Characterization of RNA Determinants Recognized by the Arginine- and Proline-Rich Region of Us11, a Herpes Simplex Virus Type 1-Encoded Double-Stranded RNA Binding Protein That Prevents PKR Activation

The herpes simplex virus Us11 gene product inhibits activationof the cellular PKR kinase and associates with a limited numberof unrelated viral and cellular RNA molecules via a carboxyl-terminal68-amino-acid segment rich in arginine and proline. To characterizethe determinants underlying the recognition of an RNA targetby Us11, we employed an in vitro selection technique to isolateRNA ligands that bind Us11 with high affinity from a populationof molecules containing an internal randomized segment. Bindingof Us11 to these RNA ligands is specific and appears to occurpreferentially on conformational isoforms that possess a higher-orderstructure. While the addition of unlabeled poly(I · C)reduced binding of Us11 to a selected radiolabeled RNA, single-strandedhomopolymers were not effective competitors. Us11 directly associateswith poly(I · C), and inclusion of an unlabeled selectedRNA in the reaction reduces poly(I · C) binding, whilesingle-stranded RNA homopolymers have no effect. Finally, Us11binds to defined, double-stranded RNA (dsRNA) molecules thatexhibit greater sequence complexity. Binding to these dsRNAperfect duplexes displays a striking dependence on length, as39-bp or shorter duplexes do not bind efficiently. Furthermore,this interaction is specific for dsRNA as opposed to dsDNA,implying that the Us11 RNA binding domain can distinguish nucleicacid duplexes containing 2' hydroxyl groups from those thatdo not. These results establish that Us11 is a dsRNA bindingprotein. The arginine- and proline-rich Us11 RNA binding domainis unrelated to known dsRNA binding elements and thus constitutesa unique recognition motif that interacts with dsRNA. The abilityof Us11 to bind dsRNA may be important for inhibiting activationof the cellular PKR kinase in response to dsRNA.

INTRODUCTION

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Introduction

Double-stranded (ds) or highly structured RNAs can be potentsignaling molecules in eukaryotic cells (reviewed in references16, 27, and 56). Indeed, it is believed that a major componentinvolved in triggering the cellular antiviral response in infectedcells is the accumulation of viral dsRNA (reviewed in references27 and 36). This large fraction of dsRNA is thought to originateeither as a by-product of the replication of RNA viruses orfrom the transcription of large DNA virus genomes in which manyopen reading frames are positioned on opposite DNA strands.The accumulation of dsRNA activates a battery of cellular enzymesimportant for establishing the antiviral state. One of theseenzymes is the cellular PKR kinase. Upon activation by dsRNAor protein effectors, PKR dimerizes, and each subunit of thedimer phosphorylates the other. The phosphorylated, activatedenzymes then can phosphorylate other substrates in trans, notablyIKB and the alpha subunit of the translation initiation factoreIF2 (reviewed in reference 25). As phosphorylated eIF2 is unableto promote the initiation of protein synthesis, PKR activationby dsRNA could potentially prevent the production of polypeptidesnecessary for viral replication and effectively arrest the virallife cycle prior to its completion, thus containing the infection.To disarm the cellular response to dsRNA, many viruses encodefunctions that inhibit PKR activation (reviewed in reference36). Adenovirus synthesizes copious amounts of VA RNAs, oneof which binds PKR and prevents activation of the enzyme (reviewedin reference 36). Other viruses, such as influenza virus, vacciniavirus, and reovirus, encode proteins that prevent PKR activationand bind dsRNA (reviewed in references 22, 48, and 49). Theinteraction of these viral polypeptides with dsRNA is thoughtto sequester the dsRNA activator, preventing PKR activationand eIF2 ${alpha}$ phosphorylation.

Recently, multiple herpes simplex virus (HSV) gene productshave been demonstrated to regulate eIF2 ${alpha}$ phosphorylation (6,20, 31, 32). The ${gamma}$ 34.5 protein is an important virulence factorthat binds the cellular PP1 ${alpha}$ and mediates the dephosphorylationof phospho-eIF2 (9, 20). This overcomes the effect of PKR activationon translation by maintaining pools of active, unphosphorylatedeIF2 ${alpha}$ . In the absence of the viral ${gamma}$ 34.5 protein, PKR is activated,phosphorylated eIF2 ${alpha}$ accumulates, and late viral protein synthesisis blocked, precluding the completion of the viral life cycle(8, 10). Genetic studies have demonstrated that expression ofthe Us11 protein, normally produced late in the viral life cycle,early in infection prevents the premature cessation of proteinsynthesis observed in cells infected with ${gamma}$ 34.5 mutants (31,32). Furthermore, biochemical analysis has shown that Us11 canprevent the activation of PKR (6, 32, 37). Us11 is found innucleoli and the cytosol of infected cells and is also packagedin the viral particle (43). A basic 68-amino-acid fragment fromthe carboxy terminus of the 21-kDa Us11 polypeptide associateswith 60S ribosomal subunits and possesses an RNA binding activity(43, 46). The only RNA targets known to interact with Us11 arean in vitro-transcribed RNA complementary to the Us11 open readingframe; the elements responsive to the transactivators Rev andRex encoded by human immunodeficiency virus and human T-cellleukemia virus; 60S rRNA; a segment derived from a region commonto the HSV type 1 (HSV-1) UL12, -13, and -14 mRNAs; and a truncatedversion of the UL34 mRNA (1, 12, 40-42). The determinants governingthe recognition of this diverse assortment of sequences by theUs11 polypeptide have not yet been expounded. Importantly, theUs11 RNA binding domain is also required to prevent PKR activation,possibly implicating RNA binding in this process (37).

