MOLECULAR AND CELLULAR BIOLOGY, Jan. 1988, p. 273-283 Vol. 8, No. 10270-7306/88/010273-11$02.00/0Copyright X 1988, American Society for Microbiology

Distinct Binding Sites for Zinc and Double-Stranded RNA in theReovirus Outer Capsid Protein u3

LESLIE A. SCHIFF,' MAX L. NIBERT,1 MAN SUNG CO,' EARL G. BROWN,1t AND BERNARD N. FIELDS12*

Department of Microbiology and Molecular Genetics, Harvard Medical School,' and Shipley Institute of Medicine andDepartment of Infectious Diseases, Brigham and Womens Hospital,2 Boston, Massachusetts 02115

Received 7 August 1987/Accepted 13 October 1987

By atomic absorption analysis, we determined that the reovirus outer capsid protein cr3, which bindsdouble-stranded RNA (dsRNA), is a zinc metalloprotein. Using Northwestern blots and a novel zinc blottingtechnique, we localized the zinc- and dsRNA-binding activities of cr3 to distinct V8 protease-generatedfragments. Zinc-binding activity was contained within an amino-terminal fragment that contained a transcrip-tion factor IIIA-like zinc-binding sequence, and dsRNA-binding activity was associated with a carboxy-terminal fragment. By these techniques, new zinc- and dsRNA-binding activities were also detected in reoviruscore proteins. A sequence similarity was observed between the catalytic site of the picornavirus proteases andthe transcription factor lIlA-Like zinc-binding site within a3. We suggest that the zinc- and dsRNA-bindingactivities of cr3 may be important for its proposed regulatory effects on viral and host cell transcription andtranslation.

The mammalian reoviruses represent the prototype of agroup of nonenveloped plant and animal viruses whosesegmented, double-stranded RNA (dsRNA) genomes aresurrounded by an inner capsid core and an outer capsidshell. Three serotypes of mammalian reoviruses have beenidentified, and the different strains of these viruses studied todate have been found to vary with respect to a number ofbiochemical markers and biological properties. As a result ofthese polymorphisms, reoviruses offer an excellent modelsystem for the study of virion morphogenesis and virus-hostcell interactions because the genetic analysis of reassortantviruses has provided a means to assign biological pheno-types and biochemical properties to particular viral genomesegments. Results of studies with reassortant viruses havesuggested that, in addition to playing structural roles, thereovirus outer capsid proteins influence the virus-host cellinteraction at a variety of stages in the viral replicative cycle.

S4, the smallest of the 10 mammalian reovirus genes,encodes the a3 protein (38, 40), a major component of thevirion outer capsid (53). The or3 protein is the only reovirusprotein that has been reported to have affinity for dsRNA(27). This is an unusual property for an outer capsid proteinwhich is removed from infecting parental virus by proteasedigestion early in infection (52, 55), and suggests that thisproperty may be one of newly synthesized a3 that operatesin the cell cytoplasm. Results of biochemical and geneticstudies have implied a role for the S4 gene product in theregulation of viral transcription (4) and translation (33, 34),the inhibition of host cell RNA and protein synthesis (50),and the establishment of persistent reovirus infection (1). Tobegin to determine whether the ability of cr3 to bind todsRNA is mechanistically important for virion morphogen-esis or any of the biological properties which have beenmapped to the S4 gene segment, we initiated a study toinvestigate the structural basis for the dsRNA-binding activ-ity of cr3.

* Corresponding author.t Present address: Influenza Section, Viral Diagnostic Services

Division, Bureau of Microbiology, Laboratory Centre of DiseaseControl, Health and Welfare Canada, Ottawa, Ontario, Canada.

A recently recognized motif for nucleic acid binding,which is exemplified in the Xenopus transcription factor IIIA(TFIIIA), involves the formation of small nucleic acid-binding domains, or "fingers," that are associated with thetetrahedral coordination of a zinc ion by cysteines andhistidines in a local region of the primary sequence (39). Thisobservation has provided new insight into the mechanism fornucleic acid binding, and has prompted Berg (6) to search forpotential metal-binding sequences in other nucleic acid-binding proteins. The proteins identified include tRNA syn-thetases and bacteriophage proteins such as the T4 gene 32protein, in which zinc atoms have been demonstrated. In thecase of the T4 gene 32 protein, zinc has been shown tocontribute to the conformation of a domain that bindssingle-stranded DNA (23). Several other viral nucleic acid-binding proteins were identified (6) that contained sequenceswhich could bind metals, including the retroviral gag pro-teins, the adenovirus ElA gene products, and the large Tantigens from simian virus 40 and polyomavirus. The poten-tial metal-binding sites of these viral proteins are in regionsthat have previously been identified to play a role in nucleicacid binding; however, studies have yet to be done whichdemonstrate the association of a metal ion with these se-quences.

In the present study we investigated the structural basisfor the RNA-binding activity of cr3 and examined the hy-pothesis that this activity is influenced by zinc. We identifieda potential zinc-binding site within the a3 sequence andshowed, by atomic absorption spectroscopy, that the a3molecule is associated with zinc. We developed a novelblotting assay to identify zinc-binding proteins and used thisassay to identify a zinc-binding proteolytic fragment of cr3.Using a Northwestern blotting technique, we identified afragment of cr3 which contains dsRNA-binding activity andshowed that this fragment is separate from the region of cr3that binds zinc. Finally, sequence analysis of the zinc-binding fragment of cr3 revealed an interesting similarity withthe picornavirus 2A and 3C proteases. This study representsthe first functional dissection of the reovirus cr3 protein, aviral capsid protein which has been proposed to have pleio-tropic effects on viral and host cell macromolecular synthe-

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274 SCHIFF ET AL.

sis. The assay techniques and results described here mayalso be of interest for the analysis of other zinc- and nucleicacid-binding proteins.

MATERIALS AND METHODS

Cells and virus. Mouse L cells were grown in suspension inJoklik modified minimal medium (Irvine Scientific, Irvine,Calif.) supplemented to contain 5% fetal bovine serum and 2mM glutamine. Reovirus T3 Dearing, Ti Lang, and T2 Joneswere the viral sL..vcks used in this study. Purified reovirusvirions were obtained by growing viral stocks in suspensioncultures of mouse L cells at 34°C and extracting virions frominfected cells as described previously (19). Virion storagebuffer consisted of 150 mM NaCI-10 mM MgCl2-10 mM Trishydrochloride (pH 7.5). Virion particle concentrations weredetermined by measuring the optical density at 260 nm andusing the conversion factor given by Smith et al. (53). Tolabel the viral proteins with 35S, either [35S]methionine (5 to12.5 ,uCi/ml) or [35S]cysteine (5 ,uCi/ml) was added to the cellsuspension 16 h after viral infection.T3 Dearing intermediate subviral particles (ISVPs) and

cores were generated by incubating purified virions at aconcentration of 2 x 1012 particles per ml (for ISVPs) or 1 x1i13 particles per ml (for cores) with 200 ,ug of tosyl lysinechloromethyl ketone treated a-chymotrypsin (Sigma Chem-ical Co., St. Louis, Mo.) per ml in virion storage buffer for 60min at 37°C. Digestion was stopped with 1 mM phenylmeth-ylsulfonyl fluoride, and the particles were isolated in a 1.25-to 1.45-g/ml preformed CsCl density gradient (SW28.1 rotor,L5-50 ultracentrifuge; Beckman Instruments, Inc., PaloAlto, Calif.) spun at 26,000 rpm for 2 h at 5°C. Particles weredialyzed against storage buffer and kept at 5°C until use.Identity of the particles was confirmed by sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis (PAGE).

