to the production of different forms of NS5A that exhibit different functions. The ability to express and purify NS5A using a bacterial system led to the observation that the mobility of NS5A as a 56-kDa protein in SDS-polyacrylamide gels was not due to phosphorylation but could be due to the proline-rich nature of the protein (11). The unphosphorylated protein exhibits specific (G/U) and nonspecific RNA-binding activity in vitro (10). Importantly, unphosphorylated NS5A exists in cells replicating HCV RNA, and so does the ability of this form of NS5A, and perhaps other forms of NS5A, to bind RNA (10). The ability of NS5A to bind RNA is a property observed in NS5A proteins from all HCV genotypes tested to date (6). Two structures of domain I of NS5A have been solved (17, 27). The fold of the protein is novel and defines a new structural class of RNA-binding proteins. The first structure showed surfaces of positive electrostatic potential that could be used for RNA binding (Fig. 1B). The second structure showed a different quaternary structure (Fig. 1C). How this conformation of the protein would bind RNA, even nonspecifically, is not obvious.
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FIG. 1. NS5A schematic and domain I crystal structure with surface potential. (A) Schematic structure of NS5A. NS5A is composed of 447 amino acids containing three domains, i.e., domain I (residues 1973 to 2185), domain II (residues 2221 to 2314), and domain III (residues 2329 to 2419), with two linkers (residues 2186 to 2220 and 2315 to 2328), as determined by prediction of the NS5A structure (26). NS5A has three highly conserved clusters of serine residues, denoted PI (residues 2194 to 2210), PII (residues 2246 to 2269), and PIII (2380 to 2409). (B) Left, ribbon diagram of the first domain I dimer; the two subdomains (1, aa 2008 to 2072; 2, aa 2073 to 2170) are colored in green and purple, respectively (27). The two zinc ions are displayed as yellow spheres; the N and C termini of each monomer are depicted as small spheres colored in blue and red, respectively. Right, the surface electrostatic potential of the dimer (rotated 90°) calculated by the APBS program contoured at ⫺1 KT (red), 0 (white), ⫹1 KT (blue). Blue and red colors indicate positively and negatively charged amino acids, respectively. (C) Left, alternate ribbon diagram of the domain I dimer using the same color scheme as in panel B (17). Right, the surface electrostatic potential of the alternate dimer as calculated based on the same parameters as for panel B.
In this study, we have performed a detailed biochemical analysis of the NS5A-RNA interaction. We define the minimal requirements of NS5A and RNA required to produce the NS5A-RNA complex, identify determinants of the uracil base (and by inference the guanine base) that are recognized by NS5A, and provide evidence for a dimer as the form of NS5A that is active in RNA binding. Therefore, this study creates a useful framework for the study of the biological role of the interaction of NS5A with G/U-rich RNA and the discovery of compounds capable of interfering with the RNA-binding activity of NS5A. MATERIALS AND METHODS Materials. All RNA oligonucleotides were from Dharmacon Research, Inc. (Boulder, CO). All restriction enzymes, T4 polynucleotide kinase, and Deep Vent DNA polymerase were from New England Biolabs, Inc. T4 DNA ligase was from Invitrogen. DNA primers for PCRs were from Integrated DNA Technologies. [␥-32P]ATP (⬎7,000 Ci/mmol) was from MP Biomedicals. All other reagents were of the highest grade available from Sigma, Fisher, or VWR. Construction of NS5A expression plasmids. Cloning methods for 2005-2419 genotype 1b Con 1 NS5A NHis or CHis were described in a previous report (11). Most NS5A deletion mutants were cloned by PCR amplifying the corresponding
regions from pHCVbart.rep1b/Ava-II (4) with the forward primer HCV-del325A-NcoI-NHis-for and reverse primers containing a HindIII site (Table 1). Primers HCV-5A-2194-2419-NcoI-NHis-for and HCV-5A-HindIII-rev were used to clone 2194-2419 NS5A NHis (Table 1). The NcoI/HindIII-digested PCR products were ligated into the pET26-Ub-NHis vector (11). Cloning for 20052221 and 2212-2419 NS5A in the pET24-6H-SUMO vector was performed as previously reported (2). Briefly, the NS5A gene segments were amplified from the above-mentioned pHCVbart.rep1b/Ava-II using forward primers SUMOBsaI-HCV-2005-2221-for and SUMO-BsaI-HCV-2212-2419-for and reverse primers SUMO-HCV-2005-2221-HindIII-rev and SUMO-HCV-2212-2419-HindIIIrev, respectively (Table 1). The BsaI/HindIII-digested PCR products were ligated into the pET24-6H-SUMO vector. Expression and purification of NS5A proteins. The NHis and CHis NS5A deletion mutants were expressed and purified as previously described (11). The SUMO-NS5A deletion plasmids were transformed into Rosetta(DE3) competent cells. Rosetta(DE3) containing the pSUMO-NS5A plasmids was grown overnight in 100 ml of NZCYM supplemented with 25 g/ml kanamycin (K25) and 20 g/ml chloramphenicol (C20). The overnight culture was used to inoculate 1 liter of K25- and C20-supplemented autoinducing medium to an optical density at 600 nm (OD600) of 0.05. The cells were grown at 37°C to an OD600 of 1.0 and then grown at 20°C for an additional 20 h. The cells were harvested by centrifugation in a Beckman JLA-16.250 rotor at 6,000 rpm for 10 min, washed once in 250 ml T10E1 (10 mM Tris [pH 8.0], 1 mM EDTA), and centrifuged again before the cell pellet was weighed. The typical yield was 20 to 22 g of cell paste per liter of culture.