The carboxyl-terminal 68 amino acids of Us11 contain 21 copiesof the triplet Arg-X-Pro, where X is preferentially an unchargedpolar or acidic amino acid. This repetitive motif is thoughtto fold into a type II poly-L-prolyl alpha helix (43, 46). Thearginine- and proline-rich Us11 RNA binding domain does notexhibit any homology to known RNA binding motifs, although alpha-helicalregions in other RNA binding proteins are known to be importantfor RNA binding. To gain insight into the types of RNA moleculesthat Us11 could potentially interact with, we isolated high-affinityRNA ligands that bind to the Us11 RNA binding domain by employingan in vitro selection procedure and characterized the interactionof Us11 with a variety of RNA substrates. We conclude that Us11is a double-stranded RNA binding protein that recognizes structuredRNA through a novel arginine- and proline-rich RNA binding element.

MATERIALS AND METHODS

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Materials and Methods

Selection of high-affinity RNA ligands that bind Us11. An oligonucleotide library that contains an internal randomizedstretch of 40 nucleotides flanked by a 5' invariant region thatcontains the T7 promoter and a 3' invariant region was a giftfrom Fenyong Liu (University of California—Berkeley) andis described in reference 51. The library was amplified (25cycles of 94°C for 1 min, 47°C for 1 min, and 72°Cfor 1 min; on the final cycle, a 10-min extension was performed)with Taq DNA polymerase using the primers JH1031 and JH1052(51). Following purification of the PCR product on a 2% agarosegel, an in vitro RNA synthesis reaction was assembled with T7RNA polymerase (NEB) using buffer supplied by the manufacturersupplemented with 0.5 mM nucleoside triphosphates, 20 U of RNasin(Promega), and 30 µCi of [ ${alpha}$ ³²P]CTP (3,000 Ci/mmol). Thereaction was incubated at 37°C for 6 h; 3 U of RNase-freeDNase (Promega) was then added, and the incubation continuedfor an additional 30 min. Samples were sequentially extractedwith an equal volume of phenol followed by chloroform and precipitatedwith ethanol (EtOH) in the presence of 2 M NH₄OAc. After thepellets were washed with 70% EtOH, they were air dried and resuspendedin RNase-free distilled H₂O (dH₂O). One nanomole of RNA wasincubated with 20 nmol of purified glutathione S-transferase(GST) protein prebound to glutothione-Sepharose beads (Pharmacia)equilibrated in 1x binding buffer (20 mM HEPES-KOH [pH 7.4],50 mM NaCl, 1 mM EDTA, 5% glycerol) at 30°C for 15 min.The samples were periodically manually agitated to ensure thatthe beads did not settle. After the beads were pelleted by briefcentrifugation, RNA in the supernatant was recovered and incubatedwith 20 nmol of a purified protein that contains the Us11 RNAbinding domain fused to GST (GST ${Delta}$ 1-87) prebound to glutathione-Sepharosebeads equilibrated in 1x binding buffer. The reaction mixtureswere incubated at 30°C for 15 min and washed five timeswith binding buffer. Following the final wash, the pellets weresuspended in 20 mM Tris-HCl (pH 8.0), 100 mM NaCl, 10 mM EDTA,1% sodium dodecyl sulfate, and 300 µg of proteinase K(Boehringer)/ml at 30°C for 30 min and manually agitatedperiodically. The beads were removed by centrifugation, andthe supernatant was sequentially extracted with phenol followedby chloroform. Glycogen was added as a carrier to a final concentrationof 0.1 mg/ml, and the RNA was precipitated with the additionof 2.5 volumes of EtOH. Following a 70% EtOH wash, the pelletwas suspended in RNase-free dH₂O; 200 pmol of the JH1052 primerwas added, and the RNA was heated to 95°C to remove anysecondary structure and then quickly cooled in an ice waterbath. Avian myeloblastosis virus reverse transcriptase (Promega)(10 to 20 U) was added along with buffer supplied by the manufacturersupplemented with 0.5 mM deoxynucleoside triphosphates and 20U of RNasin. After 1 h at 42°C, the cDNA was amplified byPCR (25 cycles as described above) using JH1031 and JH1052.The PCR product was gel purified, and the procedure was repeatedwith varying amounts of RNA and Us11 fusion protein until therewas no further increase in RNA binding activity as evaluatedby an electrophoretic mobility shift assay (12 cycles in all).In the second iteration, 10 pmol of RNA was incubated with 200pmol of protein. In the third iteration, 2 pmol of RNA was incubatedwith 40 pmol of protein. In the fourth through sixth iterations,1 pmol of RNA was incubated with 2 pmol of protein. All subsequentiterations contained 1 pmol of RNA and 0.2 pmol of protein.

To subclone the final population of PCR products, blunt endswere created by treating the products with T4 DNA polymerase(NEB) according to the manufacturer's specifications. Afterthe polymerase was heat inactivated, the PCR products were digestedwith an excess of HindIII for 3 h at 37°C. The large fragmentswere purified on agarose gels and ligated into pUC19 that hadbeen previously digested with SmaI and HindIII, and the resultingplasmid molecules were transformed into Escherichia coli DH5 ${alpha}$ .DNA sequencing was performed with a Sequenase kit (U.S. Biochemicals)according to the manufacturer's instructions.