Purification of [35SJmethionine-labeled cr3. The cr3 polypep-tide was purified by a modification of the method describedby Huismans and Joklik (27). Briefly, 109 mouse L cells wereinfected at a concentration of 106 cells per ml with 35 PFU ofreovirus T3 Dearing per cell. After 15 h at 37°C, the cellswere harvested by centrifugation and suspended at a con-centration of 3 x 106 cells per ml in medium supplemented tocontain 1 to 10 ,uCi of [35S]methionine per ml, 5% dialyzedfetal bovine serum, and 2 mM glutamine. The cells wereincubated in this labeling medium for 4 h at 37°C. At the endof the labeling period, the cells were harvested by centrifu-gation, washed in phosphate-buffered saline, and suspendedin 20 ml of RSB (0.01 M NaCl, 0.01 M Tris hydrochloride[pH 7.2], 1.5 mM MgCl2). The cells were disrupted in aDounce homogenizer, and the nuclei were removed bycentrifugation. The supernatant was made to 0.2 M withrespect to KCI and was clarified by centrifugation for 1.5 h at45,000 rpm in a fixed-angle rotor (50 Ti; Beckman). Theclarified supernatant was loaded onto a poly(IC)-CF11 cel-lulose column which was prepared by the method describedby Huismans and Joklik (27). The column was washed with3 volumes of TM buffer (0.01 M Tris hydrochloride [pH 7.8],0.05 M MgCl2) containing 0.2 M NaCl. a3 was eluted withTM buffer containing 0.5 M NaCl, and the column waswashed further with TM buffer containing 1 M NaCl. Col-umn fractions were analyzed by SDS-PAGE. The yield ofpurified cr3 varied between preparations, from 20 to 260 ,ugfrom 109 infected mouse L cells.

Preparation of N-formyl-[35Smethionine-labeled u3. Pro-teins were labeled at the amino terminus by cell-f!ee trans-lation of reovirus mRNA in the presence of N!-formyl-

[35 ]Met-tRNA Met in rabbit reticulocyte extracts. ReovirusmRNA was synthesized in vitro by transcription of virionsdigested with a-chymotrypsin as described previously (20),except for the inclusion of 10 ,uM S-adenosylmethionine.Reovirus mRNA was employed for translation as an unfrac-tionated preparation of in vitro transcripts. N-Formyl-[35S]Met-tRNA was prepared by the aminoacylation of rab-bit liver tRNA with unfractionated Escherichia coliaminoacyl synthetase extract and subsequent formylationwith the formyl ester of N-hydroxysuccinimide. The ami-noacylation conditions were essentially those described pre-viously (11), except that the reaction conditions consisted of50 mM Tris hydrochloride (pH 7.4), 10 mM ATP, 1 mMCTP, and 15 mM MgCl2, by the method described by Stanley(54). The tRNA fraction was isolated by DEAE-cellulosecolumn chromatography and then formylated. The extent offormylation of the N-formyl-[35S]Met-tRNAm"et preparationemployed in cell-free translation was greater than 99.9%.The rabbit reticulocyte translation system employed was

essentially that described by Pelham and Jackson (44),except that the reticulocyte extract was adjusted to 17 mMEGTA following treatment with nuclease. A 1-ml volume oftranslation mixture contained 80 ,ug of reovirus mRNA, 9pmol of N-formyl-[35 ]Met-tRNA Met (1,200 Ci/mmol), and 9nmol of L-methionine, in addition to the standard reactioncomponents. Reovirus mRNA translation products werefractionated by SDS-PAGE. A translation product whichcomigrated with authentic viral cr3 was excised from thepolyacrylamide gels and used for peptide mapping experi-ments.SDS-PAGE. Discontinuous SDS-PAGE was performed by

the protocol described by Laemmli (32). Gradient gels (5 to10% and 5 to 20%) were prepared by the method describedby Hames (24). Gels containing 35S-labeled proteins weretreated with Enlightning (New England Nuclear Corp.,Boston, Mass.), dried under a vacuum, and exposed to XARfilm (Eastman Kodak Co., Rochester, N.Y.) at -70°C.Alternatively, gels were dried between cellophane (Bio-RadLaboratories, Richmond, Calif.) and exposed to DEF film(Kodak) at room temperature. Both Coomassie-stained gelsdried between cellophane and autoradiograms were ana-lyzed densitometrically with an laser densitometer-inte-grator (Ultrascan XL; LKB Instruments, Inc., Bromma,Sweden). Estimates of protein relative molecular weights inSDS-PAGE were determined from multiple gels by compar-ison with protein molecular weight calibration markers (Be-thesda Research Laboratories, Gaithersburg, Md., andSigma).

Blotting procedures. Proteins were electrophoreticallytransferred to nitrocellulose (Trans-Blot; Bio-Rad) by theWestern blotting technique (56) with Tris glycine transferbuffer which contained 0.01% SDS and 20% (vol/vol) meth-anol.Northwestern blots for the detection of RNA-binding

proteins were performed by the method described by Boyleand Holmes (10). After the electrophoretic transfer of pro-teins to nitrocellulose, unreacted sites on the membranewere blocked overnight at room temperature with standardbinding buffer (10 mM Tris hydrochloride [pH 7.0], 1 mMEDTA, 50 mM NaCl, 0.04% bovine serum albumin, 0.04%Ficoll 400, 0.04% polyvinylpyrrolidone-40). The blotted pro-teins were probed with 32P-labeled reovirus genomic dsRNA(1 X 104 to 5 x 104 cpm/ml) in standard binding buffer orbinding buffer modified to contain 25 mM NaCl and 10 mMTris hydrochloride (pH 6.4; low-stringency buffer). Un-bound RNA was removed by washing the nitrocellulose with

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ZINC- AND dsRNA-BINDING SITES IN cr3 275

binding buffer at room temperature. Nitrocellulose was airdried and exposed to XAR film (Kodak) at -70°C with anintensifying screen (Cronex Lightning-Plus; E. I. du Pont deNemours & Co., Inc., Wilmington, Del.). The reovirusgenomic dsRNA to be used as a probe was purified from T3virions by phenol-chloroform extraction followed by G50column chromatography. Purified reovirus dsRNA (20 ,ug)was 3' end labeled with 200 ,uCi of [5'-32P]cytidine 3',5'-bisphosphate (New England Nuclear) to a specific activity of1 x 106 to 5 x 106 cpm/,Lg by the method described byEngland and Uhlenbeck (21) by using 20 U of T4 RNA ligase(P-L Biochemicals, Inc., Piscataway, N.J.).For the detection of zinc-binding proteins on nitrocellu-

lose, the membranes were washed in metal-binding buffer(100 mM Tris hydrochloride [pH 7.5], 50 mM NaCl) for atleast 1 h after electrophoretic transfer. The blotted proteinswere probed with 65ZnC12 (10 Ci/g; New England Nuclear) inbinding buffer (1 mCi/20 ml) for 30 to 60 min at roomtemperature. Unbound probe was removed by washing withbinding buffer for a total of 30 min with three changes ofbuffer. The 65ZnCl2 probe was reused for multiple experi-ments.