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HWANG ET AL. TABLE 1. Oligonucleotides used in this study Oligonucleotide
Sequence
HCV-del32-5A-NcoI-NHis-for .............................................5⬘-GCG GGT ACC CCA TGG ATC CTC TGG TGG AGT CCC CTT CTT C-3⬘ HCV-5A-HindIII-rev .............................................................5⬘-GCG GGT ACC AAG CTT CTA TTA GCA GCA GAC GAC GTC CTC ACT-3⬘ HCV-5A-2005-2306-HindIII-rev ..........................................5⬘-GGC AAG CTT CTA TTA GTA GTC CGG GTC CTT-3⬘ HCV-5A-2005-2245-HindIII-rev ..........................................5⬘-CCG CAA GCT TCT ATT ACT CCA CGC GGG TGA TGT T-3⬘ HCV-5A-2005-2221-HindIII-rev ..........................................5⬘-GCG CAA GCT TCT ATT AGG AGT CAT GAC GGG T-3⬘ HCV-5A-2005-2187-HindIII-rev ..........................................5⬘-TTT TTT AAG CTT CTA TTA AGC CGT CTC CGC CGT-3⬘ HCV-5A-2194-2419-NcoI-NHis-for .....................................5⬘-CGC GCC ATG GAT CCT CTG GTT CTC CCC CCT CCT TGG CC-3⬘ SUMO-BsaI-HCV-2005-2221-for.........................................5⬘-GCG GGA TCC GGT CTC AAG GTG GAG TCC CCT TCT TCT CA-3⬘ SUMO-HCV-2005-2221-HindIII-rev...................................5⬘-GCG CTC GAG AAG CTT CTA TTA GGA GTC ATG ACG GGT AGT-3⬘ SUMO-BsaI-HCV-2212-2419-for.........................................5⬘-GCG GGA TCC GGT CTC AAG GTA AGG CAA CAT GCA CTA CC-3⬘ SUMO-HCV-2212-2419-HindIII-rev...................................5⬘-CAC AAA GTG AAG CTT CTA TTA GAC GAC GTC CTC ACT AGC-3⬘
The cell pellet was suspended in lysis buffer (100 mM potassium phosphate [pH 8.0], 500 mM NaCl, 10 mM -mercaptoethanol [BME], 20% glycerol, 5 mM imidazole, 1.4 g/ml pepstatin A, and 1.0 g/ml leupeptin) supplemented with protease inhibitor cocktail tablets (Roche). The suspended cells were lysed by passage through a French press. Phenylmethylsulfonyl fluoride (PMSF) was added to a final concentration of 1 mM, and NP-40 was added to 0.1%. The extract was centrifuged in a Beckman JA-30.50 rotor for 30 min at 25,000 rpm (75,600 ⫻ g) at 4°C. The supernatant was loaded onto an Ni-nitrilotriacetic acid (NTA) column at a flow rate of 0.5 ml/min, the rate used for loading all subsequent columns. The column was washed with buffer A (100 mM potassium phosphate [pH 8.0], 500 mM NaCl, 10 mM BME, and 20% glycerol) containing 5 mM imidazole, 0.1% NP-40, 1.4 g/ml pepstatin A, 1.0 g/ml leupeptin, and 1 mM PMSF at a flow rate of 1.0 ml/min, the rate used for washing and eluting all subsequent columns. The column was washed two additional times with buffer A containing 5 and 50 mM imidazole. The protein was eluted with buffer A containing 500 mM imidazole. The collected fractions were assayed for purity by SDS-PAGE. Ulp1 was added to the pooled fractions (1 g per 2.5 mg SUMO fusion) and dialyzed overnight against buffer B (25 mM HEPES [pH 7.5], 100 mM NaCl, 5 mM BME, and 20% glycerol). The cleaved 2005-2221 NS5A protein was diluted to 85 mM NaCl and passed through a Q-Sepharose column. The protein was then loaded onto a S-Sepharose column and concentrated by stepping the protein off in 1-column-volume fractions of buffer B containing 1 M NaCl. The pooled fractions were assayed for purity by SDS-PAGE and dialyzed overnight against buffer B. The conductivity of the cleaved 2212-2419 NS5A protein was increased to 500 mM NaCl, and the protein was passed through a Talon cobalt column. The protein was diluted to 100 mM NaCl, loaded onto a Q-Sepharose column, and concentrated by stepping the protein off in 1-column-volume fractions of buffer B containing 1 M NaCl. SDS-PAGE was used to assay for purity, and the pooled fractions were dialyzed overnight against buffer B. The concentrations of both 2005-2221 and 2212-2419 NS5A were determined using Sypro Ruby protein gel stain (Molecular Probes) as performed with the His-tagged derivatives. Proteins were at least 95% pure as observed with Histagged derivatives. Proteins were aliquoted and frozen at ⫺80°C. Biotinylated RNA pulldown assay. In a typical experiment, 1 M synthetic rU7rC4-biotin was mixed with 1 M purified NS5A protein in 50 l binding buffer containing 50 mM HEPES (pH 7.5), 5 mM MgCl2, and 10 mM 〉ME. After incubation on ice for 30 min, 50 l of streptavidin magnetic beads (New England Biolabs) was added, and the mixture was incubated at room temperature for 30 min. The supernatant was removed after magnetic collection of the beads. The beads were washed twice with 100 l binding buffer. After the bound NS5A was solubilized in 1⫻ SDS sample buffer at 95°C for 10 min, the beads were spun down and the supernatant was analyzed by SDS-PAGE on a 12.5% gel. Purified proteins (0.2 pmol) were loaded along with the molecular marker. Sypro Ruby protein gel stain was used to stain the proteins prior to visualization with a Typhoon imager (Molecular Dynamics). RNA filter-binding assay. The RNA filter-binding experiments were performed as previously described (10). In a 50-l reaction mixture, various ␥-32Plabeled RNA oligomers (2 nM) were incubated with 0 to 500 nM 2005-2419 NS5A NHis in binding buffer (50 mM HEPES [pH 7.5], 5 mM MgCl2, 10 mM BME) on ice for 30 min. RNA-protein complexes were separated from free RNA by filtering the solution through polysulfone, nitrocellulose, and nylon membranes in a slot blot apparatus (Amersham). After the membranes were dried, the NS5A-bound RNA on the nitrocellulose membrane and the free RNA on the nylon membrane were quantitated with a Typhoon imager and ImageQuant software. Binding data were fit to a hyperbola (equation 1) using KaleidaGraph:
⫽ max共关P兴/关P兴 ⫹ Kd兲 ⫹ c
(1)