RNA binding assays. The RNA binding reaction mixtures contained 50,000 cpm of theselected sequence in 1x binding buffer supplemented with 1 mMdithiothreitol, 100 µg of E. coli RNA (Ambion)/ml, andvarious quantities of purified GST or GST ${Delta}$ 1-87. The reactionmixtures were assembled on ice and subsequently incubated at30°C for 10 min. In experiments with excess cold unlabeledcompetitor, protein was added last to the reaction mixtures.At the conclusion of the binding incubation, the reaction mixtureswere loaded onto a 5% native polyacrylamide gel (19:1 acrylamide-bisacrylamide)that had been prerun for 1 h at room temperature in 0.5x Tris-borate-EDTA(TBE). Bromophenol blue was loaded in adjacent lanes to serveas a marker to monitor the progress of the electrophoresis.The gels were fixed in 10% methanol-10% acetic acid, dried onWhatman paper, and exposed to Kodak XAR film. The gels werealso exposed in a PhosphorImager cassette (Molecular Dynamics),and the intensities of the bands were measured using the Imagequantsoftware package. The reaction mixtures for nitrocellulose filterbinding contained 1 mg of yeast RNA (Boehringer)/ml in placeof the E. coli RNA but were otherwise prepared and incubatedin an identical fashion. At the conclusion of the incubation,the reaction mixtures were applied to a manifold (Bio-Rad) containinga 0.45-µm-pore-size nitrocellulose sheet (Protran; Schleicher& Schuell) that had been preequilibrated in 1x binding buffer.Each sample was washed three times with 0.2 ml of 1x bindingbuffer. The filters were air dried and exposed to Kodak XARfilm. Radioactivity present in the excised spots was quantifiedby Cerenkov counting.

Preparation of RNA binding substrates. Plasmid clones containing selected inserts were linearized withKpnI and subsequently treated with 100 µg of proteinaseK/ml in 0.5% sodium dodecyl sulfate-1 mM EDTA for 30 min at50°C. Samples were extracted twice with phenol and oncewith chloroform, precipitated with EtOH, and resuspended inRNase-free H₂O. Ten micrograms of DNA was incubated with 50U of T7 RNA polymerase (NEB) in buffer supplied by the manufacturersupplemented with 20 U of RNasin and 0.5 mM ATP, UTP, and GTP.The reaction mixtures also contained 0.012 mM CTP and 25 µCiof [ ${alpha}$ -³²P]CTP (3,000 Ci/mmol). After 1 h at 37°C, RNase-freeDNase (Promega) was added and the incubation continued for anadditional 30 min. The reaction was extracted with phenol followedby chloroform and then precipitated with EtOH in the presenceof NH₄OAc. After the pellet was washed with 70% EtOH, the RNAwas resuspended in RNase-free dH₂O.

To isolate RNA conformational isoforms for RNA binding studies,radiolabeled RNA synthesis reaction mixtures were fractionatedon 5% native polyacrylamide gels, and the different bands wereexcised. After overnight elution at 4°C in 2 M NH₄OAc, theRNA was precipitated with EtOH, the pellet was washed with 70%EtOH, and the RNA was resuspended in RNase-free H₂O.

Large quantities of unlabeled RNA for competition experimentswere synthesized using a T7 Megashort script kit (Ambion) accordingto the manufacturer's instructions. To remove potential dsRNAcontaminants from labeled or unlabeled RNA preparations, a modifiedversion of the protocol described by Pe'ery and Mathews (35)was employed. The RNA was first gel purified on an 8% polyacrylamide-7M urea gel run in 0.5x TBE. Cold RNA was visualized under UVlight following staining with ethidium bromide. The excisedband was then layered onto a 10% native gel run in 0.5x TBE.The band removed from this nondenaturing gel was eluted overnightat 4°C in 10 mM Tris-HCl (pH 7.6)-1 mM EDTA. The RNA wasconcentrated by extraction with n-butanol, extracted with phenolfollowed by chloroform, precipitated with EtOH, and resuspendedin RNase-free H₂O.

To prepare labeled dsRNAs of different lengths, the plasmidpBSK II(+) was digested with either EagI, BamHI, EcoRI, HindIII,SalI, XhoI, HaeIII, or KpnI, and RNA products were synthesizedin vitro with T7 RNA polymerase in the presence of [ ${alpha}$ -³²P]CTP.Each RNA preparation was subsequently annealed to a 245-nucleotideRNA synthesized by T3 RNA polymerase from PvuII-digested pBSKII(+). Single-stranded regions were removed by treatment withRNase A and RNase T1, and the RNA duplexes were purified innondenaturing polyacrylamide gels as described previously (29).Each filter binding reaction contained 5,000 cpm of labeledsubstrate. For competition experiments, an 81-bp dsDNA derivedfrom the same sequence as the 81-bp RNA duplex was synthesizedby PCR and gel purified.

Poly(I · C) was 5'-end labeled with T4 polynucleotidekinase (NEB) according to the manufacturer's instructions. Unincorporatednucleotides were removed on a Biogel P30 spin column (Bio-Rad).Each filter binding reaction contained 50,000 cpm. All nucleotidepolymers for RNA binding and competition experiments were fromPharmacia.

RESULTS

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Results

Isolation of high-affinity RNA ligands that bind to Us11. To identify RNA molecules capable of binding with high affinityto the Us11 protein, we employed an in vitro selection techniquethat has been used previously to successfully isolate RNA ligandsthat bind to a variety of relatively simple and complex molecularstructures (2, 4, 11, 13, 45, 51, 55). This methodology allowedus to amplify specific RNA ligands from a combinatorial libraryconsisting of molecules with a randomized central region. Ourstrategy is outlined in Fig. 1. Briefly, a library of in vitro-synthesizedRNA containing a 40-nucleotide randomized internal region wasfirst incubated with purified GST bound to glutathione-agarosebeads to remove molecules that nonspecifically interact withthis polypeptide and the supporting solid matrix. The unboundRNAs were subsequently mixed with a purified hybrid proteincomprising the carboxyl-terminal 87 amino acids of Us11 fusedto GST (GST ${Delta}$ 1-87). This segment of Us11 contains a proline-and arginine-rich segment known to bind RNA and inhibit activationof the cellular PKR kinase. RNA molecules retained in this complexwere collected on glutathione-agarose beads and comprised thefirst cycle of enriched RNA ligands that bound to the Us11 RNAbinding domain. These RNAs were then reverse transcribed, thepopulation was amplified by PCR, and the process was repeated.After 12 iterations of this procedure, individual clones wereisolated and sequenced from the PCR-amplified population. Sequenceanalysis of 300 clones resulted in the isolation of nine uniquesequences within the randomized component (Fig. 2A). Althoughno substantial primary sequence homology was detected in theselected clones, their predicted secondary structures containedsome common features. Multiple short duplexed segments wereobserved, along with sequences predicted to remain single stranded.Notable among these was a stem-loop region followed by a CAor CCA nucleotide bulge present in the selected sequences ineight of nine different clones (Fig. 2B).