Peptide mapping by limited proteolysis. The protocol usedfor peptide mapping was a modification of the proceduredescribed by Cleveland et al. (16). Protein samples weresubjected to electrophoresis in a 5 to 20% SDS-polyacryl-amide gel. The gel was fixed in 30% isopropanol-10% aceticacid and then stained in a fixing solution containing 0.5%Coomassie brilliant blue R-250 (Sigma) and destained in16.5% methanol-5% acetic acid. Visualized protein bandswere excised from the gel and soaked in 125 mM Trishydrochloride (pH 6.8)-0.1% SDS for 30 min. The gel sliceswere placed into the wells of a second 5 to 20% polyacryl-amide gel and were overlaid with the sample buffers de-scribed by Cleveland et al. (16) containing 2 or 10 ng ofStaphylococcus aureus V8 protease (503 U/mg) (CooperBiomedical, Malvern, Pa.). When the samples had migratedhalfway through the stacking gel, electrophoresis wasstopped for 30 min and the samples were subjected to partialproteolysis before resumption of electrophoresis.Amino-terminal sequencing. The 24,000-Mr fragment (24K

fragment) and the 16K fragment of cr3 were electroelutedfrom gel slices by a modification of the method described byHunkapiller et al. (28). Gel fragments were loaded into thecathode chamber of an electroelution concentration cup(Isco, Lincoln, Nebr.) and overlaid with elution buffer (28).Buffer over the fragments was then underlaid with 100 ,ul of1% dithiothreitol (Sigma), and the fragments were allowed tosoak for 4 h. Protein was concentrated into the anodecollection chamber by electrophoresis at 50 V for 6 h.Excess elution buffer was removed, and the concentratedprotein sample in the collection chamber was dialyzedagainst dialysis buffer (28) at 5°C overnight. The sample (150to 300 ,il) was transferred to a microfuge tube, lyophilized todryness, suspended in 20 p,l of H20, and precipitated with180 p.1 of absolute ethanol at -20°C overnight. The precip-itated protein was obtained as a pellet by centrifugation for10 min in a microfuge, the supernatant was decanted, and theprotein pellet was lyophilized and suspended in 50 ,ul ofH20. Fractions of these final samples were analyzed bySDS-PAGE to judge their quality and quantity. Approxi-mately 300 pmol of each of the c3 fragments were used foramino-terminal sequencing.Amino-terminal sequencing was performed at the Micro-

sequencing Facility, Harvard University (Cambridge,Mass.). Edman degradation of the protein samples occurred

in a protein sequencer (470A; Applied Biosystems, FosterCity, Calif.). The released phenylthiohydantoin-derivatizedamino acids were analyzed on line with a high-performanceliquid chromatographic analyzer (120A; Applied Biosys-tems, Foster City, Calif.) and identified with an integrator(CR3A; Shimadzu, Columbia, Md.).

Atomic absorption spectroscopy. Prior to analysis, viralparticles were reisolated by CsCl grad-ent centrifugation,and both the viral particles and purified cr3 were dialyzedexhaustively against metal-depleted buffer. Trace metalswere removed from buffers by using Chelex 100 (Bio-Rad).The analysis of the zinc content of viral particles and purifiedcr3 was performed with an atomic absorption spectrometer(model 2280, The Perkin-Elmer Corp., Eden Prairie, Minn.)that was fitted with a graphite furnace (Perkin-Elmer). Theinstrument was calibrated with standard ZnCl2 solutionscontaining 5, 10, and 20 ppb (ng/ml) of Zn2+. Samples werediluted to give readings between 5 and 10 ppb of Zn2+. Theprotein concentration was determined by using the Pierceprotein assay reagent (Pierce Chemical Co., Rockford, Ill.)by the microassay procedure for protein determination (pu-rified c3) or was estimated from samples that were subjectedto SDS-PAGE (viral particles), with bovine serum albuminused as a standard.

RESULTS

Sequence analysis of the reovirus capsid protein a3 revealsa potential zinc-binding site. The suggestion that the coordi-nation of a zinc ion can contribute to the conformation ofnucleic acid-binding domains prompted us to examine theamino acid sequence of the cr3 protein of reovirus T3 Dearingfor a sequence of the form Cys-X24-Cys-X2-15-a-X24-a ora-X24-a-X2-15-Cys-X2-4-Cys, where a is either a cysteine ora histidine. These consensus sequences for metal bindingsites represent those chosen by analogy with the sequence inTFIIIA and in other proteins with structurally characterizedmetal-binding sites (6). We identified such a sequence inreovirus c3, and its relationship to the consensus sequenceof the TFIIIA zinc-binding fingers is shown in Fig. 1A. Adiagram of an extended region of the c3 sequence surround-ing the potential zinc-binding site is shown in Fig. 1B.Although the cysteines and histidines that provide the best fitto the TFIIIA consensus sequence are highlighted in Fig. 1B,this region of c3 contains nine cysteines and histidines whichcould serve as ligands for zinc ions.Examination of the amino acid sequence within this region

revealed several other interesting features. A hydrophilicregion between amino acids 63 and 72 has been suggested byGiantini and co-workers (22) to play a role in RNA bindingby cr3. The amino acids which have been reported toparticipate in nucleic acid binding (42) are indicated (Fig.1B). These residues are clustered toward the carboxy-terminal side of this region, and secondary structure predic-tions indicate that a part of this region (between amino acids63 and 68) could assume an alpha-helical conformation. Thisconformation conforms to the structure predicted for thecarboxy-terminal end of a large number of finger sequences(12). In contrast, the amino-terminal half of this potentialzinc-binding region contains a large number of hydrophobicamino acids (Fig. 1B). The TFIIIA zinc-binding fingers havebeen shown to contain invariant hydrophobic residues (37),and residues in a3 which correspond to the invariant hydro-phobic sites in TFIIIA are marked (Fig. 1B). All of thefeatures described are conserved in the cr3 protein of asecond reovirus strain, Ti Lang, as the comparison between

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276 SCHIFF ET AL.

A.

o3:

o3:

TFIIIA:

tM HOL G V V G S L Q R K L KOL P HO51 71

C X2 C

C 5 C

X12

X12

H X3 H

H X2-3 H

B.

o3: P D K M V C G G A V V40 ma.. a *

I . I ... laSa. .0. ...CNHCLGVVGS LQRKLKHLPHHRCNQQ IRHQ

a * * * * . 80

FIG. 1. (A) Amino acid sequence of a potential zinc-binding site in.the reovirus capsid protein cr3. The cr3 amino acid sequence isrepresented by a one-letter code (18). The amino acid sequence of a3 was predicted from the nucleotide sequence of the reovirus T3 DearingS4 gene, as determined by Giantini et al. (22). Residues 51 to 71 of the a3 protein are shown. The ar3 sequence was aligned with the consensussequence for metal-binding fingers of the TFIIIA type, as described by Berg (6). The conserved cysteines and histidines are circled. (B)Extended region of the amino acid sequence of cr3 surrounding the potential zinc-binding site. Residues 40 to 80 are indicated. The cysteinesand histidines in the cr3 sequence which correspond to those that are proposed to bind to a Zn2+ ion in TFIIIA are indicated with arrows (39).Potential nucleic acid-binding residues are marked with closed circles (42). Hydrophobic amino acids are indicated with closed squares, andresidues which correspond to conserved hydrophobic sites in the TFIIIA zinc fingers are marked with asterisks (39).

the sequences of Ti Lang (5) and T3 Dearing (22) revealedno differences in the region between amino acids 40 and 80.The structural similarities between the TFIIIA zinc-bindingfingers and this potential metal-binding site in cr3 led us toinvestigate whether the reovirus outer capsid protein cr3 isassociated with zinc.The reovirus outer capsid protein a3 contains zinc. To

determine whether zinc is associated with cr3 protein in thereovirus outer capsid, we took advantage of the fact thatreovirions can be converted to subviral particles with dif-ferent protein compositions by treatment with a-chy-motrypsin in vitro (8, 31, 51). A 5 to 20% SDS-polyacryl-amide gel on which samples of [35S]methionine-labeledvirions (Fig. 2A, lane 1) and subviral particles (Fig. 2A, lanes2 and 3) were run for comparison is shown. ISVPs aredistinguished from virions by the loss of cr3 as well as by theloss of the ,ulC protein and the appearance of its amino-terminal (A. K. Jayasuriya, M. L. Nibert, and B. N. Fields,Virology, in press) cleavage product 8 (Fig. 2A, lane 2). Wehave recently found that ISVPs also contain the carboxy-terminal product of ,lC cleavage (M. L. Nibert, manuscriptin preparation). Cores, on the other hand, are distinguishedfrom virions by the loss of both of the major reovirus outercapsid proteins cr3 and ,u1C, as well as the minor outercapsid protein crl. cr2, Al, and X2 remain as the majorreovirus core proteins.