where is the percentage of bound RNA, max is the maximal percentage of RNA competent for binding, [P] is the concentration of NS5A (nM), Kd is the apparent dissociation constant, and c is an offset. Fluorescence polarization assay. Experiments were performed using a Beacon2000 fluorescence polarization system (Invitrogen). Zero to 400 nanomolar 2005-2221 NS5A NHis or 2005-2187 NS5A NHis was incubated briefly with 0.1 nM 3⬘-fluorescein-labeled rU15 (FL-rU15) in the presence of 50 mM HEPES (pH 7.5), 100 mM NaCl, 5 mM MgCl2, and 10 mM BME at 25°C. The change in polarization (⌬mP) was measured by fluorescence polarization. NS5A (0 to 2000 nM) and 0.1 nM FL-rU15 or FL-rA15 were gently mixed in binding reaction buffer (50 mM HEPES [pH 7.5], 100 mM NaCl, 5 mM MgCl2, and 10 mM BME) and incubated for 30 s at 25°C. Binding of NS5A was measured by the change in polarization (⌬mP). Data were fit to a hyperbola (equation 1) using KaleidaGraph (Synergy Software). In RNA competition assays, 0.1 nM FL-rU15 was preincubated briefly with 50 nM 2005-2419 NS5A NHis in 50 mM HEPES (pH 7.5), 100 mM NaCl, 5 mM MgCl2, and 10 mM BME at room temperature (the final volume was 100 l), and then 125 nM various competitor RNAs was added. The solution was incubated at 25°C for 30 s before each measurement. The percent binding of FL-rU15 in the presence relative to in the absence of the competitors was calculated. NaCl, MgCl2, and EDTA titration assays. In a 100-l reaction mixture, 25 nM 2005-2419 NS5A NHis was preincubated briefly with 0.1 nM FL-rU15 in 50 mM HEPES (pH 7.5), 5 mM MgCl2, and 10 mM BME at room temperature. After mixing with 0 to 1 M NaCl (final concentration), the solution was incubated at 25°C for 30 s and the fluorescence polarization was measured. Similarly, 30 nM 2005-2419 NS5A NHis was preincubated with 0.1 nM FL-rU15 in 50 mM HEPES (pH 7.5) and 10 mM BME at room temperature before 0 to 20 mM MgCl2 (final concentration) was added. For EDTA titration, 60 nM 2005-2419 NS5A NHis was used and EDTA was titrated in the same 0 to 20 mM range. pH titration filter-binding assay. In the binding reactions, ␥-32P-labeled rU15 was incubated with 100 nM 2005-2419 NS5A NHis in MTCN buffers (50 mM MES [morpholineethanesulfonic acid], 25 mM Tris, 25 mM 3-[cyclohexylamino]1-propanesulfonic acid [CAPS], 50 mM NaCl) at pH 6 to 10.5. The NS5A-bound RNA and free RNA were separated by filtering the solutions through the polysulfone, nitrocellulose, and nylon membranes in a slot blot apparatus. After phosphorimager analysis (Molecular Dynamics), data were fit to equation 2: ⫽ max10pKa ⫺ pH/共1 ⫹ 10pKa ⫺ pH兲
(2)
where is the percentage of bound RNA, max is the maximal percentage of RNA competent for binding, pH is the pH of the buffer, and pKa is the acid dissociation constant. Fitting was performed using KaleidaGraph 3.5 software (Synergy Software). Western blot analysis. For detection of monomer and dimer NS5A proteins, Western blotting was performed as reported previously (10). Monomer and dimer NS5A proteins were quantified by FluorescentImager (Typhoon 9410; Molecular Dynamics). Glutaraldehyde cross-linking. 2005-2221 or 2212-2419 NS5A (0.5 M) was incubated for 5 min with the indicated concentration of fresh glutaraldehyde in the absence or presence of 5 M RNA (rU15 or rA15) in buffer containing 50 mM HEPES (pH 7.5), 100 mM NaCl, 0.5 mM TCEP [tris(2-carboxyethyl)phosphine], 5 mM MgCl2, and 0.1 mM ZnCl2. Protein was preincubated in the absence or presence of RNA at 37°C for 10 min. Glutaraldehyde was added, and the mixture was incubated at 37°C for 5 min. The cross-linking reaction was quenched by adding Tris
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FIG. 2. NS5A RNA binding is dependent on Zn2⫹, MgCl2, and ionic strength. (A) NS5A (60 nM) was incubated with 0.1 nM FL-rU15 before the addition of 0 to 20 mM EDTA in the absence of NaCl or MgCl2. The change in mP (⌬mP) measured by fluorescence polarization decreased with the increasing EDTA concentration. (B) NS5A (30 nM) was preincubated with 0.1 nM FL-rU15 before the addition of 0 to 20 mM MgCl2 in the absence of NaCl. ⌬mP increased with the increasing MgCl2 concentration and plateaued at 5 mM MgCl2. The RNA-binding activity in the presence of ⬎5 mM MgCl2 is 100% higher than that in the absence of MgCl2. (C) NS5A (25 nM) was preincubated with 0.1 nM FL-rU15 with 5 mM MgCl2 before the addition of 0 to 1 M NaCl. ⌬mP decreased with increasing NaCl concentration. The 50% inhibitory concentration (IC50) was approximately 100 mM.
(pH 7.6) to a 200 mM final concentration. The protein-RNA complex was resolved by SDS-PAGE on a 12.5% gel, followed by Western blotting. Each band was quantified using enhanced chemifluorescence (ECF) (Amersham). Reversible cross-linking and peptide fingerprinting (RCAP) analysis. Portions of NS5A that can be cross-linked to RNA were mapped using a protocol originally established by Kim et al. (13) and modified as described below. poly(U)agarose (Sigma-Aldrich) was incubated with 2 M purified recombinant NS5A protein in 50 l buffer containing 20 mM HEPES (pH 7.5), 4 mM MnCl2, and 10 mM dithiothreitol (DTT). The suspension was divided into two equal aliquots. One was treated with formaldehyde to a final concentration of 0.1%. The other was treated with a comparable amount of water but was otherwise processed in parallel to serve as a control for the first sample. After 5 min at room temperature, the reaction was terminated by the addition of glycine to a final concentration of 0.2 M, and the volume was adjusted to 50 l with 100 mM NH4CO3, pH 7.8. Trypsin (Madison, WI) was added to a final concentration of 40 nM, and the reaction mixtures were incubated at 37°C overnight. Peptides that were not bound to the poly(U)-agarose were removed by three washes with buffer containing 20 mM HEPES (pH 7.5), with each wash followed by pelleting the poly(U)-agarose by centrifugation at 2,000 ⫻ g for 2 min. The poly(U)-agarose suspensions were then washed thrice with buffer containing 20 mM HEPES (pH 7.5), 1 M NaCl, 1 mM EDTA, and 1 mM DTT. The formaldehyde cross-links were reversed by incubating the samples at 70°C for 1 h in 250 mM NaCl. The resin was pelleted by centrifugation at 3,000 ⫻ g for 5 min, and the supernatants containing the peptides were desalted and concentrated using a Ziptip (Millipore, Bedford, MA). The bound peptides were eluted in 1.5 l of 70% acetonitrile and 0.1% trifluoroacetic acid in preparation for matrix-assisted laser desorption ionization–time-of-flight (MALDI-TOF)) mass spectrometry as described previously (13).