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FIG. 1. Selection scheme for isolating RNA ligands that associate with the Us11 RNA binding domain. An oligonucleotide pool that contains a randomized central region situated between a T7 promoter (T7P) and a 3' constant region (3' CR) was amplified with specific primers. Labeled RNA synthesized from this population was first incubated with purified GST protein immobilized on Sepharose beads to remove RNA molecules that interact nonspecifically with GST. Unbound RNA was then incubated with a purified protein that contains the Us11 RNA binding domain fused to GST (GST ${Delta}$ 1-87). The bound RNA was recovered and reverse transcribed with a primer homologous to the 3' CR, and the cDNA was amplified with a primer designed to regenerate an intact T7P sequence along with the 3' CR primer. This process was repeated multiple times to enrich for RNA molecules that preferentially associate with Us11. nt, nucleotide.

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FIG. 2. Sequence alignment of selected RNA ligands that bind to the Us11 RNA binding domain. (A) Sequences corresponding to the 5' invariant region primer (left) and the 3' constant region primer (right) are boxed. (B) RNA secondary structure for L12-262 predicted using the m-fold algorithm (30, 58). The selected segment, located between the two arrows, folds into a stem-loop structure followed by a CA bulge.

The ability of RNA produced from these clones to complex withthe Us11 RNA binding domain was evaluated in an electrophoreticmobility shift assay. Three representative examples are presentedin Fig. 3. In the absence of exogenous protein, all of the selectedRNAs migrate as multiple bands in native polyacrylamide gels,suggestive of alternative RNA conformations. This pattern isunaltered by the addition of 1 µg of purified GST. Additionof as little as 30 ng of purified GST ${Delta}$ 1-87 to the binding reactionmixture results in a decrease in the amount of certain unboundconformers and the appearance of retarded complexes that representbound ligand. The intensities of these retarded complexes aregreatest in reactions programmed with L12-5 RNA, intermediatein reactions containing L12-1 RNA, and weakest in the grouprepresented by L12-3. It is difficult to reconcile these variationswith distinct binding affinities, as these clones must bindwith sufficient avidity to survive 12 iterations of the selectionprocedure. The observed differences in the extent of complexformation could be attributed to variability in the formationof active conformational isoforms capable of binding Us11 amongthe different ligands. RNA prepared from molecules in the initialunselected population did not interact with Us11 to the sameextent (Fig. 3 and data not shown). The levels of binding ofGST ${Delta}$ 1-87 to the selected RNA sequences in the absence and presence(0.1 to 5 mM) of MgCl₂ were indistinguishable. In addition,the pattern of RNA conformational isoforms observed followingelectrophoresis in a nondenaturing polyacrylamide gel was unalteredin reactions assembled either in the presence or the absenceof Mg²⁺ (not shown). This is consistent with reports demonstratingthat Mg²⁺ is not required for simple Watson-Crick pairing (5,54) but can contribute to some tertiary interactions (18). Finally,purified Us11 containing an amino-terminal His tag also boundto the selected sequences, ruling out the possibility that theGST segment of the fusion protein is required for binding tothe selected ligands (not shown).

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FIG. 3. Binding of Us11 to selected RNA ligands. Labeled RNA prepared from a representative member of either the selected (L12-5, L12-1, and L12-3) or the initial unselected population was incubated without any protein (0), with 1 µg of GST, or with increasing amounts of purified GST ${Delta}$ 1-87 (left to right, 30, 100, and 300 ng; nonselected RNA, 10, 30, 100, and 300 ng). Protein-RNA complexes were resolved by electrophoresis in native polyacrylamide gels, and the fixed dried gel was exposed to Kodak XAR film. The vertical bracket indicates the region containing protein-RNA complexes.

To further address the specificity of ligand binding, competitionexperiments were performed with a labeled RNA ligand at a singleconcentration of GST ${Delta}$ 1-87 protein in the presence of excessunlabeled specific or nonspecific challenger RNA molecules.This RNA was purified through a series of native and denaturinggels to remove any potential small double-stranded RNA contaminantsthat might affect RNA binding (35). Figure 4A demonstrates thatthe addition of increasing amounts of unlabeled L12-5 RNA substantiallyreduces the amount of bound retarded complex while simultaneouslyincreasing the amount of some of the unbound material (f_b).Quantifying these data on a PhosphorImager revealed an 80% decreasein the bound complex at 100 pmol of L12-5 competitor along witha 2.5-fold increase in the unbound form. A fraction of the unboundRNA (f_n) is apparently unable to bind protein and is not inrapid equilibrium with the unbound form, which can produce protein-RNAcomplexes. Including 100 pmol of unlabeled RNA isolated froma member of the initial unselected population, prepared in anidentical fashion, resulted in only a 28% decrease in the amountof RNA bound by the Us11 protein. Quantitative data were obtainedby using a nitrocellulose filter binding assay along with highlypurified RNA templates. Incubation of the labeled high-affinityL12-5 RNA ligand with increasing amounts of purified GST ${Delta}$ 1-87leads to the retention of greater quantities of radioactivityon the nitrocellulose filter (not shown). At a fixed concentrationof GST ${Delta}$ 1-87 protein, addition of increasing amounts of the unlabeledspecific L12-5 competitor is more effective than identical quantitiesof a nonspecific competitor at reducing binding to the labeledprobe (Fig. 4B). Furthermore, the concentration of L12-5 ligandrequired to achieve a 50% reduction in binding to the labeledprobe is approximately 10-fold less than the concentration ofnonspecific competitor necessary to generate an equivalent reductionin binding. These results demonstrate that the RNA ligands isolatedby the in vitro selection procedure bind specifically and withhigh affinity to the Us11 RNA binding domain.

