THE JOURNAL OF BIOLDCICAL CHEMISTRY 0 1994 by The American Society for Biochemistry and Molecular Biology, Ine.

Vol. 269, No. 43, Issue of October 28, pp. 27004-27014, 1994 Printed in U.S.A.

Interaction of Poliovirus Polypeptide 3CDPr0 with the 5' and 3' Termini of the Poliovirus Genome IDENTIFICATION OF VIRAL AND CELLULAR COFACTORS NEEDED FOR EFFICIENT BINDING*

(Received for publication, April 28, 1994, and in revised form, August 11, 1994)

Kevin S. Harris*§, Wenkai XiangSn, Louis AlexanderS.11, William S. Lane**, Aniko V. Paul*, and Eckard WimmerSS$ From the $Department of Molecular Genetics and Microbiology, School of Medicine, Health Sciences Center, State Universitv of New York. Stonv Brook. New York 11 794-5222 and the **Harvard Microchemistry Facility, Cambridge, Massachusetts 02138 '

Poliovirus proteinase 3CDP" by itself is not an RNA- binding protein. Two cellular proteins have been puri- fied from HeLa cells (p50 and p36) which interact with purified 3CDP" but only p36-3CDPm bind to the 5'-termi- nal 110 nucleotides of polioviral RNA genome, an RNA segment whose secondary structure resembles a clover- leaf. The identity of these factors was determined by microsequencing tryptic digests of the purified pro- teins. Host protein p60 is the eukaryotic elongation fac- tor EF-la, and p36 an N-terminal fragment thereof. p36, referred to as host factor, did not appear to interact with purified 3Cpr0 or 3DP'. Significantly, the formation of a 3CDPr"-cloverleaf complex was also observed in the pres- ence of purified poliovirus polypeptide 3AB, the precur- sor of Vpg. 3AB by itself does not stably bind to the cloverleaf. Competition experiments have demonstrated that the RNA-protein interactions are specific for the full-length cloverleaf. U V cross-linking studies were em- ployed to examine the protein components of the clover- leaf ribonucleoproteins. RNA footprinting was used to determine the site on the cloverleaf where the viral and cellular factors bind. Finally, we have discovered that 3AB-3CDP" also interacts with the 3"terminal sequence of poliovirus RNA. In contrast to the 5'-terminal clover- leaf, the 3'-terminal RNA can bind 3AB in the absence of other proteins. A model for initiation of poliovirus RNA synthesis is presented.

Replication of the poliovirus RNA genome is a multistep process, the mechanism of which is not entirely understood (1). The single-stranded genome of poliovirus is covalently bound at its 5'-end by a small viral protein, VPg, while the 3'-end is polyadenylated. Upon infection of suitable host cells such as HeLa cells, the virus releases its genome which, in turn, is

* This work was supported in part by National Institutes of Health Grants AI-15122, AI-32100, and CA-28146. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Microbiology (State University of New York at Stony Brook). Present 3 Member of a graduate training program in Molecular Genetics and

address: Fred Hutchinson Cancer Research Center, 1124 Columbia St., Seattle, WA 98104.

Biology (State University of New York at Stony Brook). fi Member of a graduate training program in Biochemistry and Cell

sity of New York at Stony Brook). Present address: Harvard Medical 11 Member of a graduate training program in Genetics (State Univer-

School, New England Regional Primate Center, 1 Pine Hill Dr., South- borough, MA 01772.

$$To whom correspondence should be addressed. Tel.: (516) 632-8800; FAX: (516) 632-8891.

cleaved from the VPg by an unknown cellular enzyme. The virion RNA whose processed 5'-end is pUpU ... serves in two crucial functions: first in viral protein synthesis as mRNA and, subsequently, as template in the early rounds of RNA replica- tion.