RESULTS Solution conditions to study the RNA-binding activity of NS5A. Our initial studies of the RNA-binding activity of NS5A used conditions that were well documented in the literature for studying the RNA helicase (NS3) and the RNA-dependent RNA polymerase (NS5B) (10). The pH was 7.5. The concentration of MgCl2 was 5 mM, and monovalent cations such as Na⫹ were omitted. We therefore performed a more systematic evaluation of the impact of solution conditions on RNA-binding activity. For these experiments, we used the fluorescence polarization assay reported previously (10). Fluorescein (FL)labeled rU15 RNA was incubated with NS5A prior to addition of a range of concentrations of the compound under investigation. The concentration of NS5A used in the different experiments was determined empirically to be the minimal concentration of NS5A required to provide sufficient signal for the experiment. The NS5A-RNA complex was in rapid equilibrium independent of the conditions used, as the addition of unla-
beled rU15 reduced the observed polarization to background levels as soon as 30 s after addition, which is the fastest time that could be measured on our instrument. Because NS5A uses a Zn2⫹ ion to stabilize the structure of domain I (Fig. 1B) (26, 27), we asked whether or not EDTA would inhibit the RNA-binding activity of NS5A. Very high concentrations of EDTA were required to inhibit RNA binding (Fig. 2A). We interpret this result to mean that the Zn2⫹ ion is bound tightly to the protein, consistent with tetracysteine coordination (27). However, NS5A molecules lacking the Zn2⫹ ion are incapable of binding RNA. Whether or not the binding reaction required Mg2⫹ was not known. We observed an increase in the amount of NS5A-RNA complex as the concentration of Mg2⫹ was increased from 0 to 5 mM (Fig. 2B). Mg2⫹ concentrations in excess of 5 mM caused no additional change in the amount of NS5A-RNA complex (Fig. 2B). It is likely that the Mg2⫹ binds to the phosphodiester backbone of the nucleic acid, which would suggest that NS5A does not interact with the phosphodiester backbone in a way that neutralizes all of the negative charge. Given the substantial positive charge associated with the NS5A dimer observed crystallographically by the Rice laboratory (Fig. 1B), there was some concern that the absence of salt would lead to a loss in the binding specificity. The presence of NaCl antagonized binding of RNA to NS5A, with a 50% reduction in RNA binding observed at 100 mM (Fig. 2C). The 2-fold reduction would still suggest an equilibrium dissociation constant (Kd) on the order of nM. Therefore, it is unlikely that the specific interaction of NS5A with RNA is solely ionic in nature. Based on these studies, all RNA-binding experiments were performed in the presence of 5 mM MgCl2 and 100 mM NaCl. The C-4 carbonyl of uridine as a determinant of specific binding to NS5A. As a part of our evaluation of the optimal solution conditions for studying NS5A-RNA interaction, we evaluated the pH dependence of the binding reaction. For this experiment, we used a filter-binding assay (10) to avoid problems with interpretation that could be caused by pH-dependent changes in fluorescein fluorescence. Formation of the NS5A-rU15 complex was dependent on an interaction(s) that exhibited a pKa value of 9.5 (Fig. 3A). Using the fluorescence polarization assay and binding to FL-rU15, we showed that the
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FIG. 3. NS5A-specific RNA binding requires hydrogen bonding. (A) RNA filter binding with ␥-32P-labeled rU15 and NS5A in MTCN buffer at various pHs. The percentage of bound RNA was plotted against pH. The pKa of NS5A rU15 binding was 9.5. The solid line is a fit of experimental data to a hyperbola (equation 1). (B and C) Fluorescence polarization assays were performed. The ⌬mP of fluorescein-labeled rU15 (B) and rA15 (C) was monitored as a function of NS5A concentration at pH 7.5 (F) or 9.5 (E). (B) The Kd values for rU15 at pH 7.5 (F) and 9.5 (E) were 50 ⫾ 5 nM and 1,000 ⫾ 200 nM, respectively. (C) The Kd values for rA15 at pH 7.5 (F) and 9.5 (E) were 300 ⫾ 50 nM and 1,000 ⫾ 200 nM, respectively. Solid lines are fits of experimental data to a hyperbola (equation 2). (D) Proton acceptance and donation of uridine have equilibrium at pKa ⫽ 9.2, similar to the pKa observed in panel A, suggesting that uracil plays a key role in NS5A RNA binding. (E) Proton donation of guanidine has equilibrium at pKa ⫽ 9.2, suggesting that guanine may confer similar properties to binding specificity.
value for the Kd increased from 50 nM at pH 7.5 to 1,000 nM at pH 9.5 (Fig. 3B). It was possible that the observed pH dependence was caused by lysine residues of the protein and/or the imino group of the uracil base. In order to distinguish between these possibilities, we evaluated the pH dependence of rA15 binding to NS5A. The only titratable proton on the adenine ring has a pKa of 3.5. Therefore, the pH dependence will be lost if caused by the uracil base but will be retained if caused by a lysine(s) of NS5A. Formation of the NS5A-rA15 complex was not as pH dependent, as only a 3-fold increase in the value of the Kd was observed in going from pH 7.5 to pH 9.5 (Fig. 3C). These data suggest that protonated N-3 imino group, the C-2 carbonyl group, and/or the C-4 carbonyl group contributes to interactions that confer not only the observed pH dependence of the binding (Fig. 3D) but also the specificity of the binding. N-1 and C-6 of the guanine base correspond to N-3 and C-4 of uracil, respectively, and may also confer the same properties to rG15 (Fig. 3E). In order to begin to define the atoms of the uracil base that interact with NS5A, we synthesized 15-nucleotide (nt) RNAs that contained a modified uracil base at positions 4, 8, and 12 of the RNA (Fig. 4A). The 4-thiouridine (4SU) modification changes the oxygen atom of the C-4 carbonyl group to a sulfur atom. The 5-bromouridine (5BrU) and 5-iodouridine (5IU) modifications serve as controls to distinguish C-4 position effects from changes in the overall size and/or electronics of the ring. The relative affinity of NS5A for these RNAs was deter-
mined by using the fluorescence polarization assay in a competitive-binding format. The 4SU-RNA was less efficient at competition than U-RNA (Fig. 4B). In contrast, both the 5BrU- and 5IU-RNAs were essentially equivalent to U-RNA (Fig. 4C). The data in Fig. 4B and C were fit to determine the RNA concentrations that yield 50% inhibition of FL-rU15 binding (Fig. 4D). Changing 3 of the 15 uridines of the RNA to 4-thiouridine weakened the binding of this RNA by an order of magnitude relative to U-RNA. We conclude that the C-4 carbonyl of the uracil base interacts with NS5A and contributes to the specificity of RNA binding. A comparable series of experiments was not possible with rG15 or related RNA sequences because of the propensity of G-rich RNA to form quadruplexes under physiological solution conditions such as those employed here (14). Substituting four guanosine residues into the context of uridine oligonucleotides also leads to the formation of intra- and intermolecular base pairing, thus precluding analysis of NS5A RNA-binding specificity in this context, as NS5A binds only single-stranded RNA (10). A 5- to 6-nt RNA site size for NS5A. Assessment of the number of specific NS5A-binding sites in viral or even cellular RNAs requires knowledge of the size of the RNA-binding site. In addition, studies of the molecular basis for the specificity of RNA binding can be simplified by using a minimal-length RNA. In order to determine the size of the RNA-binding site of NS5A, we used the filter-binding assay. A fluorescein molecule on a “long” RNA will not interact with NS5A. However,
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FIG. 4. Position 4 in uracil is involved in NS5A-specific RNA binding. (A) The molecular structures of base moieties and sequences of RNAs that were used in this study. Three substitutions were evenly distributed in rU15 (4SU, 5BrU, and 5IU). (B and C) NS5A RNA-binding competition assay. FL-rU15 (0.1 nM) competes NS5A binding with competitor RNA U (F) and 4SU (E) (B) and 5IU (f) and 5BrU (E) (C). ⌬mP was obtained by fluorescence polarization assay. (D) IC50s were obtained from panels B and C.