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FIG. 4. Selected RNA ligands preferentially associate with US11. (A) Purified GST ${Delta}$ 1-87 (100 ng) was incubated with labeled L12-5 RNA in the presence and absence of increasing amounts of unlabeled L12-5 RNA or RNA from the unselected initial population (left to right, 10, 30, and 100 pmol). Labeled L12-5 RNAs incubated without any protein (0) and with 1 µg of purified GST are also shown. Protein-RNA complexes were resolved as described in the legend to Fig. 2. GST ${Delta}$ 1-87 RNA-protein complexes migrate in the bracketed region labeled c. Free, unbound isoforms capable of binding Us11 are within the bracketed region labeled f_b, while nonbinding isoforms are labeled f_n. (B) Purified GST ${Delta}$ 1-87 (10 ng) was incubated with labeled L12-5 RNA in the presence or absence of increasing amounts of specific (L12-5 [ ${blacksquare}$ ] or nonspecific (RNA from initial unselected population [ ${diamondsuit}$ ]) competitor. The amount of radioactivity in each sample retained on a nitrocellulose filter was quantified. RNA binding in the absence of competitor was normalized to 1.0.

Preferential association of Us11 with specific RNA conformational isoforms. All of the RNA molecules enriched for by the in vitro selectionprocedure migrate as multiple species on native polyacrylamidegels (Fig. 5A). Furthermore, addition of the Us11 RNA bindingprotein to the reaction mixtures results in the appearance ofa defined set of multiple retarded bands following electrophoresisin native polyacrylamide gels. These bands could represent theassembly of multimeric protein structures on the RNA ligandsor characteristic mobility shifts of Us11 associating with aspecific RNA conformational isoform. To ascertain the natureof each species and to analyze the relative ability of eachspecies to bind to Us11, three unbound RNA species were purifiedon native polyacrylamide gels and designated class 1, class2, or class 3. All three of these purified species comigratedon denaturing polyacrylamide gels, indicating that they mayindeed represent different folded conformational isoforms ofa single primary sequence of defined length (Fig. 5B). In addition,the gel-purified species 1 and 3 each retained its characteristicmobility when subjected to subsequent electrophoresis on a nativepolyacrylamide gel, indicating that these structures are stableand do not regenerate a heterogenous population of conformationalisoforms following purification (Fig. 5A). While the class 2preparation does not contain significant amounts of the class1 isoform, some class 3 RNA is present. Due to the proximityof the class 2 and 3 bands in native gels, we are unable todetermine whether the class 2 preparation is contaminated withsome class 3 molecules or whether some of the purified class2 RNA is capable of adopting the mobility characteristic ofthe class 3 isoform. Figure 5A demonstrates that 100 and 300ng of purified GST ${Delta}$ 1-87 generates a heterogenous mixture ofseveral retarded complexes following incubation with the unfractionatedL12-262 ligand. Interestingly, only the class 1 and class 2preparations exhibit significant binding to the Us11 fusionprotein. The class 3 conformational isoform fails to demonstrateany detectable binding, suggesting that the class 2 RNA is responsiblefor the binding activity observed in the class 2 preparation(Fig. 5A). The retarded complexes generated by purified class1 isoforms migrate more slowly than the retarded complexes observedwith class 2 RNA. In addition, the complexes observed in theclass 1 and class 2 binding reactions appear to account forall of the retarded complexes observed in the unfractionatedstarting material. There are, however, multiple retarded bandsvisible in the class 1 and class 2 binding reactions, consistentwith the possible assembly of Us11 multimers on these templatesor with a Us11-induced structural change in the RNA ligand.Similar results have been obtained with the other selected RNAs(data not shown). Together, these observations suggest that,in addition to defined sequence determinants, there are significantconformational features of the RNA ligand that are importantfor recognition by the Us11 RNA binding domain.

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FIG. 5. Association of Us11 with RNA conformational isoforms. (A) Labeled RNA (L12-262) was fractionated by electrophoresis in native polyacrylamide gels, and three species (class 1, class 2, and class 3) were isolated. Each gel-purified RNA species, along with the unfractionated starting material (U), was incubated in the absence of added protein (0) or in the presence of either 1 µg of purified GST, 100 ng of GST ${Delta}$ 1-87, or 300 ng of GST ${Delta}$ 1-87. Protein-RNA complexes were resolved as described in the legend to Fig. 2. The arrowheads numbered 1, 2, and 3 to the left refer to the mobilities of the three isoforms. The numbers below the panel refer to the gel-purified isoform used in the binding reaction, and U represents the unfractionated starting material. (B) Each purified RNA species, along with the unfractionated starting material, was subjected to electrophoresis in a denaturing polyacrylamide gel. The fixed dried gel was subsequently exposed to Kodak XAR film.