Purified virions, ISVPs, and cores of reovirus T3 Dearingwere analyzed for the presence of zinc by atomic absorptionspectroscopy. In preparation for this study, the three typesof particles were reisolated by CsCl gradient centrifugationand dialyzed exhaustively against metal-depleted storagebuffer. The preparations of particles used for spectroscopywere standardized relative to one another by comparison inSDS-polyacrylamide gels. Virions, ISVPs, and cores wereall found to contain- detectable quantities of zinc (Table 1).The amount of zinc present in virions, however, was signif-icantly greater than that in either ISVPs or cores. Becausecr3 is the only major reovirus protein which is lost onconversion of virions to ISVPs, this finding suggests thatmost of the zinc in virions is associated with the cr3 protein.

Furthermore, an estimate of the concentration of each of theviral proteins present in the analyzed samples revealed thatapproximately equimolar amounts of zinc and a3 wereabsent from ISVPs relative to those amounts absent fromvi-ions (difference in zinc concentration, 0.38 ,um differencein c3 concentration, 0.35 ,uM). This result suggests that each

A B1 2 3 1 2

___ --x--- _

Am -JjiC- __

-b

FIG. 2. SDS-PAGE analysis of reovirus T3 virions, subviralparticles, and purified cr3. (A) [35S]methionine-labeled purified vi-rions of reovirus T3 Dearing (lane 1) were used to generate ISVPs(lane 2) and cores (lane 3) by treatment with a-chymotrypsin, asdescribed in the text. Samples containing 2 x 1010 particles of eachtype were loaded onto each lane. For this gel, samples of ISVPs andcores were loaded directly from digestion mixtures. (B) cr3 waspurified from reovirus T3 Dearing-infected mouse L cells by affinitychromatograpRy on a poly(IC)-CF11 cellulose column. Fractionscontaining cr3 were identified by SDS-PAGE and pooled. A fractionof the pooled pre4paration (3 .ig/ml) (lane 2) was compared with avirion marker (lane 1). All samples (panels A and B) were analyzedin a 5 to 20%o gradient SDS-polyacrylamide gel. The gel was fixed,treated with Enlightning, dried, and exposed to XAR film (Kodak)for 3 days at -70°C.

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ZINC- AND dsRNA-BINDING SITES IN a3 277

TABLE 1. Atomic absorption analysis ofzinc in reovirus particles

Zinc concnParticle A!LMb

ppb ±MC

Virion 33.0 0.51ISVP 8.5 0.13 0.38Core 6.6 0.10 0.41

a Concentration of viral proteins was estimated from virion particlessubjected to SDS-PAGE, using bovine serum albumin standards. Proteinconcentrations (micrograms per milliliter) were converted to micromolar forcomparison with zinc concentrations: c3, 12 p.g/ml (0.35 F.M); ar2, 3 4g/ml(0.08 F.M); I.1C, 15 pg/mI (0.21 ILM); A1 and X2, 10 pg/mI (0.07 FM).

b ApM represents the difference in zinc concentration with respect to wholevirions.

I The metal concentration (parts per billion) was converted to micromolarfor comparison with protein concentration. Metal content was quantitatedrelative to Zn standards.

molecule of virion-associated cr3 bound a single zinc atom.Since ISVPs and cores were found to contain approximatelyequal amounts of zinc (Table 1), the other major reovirusouter capsid protein, p1C, is unlikely to be associated withadditional amounts of this metal. The low but detectablequantity of zinc found in both ISVPs and cores, however,suggests that zinc ions are also associated with one or moreof the reovirus core proteins. Similar results to those shownin Table 1 were obtained from the spectroscopic analysis ofvirions and subviral particles of reovirus Ti Lang (data notshown).Because the results of the analysis for zinc in viral

partic'les strongly suggest that cr3 represents the major zinc-containing protein in reovirus virions, we decided to test thispossibility directly using purified c3 for atomic absorptionanalysis. cr3 was,purified from reovirus T3 Dearing-infectedmouse L cells by affinity chromatography on a poly(IC)-CF11 cellulose column by a modification of the methoddescribed by Huismans and Joklik (27). Samples of[35S]methionine-labeled T3 Dearing virions and purified cr3run on a 5 to 20% SDS-polyacrylamide gel are shown in Fig.2B. A preparation of purified cr3 was subjected to atomicabsorption analysis and found to contain 6.4 ppb (0.10 ,uM)of zinc. The purified ar3 concentration was 3 ,ug/ml (0.09p.M). These results suggest that there is a 1:1 association ofzinc atoms with purified a3 protein molecules and areconsistent with the results of the analysis of viral particlesand with the identification of a single good match to theTFIIIA-like zinc-binding consensus sequence in the aminoacid sequence of cr3.The (73 protein binds dsRNA in a Northwestern blot. To

test the hypothesis that, as in TFIIIA (25), the RNA- andzinc-binding activities of c3 are directly linked, we firstdeveloped an assay for cr3 dsRNA binding that would enableus to analyze proteolytic fragments for dsRNA-bindingactivity. In this assay, proteins from purified reovirus virionswere separated on an SDS-polyacrylamide gel, electropho-retically transferred to nitrocellulose, and probed with [5'-32P]pCp-labeled reovirus genomic RNA. A nitrocellulosefilter with proteins from reovirus T3 Dearing (Fig. 3A, lane 1)and Ti Lang (Fig. 3A, lane 2) virions that were stained withamido black is shown. An identical filter was probed with32P-labeled reovirus dsRNA, washed, and subjected to au-toradiography (Fig. 3B). The results of this Northwesternblot indicate that cr3 as well as cr2 and one or both of the Xproteins are able to bind to reovirus dsRNA. A similarexperiment with virion proteins that had been separated byelectrophoresis on a 10o SDS-phosphate-urea gel (60, 61)

allowed us to identify the dsRNA-binding A protein as Ai(data not shown).We characterized the optimal conditions for Al and a3

dsRNA binding by varying the pH (between 6.4 and 9.2) andNaCl concentration (between 0 and 250 mM) of the bindingbuffer (data not shown). The optimal pH for cr3 dsRNAbinding was observed between 6.4 and 6.8, and the ability ofcr3 to bind RNA dropped off sharply near pH 8. In contrast,Al dsRNA binding was detected over the entire pH range;however, at low pH, with increased washing, the dsRNAprobe was associated almost exclusively with the cr3 protein.The dsRNA-binding activity of a3 was found to be optimalbetween 0 and 50 mM NaCl but diminished significantlyabove 100 mM NaCl. The reactivity of Al with RNA wasdetected over the entire range of ionic conditions tested. Theoptimal conditions for cr2 dsRNA binding could not bedetermined in these experiments and are the subject offurther studies.