as the length of the RNA approaches the minimal length, the fluorescein molecule could interact with protein, modulating the affinity and/or fluorescence emission. The filter-binding assay does not suffer from this potentially confounding factor. As the length of the rU RNA was reduced from 15 nt to 10 nt and 5 nt, the observed value for the Kd increased from 50 nM to 100 nM (Fig. 5A). However, in going from rU5 to rU4, the observed value for the Kd increased to 600 nM (Fig. 5B). The error in the measurement of the value of the Kd for rU4 approached 40%. It was possible that rU4 was not efficiently retained on the nitrocellulose membrane during the washing step. Therefore, we turned to the fluorescence polarization assay in a competitive-binding format. Binding of FL-rU15 to NS5A was performed in the presence of a constant concentration of competitor RNA, and the remaining millipolarization (fraction of FL-rU15 bound in the presence of competitor) was determined. This experiment showed an apparent increase in the affinity of NS5A for rU6 relative to rU5 (Fig. 5C). Substantial differences in affinity were not observed for rU7 to rU10 relative to rU5 (Fig. 5C). We conclude that the RNAbinding site of NS5A accommodates 5 or 6 nucleotides. “Domain I-plus” (residues 2005 to 2221) as the minimal RNA-binding domain of NS5A. We used a series of deletion mutants (Fig. 6A) and an NS5A capture assay (10) to identify the minimal RNA-binding domain of NS5A. The purified de-
rivatives are shown in Fig. 6B. Biotin was attached to the 3⬘ end of an RNA oligonucleotide that contained rU7, a near-minimal-length, high-affinity NS5A-binding site, at the 5⬘ end and an rC4 spacer to which NS5A should not efficiently bind. This RNA is referred to as rU7rC4-Bio-RNA. In the absence of RNA, streptavidin did not capture NS5A (Fig. 6C, lane 1). The use of a biotinylated and essentially random-sequence DNA also failed to capture NS5A with streptavidin (Fig. 6C, lane 2). These data suggest that any capture of NS5A using this assay is mediated by rU7rC4-Bio-RNA. As expected, the amino-terminal, amphipathic helix was not essential for RNA binding (Fig. 6C, lane 3) (5). However, deletion of domain I abrogated RNA binding (Fig. 6C, lane 9). The efficiency of domain I capture was reproducibly much lower than that observed for the “full-length” derivatives (Fig. 6C, lane 8, and data not shown). Addition of the linker region containing cluster I to domain I restored binding to levels on par with full-length NS5A (Fig. 6C, lane 7). No further change in RNA binding was observed as the remainder of the carboxyterminal portion of NS5A was added back (Fig. 6C, lanes 5 and 6). We conclude that the minimal RNA domain of NS5A resides in residues 33 to 250 of the protein. This region contains domain I and the carboxy-terminal linker. We refer to this minimal NS5A derivative as “domain I-plus.” We compared the RNA-binding activity of domain I (resi-
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FIG. 5. The site size of oligo(U) binding is 5 nt. (A) Direct filterbinding assay of NS5A and rU5 (E), rU10 (f), or rU15 (F). Kd values for rU5, rU10, and rU15 binding were 100 ⫾ 20 nM, 100 ⫾ 10 nM, and 50 ⫾ 5 nM, respectively. Solid lines are fits of experimental data to a hyperbola (equation 1). (B) Direct filter-binding assay of NS5A and rU4 (F) or rU5 (f). rU4 and rU5 exhibited a 6-fold difference in binding affinity. The Kd value for rU4 was 600 ⫾ 200 nM, while the Kd value for rU5 was 100 ⫾ 20 nM. Solid lines are fits of experimental data to a hyperbola (equation 1). (C) Fluorescence polarization competition assay. NS5A was incubated with FL-rU15, and competitor RNAs were added. The competitors reduced NS5A–FL-rU15 binding to 83.2%, 56.5%, 25.8%, 32.6%, 14.9%, 25.5%, 15.1%, and 5.5% for rU4, rU5, rU6, rU7, rU8, rU9, rU10, and rU15, respectively, suggesting that five or six uridines are necessary for NS5A RNA-binding activity.