The Us11 RNA binding domain recognizes double-stranded RNA. To address the role of potential structural and sequence elementsin RNA recognition by the Us11 RNA binding domain, we evaluatedthe abilities of a variety of synthetic RNA polymers to competewith the selected ligand for binding. In the experiment presentedin Fig. 6A, Us11 binding to the gel-purified, radiolabeled conformationalisoforms class 1 and class 2 was examined in the presence oftwo concentrations of poly(C), poly(U), poly(A), or poly(I ·C). In all cases, significant effects on RNA ligand bindingwere observed only in the presence of poly(I · C), asynthetic double-stranded RNA molecule. Poly(C), a single-strandedhomopolymer containing a nucleotide component of poly(I ·C), along with poly(U) and poly(A), had no measurable effecton RNA binding. In a nitrocellulose filter binding assay, unlabeledpoly(I · C) was at least as effective as unlabeled L12-5in reducing binding to the labeled L12-5 probe (Fig. 6B). Thesimplest explanation of this observation is that Us11 has ahigh affinity for double-stranded RNA.

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FIG. 6. Recognition of selected RNA sequences by Us11 in the presence of defined single-stranded and double-stranded RNA polymers. (A) Two species of labeled selected RNA L12-262 (left, class 1; right, class 2) were incubated with 100 ng of GST ${Delta}$ 1-87 in the absence or presence of two amounts of either poly(A), poly(U), poly(C), or poly(I · C) (from left to right, 33 and 100 µg/ml). Protein-RNA complexes were resolved as described in the legend to Fig. 2. Each RNA species was also incubated with 1 µg of GST. Unbound material from each class is indicated with an arrow. (B) Purified GST ${Delta}$ 1-87 (10 ng) was incubated with labeled L12-5 in the presence of increasing amounts of unlabeled poly(I · C) ( ${blacktriangleup}$ ) or L12-5 ( ${blacksquare}$ ). The amount of radioactivity retained on a nitrocellulose filter for each sample was quantified. RNA binding in the absence of competitor was normalized to 1.0.

To directly examine the ability of Us11 to bind double-strandedRNA, radiolabeled poly(I · C) was utilized as a substratein a nitrocellulose filter binding assay. Following incubationof labeled poly(I · C) with purified GST ${Delta}$ 1-87, the bindingreaction mixtures were filtered through nitrocellulose on amanifold, and the amount of radioactivity retained on the filterwas quantified by liquid scintillation counting. As little as1 ng of purified GST ${Delta}$ 1-87 bound measurable amounts of poly(I· C) (Fig. 7A). The binding to radiolabeled poly(I ·C) was effectively competed by increasing amounts of unlabeledpoly(I · C) (Fig. 7B). Importantly, highly purified,unlabeled L12-5 selected ligand was also a potent competitorof binding to poly(I · C), while a representative RNAfrom the initial unselected population was only slightly activeas a competitor (Fig. 7B). The unlabeled single-stranded RNAhomopolymers poly(C), poly(U), and poly(A) were not effectivecompetitors when Us11 binding to labeled poly(I · C)was evaluated by nitrocellulose filter binding (Fig. 7C). Thisdirectly demonstrates that Us11 is a double-stranded RNA bindingprotein and that the RNA ligand isolated by in vitro selectionis a potent competitor in an assay that measures binding todouble-stranded RNA.

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FIG. 7. Us11 RNA binding domain directly binds double-stranded RNA. (A) Labeled poly(I · C) was incubated with increasing amounts of purified GST ${Delta}$ 1-87, and the amount of radioactivity retained on a nitrocellulose filter for each sample was determined. (B) Ten nanograms of GST ${Delta}$ 1-87 was incubated in the presence of labeled poly(I · C) in the absence of competitor or with increasing amounts of an unlabeled RNA selected to bind Us11 (L12-5 [ ${blacktriangleup}$ ]), an unlabeled RNA from the initial unselected population ( ${blacksquare}$ ), or unlabeled poly(I · C) ( ${diamondsuit}$ ). The amount of radioactivity retained on a nitrocellulose filter for each sample was determined. RNA binding in the absence of competitor was normalized to 1.0. (C) Binding to labeled poly(I · C) was as described for panel B with the modification that the reaction mixtures contained increasing amounts of either unlabeled poly(A) ( ${blacksquare}$ ), poly(C) (•), poly(U) ( ${blacktriangleup}$ ), or poly(I · C) ( ${diamondsuit}$ ).

As poly(I · C) preparations consist of two complementaryhomopolymers and vary significantly in length, we next evaluatedthe ability of Us11 to bind dsRNA molecules of defined lengthsthat possess greater sequence complexity. Labeled dsRNA moleculesderived from the E. coli cloning vector pBS and ranging in sizefrom 11 to 100 bp were prepared in vitro and incubated withincreasing amounts of purified GST ${Delta}$ 1-87 (Fig. 8A). RNA duplexesof 81 bp and above bound GST ${Delta}$ 1-87 with similar efficiencies,while binding to smaller RNA duplexes was reduced (Fig. 8B).Importantly, binding to RNA duplexes of 39 bp or shorter wasnot detected, demonstrating that the length of a perfectly duplexedRNA target can have a substantial impact on binding to Us11.Purified full-length Us11 that contained an amino-terminal tagcomposed of six histidine residues also bound efficiently todefined dsRNA molecules, exhibiting an apparent K_d of approximately12.6 nM for the 81-bp substrate in a filter binding assay (notshown).