Identification of an RNA-binding fragment of r3. In anattempt to identify the region of the cr3 protein which isinvolved in binding dsRNA, we next characterized theproteolytic fragments of a3 that are generated by digestionwith S. aureus V8 protease. Bands of [35S]methionine-labeled c3 from reovirus T3 Dearing virions were excisedfrom an SDS-polyacrylamide gel and subjected to partialproteolysis by the method described by Cleveland et al. (16)(Fig. 4A). Digestion of cr3 from 2 x 1011 virions with 10 ng ofS. aureus V8 protease (Fig. 4A, lane 3) resulted in thecomplete digestion of c3, yielding only two major fragmentswith estimated Mrs of 24,000 and 16,000. The sizes of thesefragments were consistent with that of the cr3 protein (se-quence-predicted molecular mass, 41,164 daltons; Mr Qf39,000) that underwent a single endoproteolytic cleavageunder these conditions. We used cr3 protein generated by in

BA1 2 1 2

--_--

__ -j 1C

_ - 2 -'

S

FIG. 3. Reovirus dsRNA binds to reovirus proteins in a North-western blot. Viral proteins from 5 x 1011 purified reovirus T3Dearing (lanes 1) and Ti Lang (lanes 2) virions were separated on a5 to 201% gradient SDS-polyacrylamide gel and electrophoreticallytransferred to a nitrocellulose filter. (A) One portion of the nitrocel-lulose was stained with amido black immediately after electropho-retic transfer. (B) Additional protein-binding sites were blocked ona second portion of the nitrocellulose, and the filter was probed with5 x 104 cpm of [5'-32P]pCp-labeled reovirus genomic RNA per ml.The filter was washed in low-stringency buffer (25 mM NaCl [pH6.4]) and exposed to XAR film (Kodak) for 24 h at -70°C with anintensifying screen.

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278 SCHIFF ET AL.

A12 3

a.-- *4

24 - - _

16-

B1 2

24- -4

16- t

FIG. 4. SDS-PAGE analysis of [35SJmethionine and N-formyl-[35S]methionine-labeled a3 cleaved with S. aureus V8 protease. (A)Bands containing [35S]methionine-labeled cr3 from 2 x 1011 T3Dearing virions were excised from a 5 to 20%to gradient SDS-polyacrylamide gel and subjected to partial proteolysis with 0 (lane1), 2 (lane 2), or 10 (lane 3) ng of S. aureus V8 protease. The cr3cleavage products were analyzed on a second 5 to 20%'o gradientSDS-polyacrylamide gel, which was fixed, treated with Enlightning,dried, and exposed to XAR film (Kodak) at -70°C. (B) T3 Dearingcr3 protein that was labeled at its amino terminus with N-formyl-[35S]methionine was synthesized in vitro and purified by SDS-PAGE. Bands containing N-formyl-[35S]methionine-labeled cr3 (lane1) and gel-purified cr3 from virions that were metabolically labeledwith [35S]methionine (lane 2) were excised from a 10% polyacryl-amide gel and subjected to partial proteolysis with 2 ng of S. aureusV8 protease. The cr3 cleavage products were analyzed on a 15%SDS-polyacrylamide gel. The Mrs (103) of the cr3 cleavage productsare indicated and were determined as described in the text.

vitro translation of reovirus mRNA in a similar experimentto determine which of these two fragments contained theamino terminus of cr3 (Fig. 4B). The S. aureus V8 proteasecleavage products of N-formyl-[35S]methionine-labeled cr3(Fig. 4B, lane 1) or uniformly [35S]methionine-labeled cr3(Fig. 4B, lane 2) are shown. While both the 24K and the 16Kfragments were evident in samples generated from uniformlylabeled protein, only the 24K fragment of cr3 was labeledwith N-formyl-[35S]methionine.To confirm the locations of the 24K and 16K fragments in

the sequence of cr3, we performed a quantitative analysis ofthe cysteine content of the two fragments. Based on theestimated sizes of the S. aureus V8 protease-generatedfragments and an examination of the predicted amino acidsequence of a3 (22), we predicted that the 24K fragmentshould contain five cysteine residues (cysteines at positions4, 45, 51, 54, and 73), whereas the 16K fragment shouldcontain only one cysteine residue (at position 280). c3 fromvirions that were metabolically labeled with [35S]cysteinewas subjected to digestion with 10 ng of S. aureus V8protease as described above. Densitometric analysis of[35S]cysteine incorporation into the two proteolytic frag-ments of c3 revealed that the 24K fragment containedapproximately 5 times the radioactivity of the 16K fragment,confirming the suggested orientation of these fragments inthe cr3 protein sequence (data not shown). The 24K fragmentis therefore expected to contain the entire potential zinc-binding site.

Using the conditions of S. aureus V8 protease digestionthat result in the generation of the amino- and carboxy-terminal fragments of cr3, we localized the dsRNA-bindingfragment of cr3 by the Northwestern blotting technique. Anautoradiogram of a nitrocellulose filter that was probed with32P-labeled reovirus dsRNA is shown in Fig. SB, and asimilar filter that was stained with amido black is shown inFig. SA. Undigested c3 (Fig. 5B, lane 1) was detected withthe labeled probe, as was the 16K carboxy-terminal fragment

generated by digestion of a3 with 2 ng (Fig. 5B, lane 2) or 10ng (Fig. 5B, lane 3) of S. aureus V8 protease. No reactivityof the 24K amino-terminal fragment of cr3 with the RNAprobe was ever observed. The elimination of EDTA from thebinding buffer and the inclusion of ZnCl2 did not reveal anyreactivity of the RNA probe with the 24K fragment (data notshown). Thus, although the amino-terminal fragment con-tained the predicted zinc-binding site within cr3, the carboxy-terminal fragment contained the dsRNA-binding activitydetected in this assay.To further the characterization of this RNA-binding do-

main, we determined the sequence of the five amino-terminalresidues of the S. aureus V8 protease-generated carboxy-terminal fragment of c3. The 24K and 16K fragments wereisolated from preparative gels and electroeluted from the gelslices by using a modification of the method described byHunkapiller et al. (28). The quantity and purity of theelectroeluted fragments were analyzed by SDS-PAGE (Fig.6, inset) before they were subjected to amino-terminal se-quencing in a gas-phase sequenator. The amino-terminalsequence of the electroeluted 16K fragment was determinedto be Trp-Gly-Val-Met-Val, which is consistent with thesequence of amino acid residues 218 to 222 predicted fromthe nucleotide sequence of the T3 Dearing S4 gene (22). Thelocation of t:iis sequence in the cr3 molecule relative to thepredicted zinc-binding site is diagrammed in Fig. 6. Theseresults indicate that the RNA-binding domain of a3 whichwas identified in these experiments lies between the Trp atposition 218 and the carboxy terminus of the molecule. Theamino terminus of the 24K fragment was determined to beblocked, which is consistent with the fact that it is theamino-terminal fragment of a3 (46).&3 binds zinc in a zinc blot. To provide positive evidence

for the occurrence of zinc and dsRNA binding in two distinct

AV 1 2 3

B1 2 3

- _ -_ -Oi --

-24

-16-

FIG. 5. Reovirus dsRNA binds to the 16K carboxy-terminalfragment of cr3. Bands containing cr3 from 2 x 1011 T3 Dearingvirions were excised from a 5 to 20% gradient SDS-polyacrylamidegel and subjected to partial proteolysis with 0 (lanes 1), 2 (lanes 2) or10 (lanes 3) ng of S. aureus V8 protease. The digestion productswere analyzed in a second 5 to 209o gradient SDS-polyacrylamidegel. After electrophoresis the proteins and protein fragments wereelectrophoretically transferred to a nitrocellulose filter. (A) Oneportion of the filter, containing an additional marker lane (lane V) inwhich proteins from 2 x 1011 T3 Dearing virions were analyzed, wasstained with amido black immediately after electrophoretic transfer.(B) Additional protein-binding sites were blocked on a secondportion of the nitrocellulose, and the filter was probed with 5 x 104cpm of [5'-32P]pCp-labeled reovirus genomic RNA per ml for 1 h atroom temperature. The filter was washed in standard binding bufferand exposed to XAR film (Kodak) for 2 h at -70°C with anintensifying screen.