dues 2005 to 2187) to that of domain I-plus (residues 2005 to 2221) directly by using the fluorescence polarization assay. The outcome of this experiment was consistent with the NS5A capture experiment. The observed value of the Kd for domain I-plus was equivalent to that for the full-length protein (Fig. 7) and at least 8-fold higher than that measured for domain I (Fig. 7). The endpoint of the truncated derivative is lower than that observed for full-length NS5A (Fig. 3D) because of the reduced mass of the complex. Unlike the full-length derivatives, domains I and I-plus did not hold on to the Zn2⫹ ions well. Long-term storage of these proteins often led to inactivation of the RNA-binding activity. These complications were alleviated by storage in the presence of phosphine (500 M) and Zn2⫹ (100 M). RNA-induced and/or stabilized NS5A dimers. Although the first crystal structure of domain I of NS5A showed a dimer with
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a channel of positive electrostatic potential (Fig. 1B) (27), NS5A dimers have not been observed in solution by us (data not shown) or others (27). Development of structure-based hypotheses for the NS5A-RNA complex would benefit from knowledge of the form of NS5A (monomer or dimer) to which RNA binds. Our inability to resolve dimers in solution by methods such as gel filtration could reflect the transient nature of the dimer. We used glutaraldehyde cross-linking to trap dimers in solution in the absence of RNA. Dimeric forms of both full-length and domain I-plus NS5A were observed only in the presence of glutaraldehyde (compare lanes 1 to 4 to lanes 5 to 8 in Fig. 8A). The identity of the cross-linked species was confirmed by Western blotting (data not shown). Under the conditions employed, the presence of RNA did not appear to have an impact on the level of the NS5A dimer (compare lane 1 to lane 3 or lane 2 to lane 4 in Fig. 8A). We pursued a more systematic, sensitive, quantitative analysis of the cross-linking experiment using domain I-plus. We observed a time (data not shown) and glutaraldehyde concentration dependence to the cross-linking (⫺ RNA in Fig. 8B). Dimer was present in the absence of glutaraldehyde, which may have been caused by oxidation of two cysteines to an intermolecular cystine during sample preparation for electrophoresis. As the concentration of glutaraldehyde was increased, there was also an increase in the amount of dimer observed, although the fraction of protein that was cross-linked never exceeded 50% (Fig. 8B). Under these conditions, U-rich RNA stimulated formation of the dimeric species (⫹ rU15 in Fig. 8B and C), but nonspecific RNA did not (⫹ rA15 in Fig. 8B and C). When the U-rich RNA was used, higher-order oligomers were also observed (⫹ rU15 in Fig. 8B). These data are consistent with NS5A existing in an equilibrium between monomer and dimer. RNA shifts this equilibrium in favor of dimer, consistent with the dimer being the form of the protein to which RNA binds. As a control for cross-linking specificity, we performed the same experiments described above with the domain II-III protein. Dimer was not observed in the absence or presence of any RNA (Fig. 8D). Identification of sites on NS5A in (in)direct contact with U-rich RNA by using mass spectrometry. In order to test the predictions of our model for the minimal determinants of the NS5A-RNA interaction in the context of “full-length” NS5A (construct b in Fig. 6A) and “long” RNA, we used the reversible cross-linking and peptide fingerprinting (RCAP) described previously for elucidation of sites on NS5B that interact with RNA (13). Briefly, the RCAP method uses the reversible bifunctional cross-linker formaldehyde to form protein-RNA cross-links, followed by proteolysis of the protein with trypsin. The RNA is then purified along with covalently attached peptides. After reversal of the cross-links with heat, the peptides are subjected to mass spectrometry and the results are compared to those for the peptides generated from a theoretical digest of the protein. For proteins with known structures, the peptides can be immediately mapped back to the three-dimensional (3D) model of the protein (3, 21, 30). Recombinant NS5A protein was subjected to the RCAP assay, and clear signals were observed in the mass spectra compared to control reactions (Fig. 9A). Nearly identical sig-
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FIG. 6. The optimal RNA-binding domain of NS5A is residues 2005 to 2221. (A) Deletion mutants of NS5A were generated to determine the RNA-binding domain (b to g). Removal of the ␣-helix structure in the N terminus (a) increased NS5A solubility. Conserved clusters of serine residues are indicated as PI, PII, and PIII. (B) SDS-PAGE analysis of the input NS5A deletion mutants on a 12.5% gel stained with Sypro Ruby. (C) Biotinylated rU7rC4 pulldown assay. In the absence of RNA or in the presence of a nonspecific biotinylated DNA oligonucleotide, NS5A deletion mutant b was not pulled down (lanes 2 and 3, respectively). In the presence of rU7rC4-Biotin, mutants a to f were pulled down (lanes 3 to 8), but mutant g was not (lane 9). The f derivative (domain I) showed less binding than the e derivative (domain I-plus) (compare lanes 7 and 8), suggesting that residues in PI enhance RNA-binding activity.
nals were observed with a preparation of the NS5A protein containing a different affinity purification tag, indicating that the histidine tag did not affect the results. Several of the peptides identified contained one or more arginines or lysines that were not cleaved by trypsin, as would be expected should the RNA prevent cleavage through steric hindrance or through covalent attachment to NS5A (Fig. 9B). The results can thus be interpreted in terms of NS5A-RNA contact. The peptides were distributed along domains I and II of NS5A and the intervening sequences between the three domains (see the schematic associated with Fig. 9B). Within domain II, residues 263 to 311 contain a region that contacts the poly(U) RNA. No peptides from domain III were discov-
ered. Notably, the linker region between domains I and II cross-linked to RNA. The cross-linked peptides were mapped on the two structural models for the NS5A dimer (white surfaces in Fig. 9C and D). The location of the carboxy terminus of each subunit is indicated in red. Cross-linkable peptides lined the positively charged cleft of the dimer reported by Tellinghuisen et al. (27) (Fig. 9C). However, the positively charged surface on the back of the protein did not cross-link to the RNA, consistent with the cross-linking being mediated by interactions other than ionic interactions of a nonspecific nature (Fig. 9C). In this dimer, the linker and domain II would be on the same face as the RNA (Fig. 9C). In addition, several cross-linked peptides (those that contain amino acids [aa] 42 to 45, 109 to 110, 120 to 123, and 164 to 170) have side chains exposed within the concave inner surface of the dimer, suggesting that the RNA could contact both the inner surface and the outer surface of the dimer (Fig. 9C). Cross-linked peptides formed a ribbon around the alternative NS5A dimer that is completely symmetrical (Fig. 9D). One might imagine that for this conformation to function in RNA binding, the RNA would lie atop the cross-linked residues. In this conformation, the carboxyl terminus is remote from the site of cross-linking (Fig. 9D).
FIG. 7. Binding of NS5A to RNA is enhanced by residues in cluster I. Kd values for 2005-2221 NS5A (domain I-plus) and 2005-2187 NS5A (domain I) were 100 ⫾ 5 nM and 800 ⫾ 50 nM, respectively. Solid lines are fits of experimental data to a hyperbola equation (equation 2).
DISCUSSION This study was initiated to define the physical/chemical properties driving the high-affinity, sequence-specific interaction of
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FIG. 8. Dimerization of NS5A. (A) NS5A or domain I-plus was incubated in the absence or presence of RNA (rU7) with or without 1 mM glutaraldehyde treatment. Samples were resolved on a 4 to 20% gradient SDS-polyacrylamide gel and stained with Sypro Ruby stain. The asterisks indicate dimerization (lanes 1 to 4). (B) Domain I-plus was incubated in the absence (⫺ RNA) or presence (⫹ rU15 and ⫹ rA15) of RNA and treated with glutaraldehyde to a 0.1 to 1 mM final concentration. The presence of specific RNA (rU15) promoted dimerization of domain I-plus, whereas nonspecific RNA did not. (C) Quantification of the amounts of domain I-plus monomer and dimer at 0.5 mM glutaraldehyde observed in panel B. The quantitation derives from at least three independent trials, with the error bars indicating the standard deviation. (D) Domain II-III does not dimerize in the absence or presence of RNA (performed under the same conditions as for panel B).