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FIG. 8. The Us11 RNA binding domain exhibits length-dependent binding to dsRNA and prefers RNA duplexes to DNA duplexes. (A) Labeled RNA duplexes of defined lengths were prepared as described in Materials and Methods. A sample of each gel-purified dsRNA was resolved by electrophoresis in a nondenaturing polyacrylamide gel. An exposure of the fixed, dried gel is shown. The numbers on the top refer to the length in base pairs of the purified RNA duplex. The numbers to the right indicate the migration of dsDNA markers. (B) Labeled dsRNA of defined length was incubated with increasing amounts of purified GST ${Delta}$ 1-87 and subsequently filtered through a nitrocellulose membrane. Radioactivity retained on the membrane was quantified. ${diamondsuit}$ , 100-bp dsRNA; ${circ}$ , 81-bp dsRNA; ${blacktriangleup}$ , 63-bp dsRNA; ${blacksquare}$ , 51-bp dsRNA; (x) 39-, 33-, or 11-bp dsRNA. (C) Ten nanograms of GST ${Delta}$ 1-87 was incubated with a labeled 81-bp dsRNA substrate in the presence of increasing amounts of unlabeled dsRNA ( ${diamondsuit}$ ) or dsDNA (•). Both nucleic acid duplexes were 81 bp in length and were derived from the same sequence.

To determine if Us11 binding to a nucleic acid duplex was specificfor RNA, the binding of GST ${Delta}$ 1-87 to an 81-bp dsDNA fragmentand its binding to an 81-bp dsRNA duplex with an identical sequencewere compared. Increasing amounts of unlabeled dsRNA or dsDNAwere added to reaction mixtures that contained a fixed amountof radiolabeled dsRNA. Following the addition of GST ${Delta}$ 1-87, thereaction mixtures were incubated for 10 min at 30°C, RNA-proteincomplexes were collected on nitrocellulose filters, and thefractions of radioactivity retained on the filters were quantifiedby counting in liquid scintillant. Figure 8C demonstrates thatunlabeled dsRNA is significantly more effective than dsDNA atreducing the binding of GST ${Delta}$ 1-87 to the labeled dsRNA probe.This demonstrates that the Us11 RNA binding domain preferentiallybinds dsRNA duplexes of 51 bp or longer and can effectivelydiscriminate between dsRNA and dsDNA.

DISCUSSION

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Discussion

Us11 binds RNA through a novel 68-amino-acid domain that containsa repetitive arginine- and proline-rich motif (43, 46). As theUs11 RNA binding domain is not homologous to any known RNA recognitionmotif and the few RNAs known to interact with Us11 are unrelatedat the level of primary sequence, we proceeded to characterizefeatures of RNA molecules that associate with Us11. After isolatingRNA ligands that bound Us11 from a combinatorial library consistingof molecules that contained a randomized segment, we demonstratedthat Us11 bound specifically to these selected molecules andpreferentially associated with discrete conformational isoforms.While the addition of unlabeled poly(I · C) reduced thebinding of Us11 to a radiolabeled selected RNA, single-strandedhomopolymers were not effective competitors. Our study establishesthat Us11 binds directly to dsRNA polynucleotides and stablyassociates with perfect RNA duplexes of 51 bp or longer; furthermore,Us11 preferentially associates with nucleic acid duplexes thatcontain 2' hydroxyl groups, since dsDNA duplexes of identicalsequence and length do not reduce binding to dsRNA. This isthe first demonstration that the Us11 RNA binding domain canrecognize dsRNA.

While earlier studies reported that the Us11 polypeptide associateswith a synthetic RNA probe complementary to the 5' end of theUs11 mRNA and an internal segment of the Us12 mRNA, they didnot adequately address the RNA determinants involved in nucleicacid recognition (40). Thus, their observed reduction in Us11binding to RNA probes with 80-nucleotide deletions at their3' ends may not reflect binding to a specific site or sequencebut rather the global alteration of an RNA structure requiredfor binding that involves sequences from disparate parts ofthe molecule. Similarly, the changes in the distribution ofRNA conformational isoforms they described does not establishthat Us11 is a conformation-specific RNA binding protein, asthe experiments were all performed with crude extracts preparedfrom HSV-1-infected cells. Other activities in the extract inconjunction with Us11 could be responsible for modifying thestructure of the RNA probe. Finally, the ability of Us11 toassociate with the different RNA conformational isoforms wasnever examined, as the conformers were in rapid equilibriumand could not be isolated. Our study, in contrast, was performedwith both purified protein and RNA conformational isoforms;furthermore, we have established that Us11 associates with dsRNAand structured RNA as opposed to single-stranded polynucleotidesor dsDNA.

Numerous proteins that bind dsRNA contain a 65- to 75-amino-acidmotif (dsRNA binding motif [dsRBM]) that either folds into oris predicted to adopt a tertiary structure where two alpha helicesflank a three-stranded antiparallel beta sheet ( ${alpha}$ -ß-ß-ß- ${alpha}$ ).This domain comprises a dsRNA recognition motif (reviewed inreference 15). Multiple copies of this motif are found in manydsRNA binding proteins, although a single copy, as in the caseof the vaccinia virus E3L protein, is sufficient. Recognitionof dsRNA, however, is not limited to proteins that contain thedsRBM structure. Alternative dsRNA binding structures that haveno homology to each other or to the dsRBM have been observedin several viral proteins. Multiple basic residues that do notappear to fold into an independent domain or assume a structureon their own are distributed along the side of the sigma 3 polypeptideencoded by reovirus. Recent structural studies propose thatsigma 3 binds to RNA as a dimer via a positively charged surfacepatch on one side of the structure. This charged region is formedby the association of sigma 3 monomers and spans the interfacebetween the two subunits (34). Binding to dsRNA by the influenzavirus NS-1 protein also involves dimerization. In this case,structural studies revealed that the dimer contains six helicesthat contribute to multimerization and RNA binding (52). Althoughthe NS1 protein lacks a canonical dsRBM, it can also associatewith a cellular protein, Staufen, that contains this motif (14).In addition to its dsRNA binding activity, NS-1 also recognizespoly(A) stretches and U6 snRNA (19, 38, 39). We have now establishedthat the RNA binding domain in the HSV-1 Us11 protein constitutesyet another class of elements that can bind dsRNA. While thestructure of the Us11 RNA binding domain has not been determined,it is predicted to adopt the conformation of a type 2 polyprolylalpha helix built up of a series of repeats of the amino acidtriplet Arg-X-Pro (43, 46).