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ZINC- AND dsRNA-BINDING SITES IN (x3 279

24K

ProposedZn2l Binding

V8WGVMV-0;C:

RNA Bin Northwe

I2 3

4 -16

f6K

3indingastern Blot

FIG. 6. Amino-terminal sequence of the 16K fragment of cr3.Bands containing a3 from 1.4 x 1013 reovirus T3 Dearing virionswere excised from a 5 to 20% gradient SDS-polyacrylamide gel andsubjected to partial proteolysis with 10 ng of S. aureus V8 protease.The digestion products were separated on a 10 to 20% gradientSDS-polyacrylamide gel, and bands containing the 24K and 16Kfragments were excised. The protein fragments were electroelutedfrom the gel slices, lyophilized, and ethanol precipitated. Prior toamino-terminal sequence analysis, fractions of the electroeluted16K (inset, lane 1) and 24K (inset, lane 2) fragments were analyzedby SDS-PAGE on a 5 to 20% gradient gel. A marker lane (inset, lane3) contains the cleavage products obtained by digestion of gel-purified cr3 from 2 x 1011 reovirus T3 Dearing virions with 2 ng of S.aureus V8 protease. The proteins and protein fragments werevisualized by staining the gel with Coomassie blue. The electroelut-ed samples were subjected to five cycles of Edman degradation in agas-phase sequenator. The five amino-terminal residues of the 16Kfragment and their location in the cr3 molecule are diagrammed.

regions of a3, we developed a method to identify zinc-binding proteins and protein fragments (L. A. Schiff, manu-script in preparation). This technique involved separatingproteins by SDS-PAGE, transferring them to nitrocellulose,and then incubating the nitrocellulose with a radioactiveprobe, 65ZnC12. A similar method for detecting calcium-binding proteins has been described previously (37). Evi-dence for the relative specificity of the zinc blot is shown inFig. 7A. Molecular weight marker proteins (Pharmacia FineChemicals, Piscataway, N.J.) that included the zinc-bindingprotein, carbonic anhydrase, and the calcium binding proteina-lactalbumin, were separated by electrophoresis on a 5 to20% SDS-polyacrylamide gel, electrophoretically trans-ferred to nitrocellulose, and probed with 65ZnC12. In anautoradiogram of this filter, the only protein found to bind adetectable level of 65Zn was carbonic anhydrase (Fig. 7A,lane 2). The other proteins present in this preparation wereevident on an identical filter that was stained with amidoblack (Fig. 7A, lane 1). We have determined that otherzinc-binding proteins (for a review, see reference 59) canalso bind 65Zn in this assay (data not shown).The zinc blotting technique was applied to the proteins

from reovirus strains Ti Lang (Fig. 7B, lanes 1 and 3) and T3Dearing (Fig. 7B, lanes 2 and 4). Whereas all of the virionstructural proteins were detected on a nitrocellulose filterthat was stained with amido black (Fig. 7B, lanes 1 and 2),only the cr3 and A proteins were seen on the autoradiogram ofthe filter that was probed with 65ZnCl2 (Fig. 7B, lanes 3 and4). The relative specificity of this assay was supported by thelack of binding of 65Zn to the reovirus ,lC protein, whichhas been shown to be present in reovirus virions in approx-imately equal numbers to cr3 (Jayasuriya et al., in press) or tothe cr2 core protein, which is rich in cysteine (14). In thisassay the identification of cr3 as the major zinc-bindingprotein in reovirus virions is consistent with the results of

atomic absorption analysis described above. The observa-tion of 65Zn associated with the K proteins suggests that thezinc-binding protein(s) in reovirus cores identified by spec-troscopy is likely to be one or both of the major X proteins.

Identification of a zinc-binding fragment of (73. After weshowed that the cr3 protein can bind zinc in a zinc blot, weidentified those S. aureus V8 protease-generated cr3 frag-ments that contain the zinc-binding activity detected in thisassay. Bands containing cr3 from T3 Dearing virions wereexcised from an SDS-polyacrylamide gel and subjected topartial proteolysis with S. aureus V8 protease as describedabove. The protein fragments were electrophoretically trans-ferred to nitrocellulose, and identical filters were eitherstained with amido black (Fig. 8A) or probed with either65ZnC12 (Fig. 8B) or 32P-labeled reovirus dsRNA (Fig. 8C).On the filter that was probed with 65ZnC12, undigested cr3(Fig. 8B, lanes 1 and 2) and the 24K amino-terminal fragment(Fig. 8B, lanes 2 and 3) were detected. On the filter probedwith 32P-labeled reovirus dsRNA, undigested cr3 (Fig. 8C,lane 1) and the 16K amino-terminal fragment (Fig. 8C, lanes2 and 3) were detected. Undigested cr3, the 24K fragment,and the 16K fragment were all seen on the stained filter (Fig.8A). Thus, the results of this experiment indicate that theamino-terminal fragment of cr3, which contains a TFIIIA-likezinc-binding sequence, is able to bind 65Zn. Furthermore,the 65Zn-binding fragment of c3 is clearly distinct from thefragment that can bind dsRNA.The amino-terminal fragment of r3 contains a region with

sequence similarity to the picornavirus 2A and 3C proteases.The results of the experiment described above suggest thatthe ability of cr3 to bind zinc may be important for a function

A1 2

B1 2 3 4

94--A- __67-_

43- Pic- __

30-_ -

14-

FIG. 7. Zinc-binding proteins can be detected in a zinc blot. (A)Molecular weight marker proteins (low-molecular-weight calibrationkit; Pharmacia) consisted of phosphorylase b (94,000), bovine serumalbumin (67,000), ovalbumin (43,000), carbonic anhydrase (30,000),soybean trypsin inhibitor (20,100), and bovine a-lactalbumin(14,400). The marker proteins were subjected to electrophoresis ona 5 to 20%o gradient SDS-polyacrylamide gel and electrophoreticallytransferred to nitrocellulose. Each protein in the mixture waspresent at a concentration of 1 p.g/plJ, and 3 RIl was loaded per lane.Portions of the nitrocellulose were either stained with amido black(lane 1) or incubated with 65ZnC12 for 30 min at room temperature,washed, and exposed to XAR film (Kodak) for 29 h at -70°C with anintensifying screen (lane 2). (B) Virion proteins from 2 x 1011reovirus Ti Lang (lanes 1 and 3) and T3 Dearing (lanes 2 and 4) wereanalyzed. Proteins were subjected to electrophoresis on a 5 to 20%ogradient SDS-polyacrylamide gel and electrophoretically transferredto nitrocellulose. Portions of the nitrocellulose filter were eitherstained with amido black (lanes 1 and 2) or incubated with 65ZnC12for 30 min at room temperature, washed, and exposed to XAR film(Kodak) for 2 h at -70°C with an intensifying screen (lanes 3 and 4).

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280 SCHIFF ET AL.