NS5A with RNA and to elucidate the minimal requirements thereof. NS5A appears to exist in equilibrium between monomer and dimer, with the monomer being favored (Fig. 8). Binding of “specific” (G/U) RNA shifts the equilibrium in favor of dimer; binding of other RNAs does not (Fig. 8). Residues 2005 to 2221 of NS5A are sufficient for specific RNA binding (Fig. 6). This minimal RNA binding encompasses domain I (residues 2005 to 2187) and the carboxy-terminal linker region (residues 2188 to 2221) (Fig. 1). We refer to this minimal RNA-binding domain of NS5A as domain I-plus. Mass spectrometry data support domain I-plus as the minimal RNAbinding domain but also demonstrate that domain II is also capable of interacting with RNA as well (Fig. 9). Domain II, and perhaps even domain III, could contribute to binding of long RNAs. A recent report from the Harris laboratory has shown that all three domains of NS5A can bind to RNA (6). RNA binding requires Zn2⫹, as EDTA is inhibitory (Fig. 2A). Uridine oligonucleotides 5 to 6 nt in length are sufficient for high-affinity binding to NS5A (Fig. 5). As the length of the RNA increases, the affinity does not change and cooperative binding is not observed (Fig. 5), despite the presence of multiple NS5A dimers on “long” RNA (Fig. 8). G/U RNA is recognized, in part, by the keto group at position 4 of the uracil base, with the equivalent keto moiety at position 6 of the guanine base (Fig. 3 and 4). It is possible that the imino groups of uracil (position 3) and guanine (position 1) bases also contribute to G/U RNA recognition. Interaction with these positions of the G/U bases would explain the observed pH dependence of the binding (Fig. 3) and the sensitivity of the binding to substitution of O-4 atom of uracil with a sulfur atom (Fig. 4).
The use of hydrogen bonding to augment specific binding is consistent with the robustness of specific RNA binding in the presence of salt (Fig. 2C). Stimulation of RNA binding by Mg2⫹ (Fig. 2B) suggests that interaction with the phosphodiester backbone may be indirect. Structural studies of NS5A have revealed the existence of dimers (17, 27). The fact that two different structures yielded two different dimers leads to the question of whether either or both dimers have function. To date, there are no biochemical or biophysical data to corroborate the structure. The ability to trap dimers by glutaraldehyde cross-linking (Fig. 8) represents the first support for the existence of a dimer in solution. The need to use cross-linking may reflect a high equilibrium dissociation constant for the dimer. The high concentration of protein in the crystal could compensate for a low-affinity dimer. This initial cross-linking study was not able to provide any insight into which of the two dimers occurs in solution. However, identification of a cross-linked peptide(s) by mass spectrometry should provide some insight into the interactions responsible for dimerization in solution. Of the two NS5A dimer structures, that from the Rice laboratory is most consistent with our biochemical data (27). The Rice NS5A dimer has a cleft of positive electrostatic potential that is 33 Å long and, minimally, 16 Å wide (Fig. 1B). In addition to basic amino acid residues, the cleft contains aromatic amino acids and residues capable of hydrogen bonding to the keto and imino groups of the uracil and guanine bases. Peptides capable of cross-linking to RNA decorate the ridges surrounding this cleft (Fig. 9C). There is also a positively charged surface on the back of this dimer; however, this sur-
FIG. 9. Interaction of NS5A with RNA evaluated by RNA cross-linking and mass spectrometry. (A) Sample mass spectra used to assign the peptides derived from NS5A. Left, spectrum from a mock cross-linking reaction; right, peaks observed in a reaction where NS5A was cross-linked to the poly(U)-agarose. The reactions were performed as described in Materials and Methods. The ions were resolved with a Bruker Autoflex III MALDI-TOF mass spectrometer set in reflectron mode. All ions that are more than 150% above the comparable one in the background that contained the proper isotope ratios were used for analysis. (B) Summary of the most prominent ions observed in the mass spectra containing NS5A peptides reversibly cross-linked to the poly(U)-agarose. Only the peptides whose masses are within 1 Da of a peptide from a theoretical digest are shown. In addition, all peptides were analyzed for proper natural abundance of carbon isotope. Obs, observed molecular mass, in daltons; Theor, expected masses of peptide fragments generated by a theoretical digestion with trypsin; AA #, amino acid number of the NS5A protein. The peptide sequences are shown in the standard one-letter code. Where a cleavage was missed, the cognate lysine or arginine N terminal to the expected scissile bond is underlined. Peptides that overlap due to one or more missed cleavage events are aligned and shaded. The rightmost schematic shows the domains from NS5A from which the peptides were derived. Domain III did not contribute any peptides to the RCAP analysis and is left incomplete at the bottom of the schematic. (C and D) Locations of the RNA-cross-linked peptides mapped onto the dimers of the NS5A crystal structures. Panel C corresponds to PDB code 1Zh1, which consists of residues 36 to 198 (27), and panel D corresponds to PDB code 3FQM, which consists of residues 32 to 191 (17). The dimers are displayed as surface (black) with the peptides identified in domain I as listed in panel B highlighted in white. Note that peptides 221 to 240 and 241 to 2466 are missing from the crystal structures. The C termini are highlighted in red to indicate the positions of residues missing from the structures. Both dimers are shown in two views, related by 180° rotation around the y axis. 12489
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face does not appear to contribute to additional or nonspecific interactions of NS5A with RNA, as peptides from this surface could not be cross-linked to RNA (Fig. 9C). This surface could permit stacking of NS5A dimers, which may give rise to the oligomers observed in the glutaraldehyde cross-linking experiment (Fig. 8). The reason for the requirement of NS5A residues 2188 to 2221 for high-affinity G/U RNA binding (Fig. 7) is not clear; however, this linker region clearly cross-links to RNA (Fig. 9B). This region of the protein was not in the derivative used for structural studies (17, 27) and is not thought to have defined structure (26). However, this region contains serine residues that are known to be required for production of the p58 form of NS5A (1, 20). In addition, mutations that adapt HCV replicons for persistent replication in cell culture map to this region of the protein, although the S2204I substitution does not appear to alter the RNA-binding activity of the protein (10). The use of the keto and imino moieties of