Copious amounts of Us11 are synthesized at late times in theinfectious cycle. HSV-1-infected cells have been demonstratedto contain a large amount of dsRNA, and dsRNA is a potent activatorof cellular enzymes poised to execute an antiviral response(23). The dsRNA binding domain of Us11 may therefore functionstoichiometrically to sequester viral dsRNA and prevent it fromengaging cellular dsRNA binding proteins such as PKR. Alongthese lines, the minimum lengths for RNA duplexes to efficientlyactivate PKR and to bind to Us11 are strikingly similar (29).RNA-dependent and -independent interactions involving Us11 andPKR may also reduce levels of activated PKR (reference 7 andunpublished observations). A related mechanism invoking bothdsRNA binding and a physical association with PKR has been proposedfor the vaccinia virus dsRNA binding protein, E3L (44).

Although dsRNA is thought to accumulate in HSV-1-infected cells,the activities of the Us11 protein are not essential for viralreplication in cultured cells or pathogenesis in mice, sinceUs11 is one of two known HSV-1 proteins capable of modulatingthe cellular antiviral response (21, 28, 33). Should viral dsRNAactivate the cellular PKR kinase and lead to the accumulationof phosphorylated eIF2 ${alpha}$ , the viral ${gamma}$ 34.5 gene product, in associationwith the cellular protein phosphatase 1 ${alpha}$ , can mediate the dephosphorylationof phospho-eIF2 ${alpha}$ and prevent the premature cessation of proteinsynthesis that would otherwise occur (20). Us11 is also packagedin the virus particle and therefore is delivered into the cellat times that precede the expression of viral immediate-earlygenes (42). While the role of Us11 in the virion is not understood,RNAs have not been found packaged in a Us11-dependent manner(47). The smaller quantities of Us11 delivered by the virusparticles could suffice to sequester the reduced amounts ofdsRNA formed early in the infection. In addition, Us11 has beenfound in nucleoli and associated with ribosomes. As PKR hasalso been detected in polysomes and nucleoli, similar subcellularlocalization of Us11 may be important in preventing PKR activation(24, 53, 57).

The selected RNA ligands that bound Us11 all formed a seriesof conformational isoforms. Several of these isoforms were stableupon gel purification, and Us11 binding activity was limitedto those isoforms that possessed increased structure. We donot know the precise nature of these putative structural determinants,as they may be either intramolecular, intermolecular, or a combinationof these alternatives. While Us11 prefers to bind perfect RNAduplexes of 51 bp or longer, the selected RNA molecules arepredicted to contain duplex regions substantially shorter than51 bp. Yet the selected RNAs associate with Us11, and a 39-bpdsRNA perfect duplex does not. As both of these templates arecomposed of approximately 80 nucleotides, this suggests thatthe selected RNAs contain binding determinants that are distinctfrom perfectly base paired regions. These determinants coulddepend solely on structure, primary sequence, or a combinationof both. Along these lines, eight of the nine selected sequencesare predicted to contain a CA or CCA bulge in the stem of adsRNA segment, and this element could conceivably be importantfor binding to Us11. All of the RNAs reported by others to associatewith Us11 are quite large, ranging from 200 to 600 nucleotidesin length (1, 12, 40, 41, 43, 46). While these studies convincinglydemonstrate the binding of Us11 to specific RNA molecules, theyfall short of establishing sequence-specific binding. In fact,the interaction of Us11 with RNA observed in published reportscould be explained either entirely or in part by recognitionof RNA structure. Only the Rev- and Rex-responsive elements,both of which bind Us11, have been determined to contain extensiveregions of secondary structure, although the involvement oftheir structured segments in binding to Us11 has not been explored(3, 17, 26, 50). The remaining lengthy RNA molecules reportedto bind Us11, some of which have a high GC content characteristicof herpesvirus mRNAs, would invariably possess a large degreeof predicted secondary structure, making it difficult to distinguishrecognition of structural determinants from recognition of sequencedeterminants. Indeed, the relative contributions of sequenceand structure are not evident and are worthy of future investigation.Is there, for example, a role for specific RNA sequence recognitionby Us11, or is the role of primary sequence simply to createa secondary structure that is effectively recognized by theUs11 RNA binding domain? Similarly, although simple RNA homopolymericsequences did not compete effectively for Us11 in binding experimentswith labeled poly(I · C), this does not conclusivelyprove that Us11 could not recognize discreet unstructured RNAmolecules of greater sequence complexity. Our isolation of asmall, defined RNA sequence that binds with high affinity toUs11 will make it possible to approach these questions experimentally.Finally, Us11 might execute several different functions, dependingon the nature of its RNA ligand. Complexes with dsRNA may functionto prevent PKR activation, while other unidentified sequence-specifictargets may be important for additional activities that remainto be discovered.

ACKNOWLEDGMENTS

We are grateful to Fenyong Liu for generously providing thecombinatorial library. We thank Lisa Parker and Tsafi Pe'eryfor advice concerning the RNA binding procedures, Joel Belascoand Matt Mulvey for their critical comments on the manuscript,and Joel Belasco for numerous discussions concerning RNA structure.

This work was supported by a grant from the NIH to I.M.

FOOTNOTES

* Corresponding author. Mailing address: Department of Microbiology and Kaplan Comprehensive Cancer Center, NYU School of Medicine, 550 First Ave., MSB 214, New York, NY 10016. Phone: (212) 263-0415. Fax: (212) 263-8276. E-mail: ian.mohr{at}med.nyu.edu.

REFERENCES

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