Av 12 3

B Ci 2 3 1 2 3

_ --*a- -

_ "-16.%

16-

FIG. 8. Zinc-binding activity of the 24K fragment of a3. Bandscontaining ar3 from 4 x 1011 T3 Dearing virions were excised from a5 to 20% gradient SDS-polyacrylamide gel and subjected to partialproteolysis with 0 (lanes 1), 2 (lanes 2), or 10 (lanes 3) ng of S. aureusV8 protease. The digestion products were resolved on a 5 to 20%gradient SDS-polyacrylamide gel and electrophoretically transferredto a nitrocellulose filter. (A) A portion of the nitrocellulose contain-ing an additional marker lane (lane V) in which the proteins from 2x 1011 reovirus T3 Dearing virions were analyzed was stained withamido black. (B) A second portion of the nitrocellulose was probedfor 1 h with 65ZnCI2, washed, and exposed to XAR film (Kodak) for15 h at -70°C with an intensifying screen. (C) A third portion of thenitrocellulose was blocked, probed for 1 h with 5 x 104 cpm of[5'-32P]pCp-labeled reovirus genomic RNA per ml, washed, andexposed to XAR film (Kodak) for 16 h at -70°C with an intensifyingscreen.

other than nucleic acid binding. We analyzed the amino acidsequence of the putative zinc-binding site of c3, and discov-ered that there was an interesting similarity to the picorna-virus 2A and 3C proteases (Fig. 9). These proteases areencoded by the genomes of polioviruses and several otherpicornaviruses and are responsible for all but one of theproteolytic cleavages of the polyproteins of these viruses(41). The region of sequence with which cr3 shows similarityis conserved in the 2A and 3C proteases of a number ofdifferent picornavirus strains (2, 35). This observation, alongwith the results of experiments in which protease inhibitorswere used (43), has led to the suggestion that many picorna-virus proteases are of the thiol class. In fact, the Cys residueat position 108 and the His residue at position 116 ofpoliovirus 2A and their analogs in other 2A and 3C proteinshave been suggested to act as catalytic residues for proteo-lysis (2, 58). These putative catalytic residues are conservedin the homologous region of a3 (Fig. 9).

DISCUSSION

In this report, we have presented the results of studies inwhich we investigated the structural basis for the dsRNA-binding activity of the reovirus outer capsid protein cr3.Using a filter-binding assay, Huismans and Joklik (27) iden-tified cr3 as the only reovirus protein that has affinity fordsRNA and determined that cr3 recognizes dsRNA in anonspecific fashion since it binds both to reovirus genomicRNA and to unrelated and synthetic dsRNA molecules. Wedemonstrated here that a Northwestern blotting techniquealso identifies a dsRNA-binding activity in cr3. Blottingtechniques have been used by other investigators to identifyviral DNA-binding proteins (9, 29, 36) and viral and cellularRNA binding proteins (10, 47, 49). The optimal conditions

determined for cr3 dsRNA binding in this assay (pH 6.4 to6.8; 0 to 50 mM NaCI) were very similar to those for theRNA binding of rotavirus VP2 (10) and the DNA binding ofbovine papillomavirus E2 protein (36), both of which havebeen characterized as sequence-independent interactions.

Northwestern blots performed on protein fragments gen-erated by digestion with S. aureus V8 protease localized adsRNA-binding activity of cr3 to the 16K carboxy-terminalfragment. This fragment contains several stretches of poten-tial nucleic acid-binding residues (42), including one betweenamino acids 325 and 330 and another larger region betweenamino acids 287 and 297. In addition, Giantini and co-workers (22) have reported that the hydropathicity profile ofc3 in the region between residue 318 and the carboxyterminus shows strong similarity to orNS, a reovirus single-stranded RNA-binding protein, and have suggested that thisfeature reflects a shared nucleic acid-binding function ofthese proteins.

Berg (6) has recently proposed that a number of nucleicacid-binding proteins use a structural motif analogous to thatdescribed for the Xenopus 5S RNA transcription regulatoryprotein TFIIIA, in which two invariant pairs of cysteines andhistidines provide ligands for the tetrahedral coordination ofa zinc ion (25, 39). We identified a similar sequence in a3 andconfirmed that c3 is a zinc metalloprotein by atomic absorp-tion spectroscopy of extensively dialyzed reovirions, sub-viral particles, and purified c3 protein. We also showed thatc3 can bind zinc by using a modification of the Westernblotting technique, in which 65ZnCI2 was used to probeproteins that have been immobilized on nitrocellulose filtersfollowing SDS-PAGE. The zinc blot allowed us to show thatthe amino-terminal fragment of c3, which contains theTFIIIA-like zinc-binding sequence, is able to bind 65Zn.Thus, these results may indicate that the zinc- and nucleicacid-binding paradigm (6) is not relevant to c3. On the otherhand, the amino-terminal zinc-binding site in c3 may possessa nucleic acid-binding activity, perhaps a sequence-specificone, which we have so far failed to detect and which isdistinct from the characterized carboxy-terminal function.Consistent with the possible separation of these bindingfunctions in a3 are the results of a recent report on DNAbinding by the human glucocorticoid receptor (26). Resultsof that study indicated that mutations in a putative zinc-binding site did not affect a nonspecific DNA-binding activ-ity of the protein but did eliminate sequence-specific DNA

a A a a* a45 C G G A V V C M H 53

108 C C G I L R C H H 116

108 C G G I L R C Q H 116

109 C G G I L R C I H 117

110 C G G I L R C E H 118

147 C G G V I T C T G 155

o3: Reovirus T3 Dearing, Ti Lang

2A protease: Poliovirus Ti Mahoney

Poliovirus T3 Sabin

Rhinovirus 14

Coxsackievirus 81

3C protease: Poliovirus Ti Mahoney

FIG. 9. A region of similarity between cr3 and picornavirusproteases. A region of amino acid sequence in reovirus cr3 proteinswas aligned with a homologous region found in the amino acidsequences of the 2A and 3C proteases of some picornaviruses. Thenumbers of the first and last amino acids shown for each protein areindicated. (The amino acid sequence for coxsackievirus 2A is thatpredicted by lizuka et al. [30]. The amino acid sequences for otherpicornaviruses were obtained from Toyoda et al. [57] and Callahanet al. [131.) Amino acid identities between cr3 and the 2A proteasesare indicated with underlined boxes; similarities are indicated withboxes.

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binding. It has also been suggested that TFIIIA has asequence-independent nucleic acid-binding activity which isnot influenced by zinc (25).The zinc blot described in this study should be applicable

to the characterization of other zinc-binding proteins. Unlikeatomic absorption spectroscopy, the zinc blot does notrequire extensive sample preparation and can be used toidentify zinc-binding proteins in biological mixtures and toidentify protein fragments which have zinc-binding activity.Although in this assay (as well as in the previously describednucleic acid-protein blots) proteins are denatured beforethey are incubated with the probe, enough native proteinstructure is apparently assumed after blotting to allow spe-cific binding interactions to occur. The lack of binding of65Zn to a-lactalbumin, a calcium-binding protein, as well asthe observation that the binding to authentic zinc-bindingproteins was not eliminated by lowering the pH of thewashing buffer (data not shown), implies that the ability of65Zn to bind to proteins in this assay was not simply theresult of a nonspecific charge interaction between the posi-tively charged ion and acidic proteins. We are currentlyevaluating the ability of other divalent metal ions to competewith 65Zn binding in this assay. Given the evidence for therelative specificity of binding in the zinc blot, we have beguna study using this technique to test the prediction by Berg (6)that a class of nucleic acid-binding proteins exists which usezinc-binding sites as an element of structural organization.A potential role for the zinc associated with the cr3 protein,