the uracil and guanine bases for the G/U-specific interaction of NS5A with RNA (Fig. 3 and 4) is consistent with information available for other GU-rich RNA-binding proteins (19). Essentially all of the known GU-rich RNA-binding proteins use the well-described RNA recognition motif (RRM) for binding (28). HCV has selected a novel fold instead of the RRM to bind RNA. The reason for this departure from the more conventional mechanism is not clear, but it suggests that cellular G/U RNAbinding proteins will not be off targets for inhibitors of the G/U RNA-binding activity of NS5A. Very effective compounds targeting domain I of NS5A have recently been reported (7, 15). The mechanism of action of these compounds is not clear. All of the inhibitors reported harbor keto and imino groups. It will be interesting to compare the binding of these compounds to NS5A to that of RNA. There are very few G/U RNA sequences of greater than 5 nt present in established or putative cis-acting elements of HCV plus- or minus-strand RNA (10). Those that do exist appear to be placed strategically to possibly regulate translation or replication (10). It is now known that the poly(rU) tract in the 3⬘ nontranslated region (NTR) of plus-strand RNA is recognized by RIG-I for activation of the innate immune response (23), by the cellular LSm1 to -7 proteins for translation (24), and by viral and/or cellular factors for RNA replication. It is worth noting that interruption of a 26-nt poly(rU) tract by introduction of a cytidylate or adenylate residue every 4 to 6 nt substantially impairs replication of HCV RNA (31). These data are very consistent with outcomes expected for the poly(rU) tract if NS5A targets these sites. The ability of NS5A to target the poly(rU) tract in infected cells could antagonize RIG-I activation, as well as coordinate the switch from translation to genome replication. GU-rich elements (GREs) are now known to function in posttranscriptional regulation of gene expression in mammalian cells (19). GREs reduce the half-life of cellular mRNA (29). Reduced stability is achieved by virtue of CUGBP1 binding to the GRE and recruiting the deadenylase, leading to loss of the poly(rA) tail and degradation by the exosome (29). Cellular mRNAs regulated by GREs are involved in a variety of pathways, including cell growth, cell motility, and apoptosis
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(22). Binding of NS5A to GREs has the potential to alter the regulation of these pathways. The roles of the NS5A-RNA interaction in the HCV life cycle and the interaction of HCV with the cell clearly merit additional study. Inhibitors of NS5A G/U RNA binding could interfere with multiple steps in the HCV life cycle, stimulate the innate immune response to HCV infection, and restore other aspects of normal cellular function, akin to the case for NS3 protease inhibitors. The studies described here should assist in the development of directed approaches for the discovery of such inhibitors. ACKNOWLEDGMENTS We thank Ibrahim Moustafa for contributing to the preparation of Fig. 1B and 9C and D. Y. Chen for providing some preparations of purified NS5A proteins for the RCAP assay, and Jamie Arnold and Eric Smidansky for comments on the manuscript. This work was supported in part by grant GM089001 to C.E.C. and K.D.R. from NIGMS/NIH and grants AI075015 and AI073335 to C.C.K. from NIAID/NIH. REFERENCES 1. Appel, N., T. Pietschmann, and R. Bartenschlager. 2005. Mutational analysis of hepatitis C virus nonstructural protein 5A: potential role of differential phosphorylation in RNA replication and identification of a genetically flexible domain. J. Virol. 79:3187–3194. 2. Arnold, J. J., A. Bernal, U. Uche, D. E. Sterner, T. R. Butt, C. E. Cameron, and M. R. Mattern. 2006. Small ubiquitin-like modifying protein isopeptidase assay based on poliovirus RNA polymerase activity. Anal. Biochem. 350:214–221. 3. Bhardwaj, K., S. Palaninathan, J. M. Alcantara, L. L. Yi, L. Guarino, J. C. Sacchettini, and C. C. Kao. 2008. Structural and functional analyses of the severe acute respiratory syndrome coronavirus endoribonuclease Nsp15. J. Biol. Chem. 283:3655–3664. 4. Blight, K. J., A. A. Kolykhalov, and C. M. Rice. 2000. Efficient initiation of HCV RNA replication in cell culture. Science 290:1972–1974. 5. Brass, V., E. Bieck, R. Montserret, B. Wolk, J. A. Hellings, H. E. Blum, F. Penin, and D. Moradpour. 2002. An amino-terminal amphipathic alphahelix mediates membrane association of the hepatitis C virus nonstructural protein 5A. J. Biol. Chem. 277:8130–8139. 6. Foster, T. L., T. Belyaeva, N. J. Stonehouse, A. R. Pearson, and M. Harris. 2010. All three domains of the hepatitis C virus nonstructural NS5A protein contribute to RNA binding. J. Virol. 84:9267–9277. 7. Gao, M., R. E. Nettles, M. Belema, L. B. Snyder, V. N. Nguyen, R. A. Fridell, M. H. Serrano-Wu, D. R. Langley, J. H. Sun, D. R. O’Boyle II, J. A. Lemm, C. Wang, J. O. Knipe, C. Chien, R. J. Colonno, D. M. Grasela, N. A. Meanwell, and L. G. Hamann. 2010. Chemical genetics strategy identifies an HCV NS5A inhibitor with a potent clinical effect. Nature 465:96–100. 8. Hanoulle, X., A. Badillo, D. Verdegem, F. Penin, and G. Lippens. 2010. The domain 2 of the HCV NS5A protein is intrinsically unstructured. Protein Pept Lett. 17:1012–1018. 9. Hanoulle, X., D. Verdegem, A. Badillo, J. M. Wieruszeski, F. Penin, and G. Lippens. 2009. Domain 3 of non-structural protein 5A from hepatitis C virus is natively unfolded. Biochem. Biophys. Res. Commun. 381:634–638. 10. Huang, L., J. Hwang, S. D. Sharma, M. R. Hargittai, Y. Chen, J. J. Arnold, K. D. Raney, and C. E. Cameron. 2005. Hepatitis C virus nonstructural protein 5A (NS5A) is an RNA-binding protein. J. Biol. Chem. 280:36417– 36428. 11. Huang, L., E. V. Sineva, M. R. Hargittai, S. D. Sharma, M. Suthar, K. D. Raney, and C. E. Cameron. 2004. Purification and characterization of hepatitis C virus non-structural protein 5A expressed in Escherichia coli. Protein Expr. Purif. 37:144–153. 12. Kaneko, T., Y. Tanji, S. Satoh, M. Hijikata, S. Asabe, K. Kimura, and K. Shimotohno. 1994. Production of two phosphoproteins from the NS5A region of the hepatitis C viral genome. Biochem. Biophys. Res. Commun. 205:320–326. 13. Kim, Y. C., W. K. Russell, C. T. Ranjith-Kumar, M. Thomson, D. H. Russell, and C. C. Kao. 2005. Functional analysis of RNA binding by the hepatitis C virus RNA-dependent RNA polymerase. J. Biol. Chem. 280:38011–38019. 14. Lane, A. N., J. B. Chaires, R. D. Gray, and J. O. Trent. 2008. Stability and kinetics of G-quadruplex structures. Nucleic Acids Res. 36:5482–5515. 15. Lemm, J. A., D. O’Boyle II, M. Liu, P. T. Nower, R. Colonno, M. S. Deshpande, L. B. Snyder, S. W. Martin, D. R. St Laurent, M. H. Serrano-Wu, J. L. Romine, N. A. Meanwell, and M. Gao. 2010. Identification of hepatitis C virus NS5A inhibitors. J. Virol. 84:482–491.
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