other than one in nucleic acid binding, is suggested by asequence similarity between the proposed zinc-binding siteof cr3 and the proposed catalytic site of the picornavirusproteases. Many picornavirus 2A and 3C proteins appear tobe thiol proteases which are subject to inhibition by zinc(43). One interesting possibility is that the reovirus cr3protein also possesses proteolytic activity and that thisactivity is regulated by the ability of cr3 to bind zinc with ahigh affinity. Another is that a region of primary sequencewhich exhibits a low affinity for zinc in the picornavirusproteases occurs in cr3 as part of a high-affinity binding sitethat is used for some other purpose.When it is considered that reovirus virions contain a

dsRNA genome that is enclosed in a protein inner capsidcore, the identification by Huismans and Joklik (27) ofdsRNA-binding activity in an outer capsid protein and thelack of such an activity in core components is intriguing.Using a Northwestern blot, we detected dsRNA-bindingactivity in two of the three major reovirus core proteins, cr2and Xl. It is possible that the denaturing conditions used toseparate the virion proteins prior to electrophoresis in thisstudy revealed cryptic RNA-binding sites in the core pro-teins that were inaccessible under the isolation conditionsused in previous studies. In addition, since the probe used inour studies was reovirus genomic dsRNA, our assay mayhave detected sequence-specific interactions between one orboth of the core proteins and reovirus dsRNA. In fact,experiments in which the optimal conditions for protein-dsRNA binding in this assay were addressed revealed thatthe X1-dsRNA binding is less condition dependent than thatof cr3 or other characterized sequence-independent nucleicacid-protein interactions. In future experiments we willaddress the nature of the interaction between the coreproteins and dsRNA. Our current findings are consistentwith a role for the cr2 and A1 proteins in the packaging of viralgenome segments or in the transcriptase activity that isassociated with the reovirus core.Using the zinc blot, we also were able to assign zinc-

binding activity to one or more of the A core proteins. Thisfinding agrees with the results of atomic absorption spectros-copy which indicated that zinc is present in the reoviruscore. The lack of sequence information for the X proteinsmakes it impossible as yet to determine whether any of theseproteins contain sequences similar to the TFIIIA zinc fin-gers. We are currently investigating whether the RNA-binding activity of the reovirus Al protein colocalizes withzinc binding. Although specific biochemical activities havenot been identified for all of the components of the viral core,the reovirus X2 protein, which constitutes the virion corespike (60), has been reported to possess guanylyltransferaseactivity (15), and the X3 protein, a minor core component,has been suggested to be the viral polymerase (20). Theability of zinc to bind to these proteins could reflect a role forthe ion in their enzymatic activities, as zinc has beenreported to play a catalytic role in a number of transferasesand polymerases (for a review, see reference 59).

In summary, using a protein-nucleic acid blotting tech-nique, we located a dsRNA-binding activity in a 148-residuecarboxy-terminal fragment of the reovirus cr3 protein. Usingatomic absorption spectroscopy and a novel protein-zincblotting technique, we further identified that cr3 is a zincmetalloprotein and that it binds zinc in a 217-residue amino-terminal fragment, probably in a region of sequence betweenresidues 40 and 80, which is similar to the putative zinc-binding locus of Xenopus TFIIIA. During the course of thisstudy, we were also able to identify new RNA- and zinc-binding activities associated with particular reovirus coreproteins. We have undertaken a biochemical investigation ofthe cr3 protein primarily to relate the biochemical propertiesof this protein to more complicated biological phenotypes.Present evidence suggests a role for cr3 in the inhibition ofhost cell protein and RNA synthesis (50), the regulation ofviral translation (33, 34), and the establishment of persistentinfection in cell culture (1). It is easy to envision that manyof these biological attributes might be based in transcrip-tional and/or translational regulatory effects of cr3. For sucha function, binding of cr3 to viral and/or cellular dsRNA(rRNA, for example) would not be unexpected. It is alsointeresting that the transcriptional and translational regula-tory proteins of lentiviruses, e.g., the tat protein of humanimmunodeficiency virus (17, 45, 48), have been found tocontain a region of primary sequence rich in cysteine andhistidine that is postulated to mediate binding to zinc ions(3). In addition, the poliovirus 2A protein, with which cr3shows a sequence similarity, has been implicated in effectson host protein synthesis (7). We anticipate that studies ofthe reovirus cr3 protein will contribute to a general under-standing of viral effects on host cell metabolism. Sequenceanalysis of S4 genes from biologically variant reovirusstrains should provide additional clues as to the importanceof zinc and dsRNA binding for the functions of cr3.

ACKNOWLEDGMENTSWe thank Bill Lane for performing the amino-terminal amino acid

sequence analysis, Temple Smith for useful discussion concerningamino acid sequence homologies, and Robert Shapiro for perform-ing the atomic absorption spectroscopy.

This study was supported by Public Health Service grant A113178from the National Institute of Allergy and Infectious Disease.M.L.N. was supported by the Public Health Service NationalResearch Service Award 5 T32 GM07753-09 from the NationalInstitute of General Medical Sciences. M.S.C. was supported byPublic Health Service New Investigator Research Award DK36837from the National Institute of Diabetes and Digestive and KidneyDiseases.

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LITERATURE CITED1. Ahmed, R., and B. N. Fields. 1982. Role of the S4 gene in the

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2. Argos, P., G. Kamer, M. J. H. Nicklin, and E. Wimmer. 1984.Similarity in gene organization and homology between proteinsof animal picornaviruses and a plant comovirus suggest com-mon ancestry of these virus families. Nucleic Acids Res.12:7251-7276.

3. Arya, S. K., C. Guo, S. F. Josephs, and F. Wong-Staal. 1985.Trans-activator gene of human T-lymphotropic virus type III(HTLV-III). Science 229:69-73.

4. Astell, C., S. C. Silverstein, D. H. Levin, and G. Acs. 1972.Regulation of the reovirus RNA transcriptase by a viral capso-mere protein. Virology 48:648-654.

5. Atwater, J. A., S. M. Munemitsu, and C. E. Samuel. 1986.Biosynthesis of reovirus-specified polypeptides. MolecularcDNA cloning and nucleotide sequence of the reovirus serotype1 Lang strain s4 mRNA which encodes the major capsid surfacepolypeptide u3. Biochem. Biophys. Res. Commun. 136:183-192.

6. Berg, J. M. 1986. Potential metal binding domains in nucleicacid binding proteins. Science 232:485-487.

7. Bernstein, H. D., N. Sonenberg, and D. Baltimore. 1985. Polio-virus mutant that does not selectively inhibit host cell proteinsynthesis. Mol. Cell. Biol. 5:2913-2923.

8. Borsa, J., T. P. Copps, M. D. Sargent, D. G. Long, and J. D.Chapman. 1973. New intermediate subviral particles in the invitro uncoating of reovirus virions by chymotrypsin. J. Virol.11:552-564.

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10. Boyle, J. F., and K. V. Holmes. 1986. RNA-binding proteins ofbovine rotavirus. J. Virol. 58:561-568.

11. Brown, E., and L. Prevec. 1982. Linear mapping of tryptophanresidues in Vesiculovirus M and N proteins by partial chemicalcleavage. J. Virol. 42:311-316.

12. Brown, R., and P. Argos. 1986. Fingers and helices. Nature(London) 324:215.

13. Callahan, P. L., S. Muzutani, and R. J. Colonno. 1985. Molec-ular cloning and complete nucleotide sequence determination ofRNA genome of human rhinovirus type 14. Proc. Natl. Acad.Sci. USA 82:732-736.

14. Cashdollar, L. W., J. Esparza, G. R. Hudson, R. Chmelo,P. W. K. Lee, and W. K. Joklik. 1982. Cloning the double-stranded RNA genes of reovirus: sequence of the cloned S2gene. Proc. Natl. Acad. Sci. USA 79:7644-7648.

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