Translation of poliovirus RNA, a process initiated through the function of an IRES element (Fig. 1) in a cap-independent fashion (11, leads to the synthesis of a single polypeptide, the polyprotein, that is co-translationally processed, yielding struc- tural and non-structural proteins (2, 3). All but one cleavage are catalyzed by the virus-encoded proteinases ZAP'", 3Cpr0, and 3CDP'" (2,3). Interestingly, some cleavage intermediates with a relatively long half-life have functions distinct from those of their cleavage products. Examples relevant to the experiments reported here are 3AB, the precursor for VPg, and 3CDP", the precursor for 3Cp" and 3DP"l, the viral primer-dependent RNA polymerase (Fig. 1). In parallel studies, we report that 3AB and 3CDPr0, and 3AB and 3DP"' form a tight complex (41.' Indeed, interaction between 3AB, 3DPo1, and template/primer leads to a nearly 100-fold stimulation of the polymerase activity (61.'

Purified 3CDPr0, a proteinase thought to be essential for proc- essing of the P1 capsid precursor (7-9), expresses no RNA po- lymerase activity (9). Genetic evidence has revealed that, sur- prisingly, 3CDP" has the propensity to bind the 5"terminal cloverleaf of poliovirus RNA (Fig. 2 A ) (10, ll), but this occurs only in the presence of a 36-kDa host factor (HF)' of uninfected HeLa cells (11, 12). Mutations that destroyed cloverleaf struc- ture abrogated both HF-3CDP" binding and viral replication, an observation suggesting that 3CDPm binding is necessary for RNA replication (11, 12). The nature of HF has not been re- ported.

Using gel shift assays we have purified two proteins (p50 and p36) that interact with the cloverleaf RNA, but only p36 was found to promote binding of 3CDPr0 to the probe. p50 and p36 were microsequenced and determined to be eukaryotic elonga- tion factor 1 a (EF-la) and an amino-terminal proteolytic frag- ment of EF-la, respectively.

The tight interaction between 3AB and 3CDP" (41, the pri- mary cleavage products of the P3 region of the poliovirus polyprotein (Fig. 11, and between 3AB and 3DP1(6),' prompted us to investigate RNA binding properties of these proteins to fragments of the poliovirus genome. We report here the inter- action of the 3AB-3CDPr0 complex with cloverleaf RNA. More-

l A. V. Paul, X. Cao, K. s. Harris, J. Lama, and E. Wimmer, in press.

linoJethanesulfonic acid; Dm, dithiothreitol; cpm, countdminute; nt, 2The abbreviations used are: HF, host factor; MES, 2-[N-morpho-

nucleotide(s); PMSF, phenylmethylsulfonyl fluoride; MOPS, 4-morpho- lineethanesulfonic acid; RF, replicative form; NTR, non-translated re- gion; EMCV, encephalomyocarditis virus; PK, pseudoknot.

27004

Polioviral Polypeptide 3 C L P 27005

A 9 StrudurdRegion Non-structural Region

V

B P3: 3A [3B[ 3C 3D

A A A

1 , Proteolytic processing at the 3B/3C cleavage site

3 c 3D 1 FIG. 1. Structure of polioviral genomic RNA. A, domains of the

5'-NTR are indicated by Roman numerals. Stars indicate the position of noninitiating AUG triplets. B, enlargement of the P3 non-structural precursor. fiiangles indicate Q/G cleavage sites.

over, we have found that the 3AB-3CDP" complex binds to 3'-terminal RNA fragments of the poliovirus genome. Based on these and previous results (4-6, 12-15) we have developed a model for the initiation of poliovirus RNA replication that does not take cellular proteins into account. Our model does not exclude the possibility, however, that cellular proteins partici- pate in poliovirus genome replication.

MATERIALS AND METHODS RNA Binding Assays-RNA gel shift analysis was carried out as

described before (11) with a few modifications. The RNA probe was incubated in MMK buffer (20 mM MOPS, pH 7.4, 60 mM KCl, 2 m MgC1,) at 45 "C for 30 min prior to its addition to the binding reaction. The binding buffer consisted of 5 mM MOPS, pH 7.4, 25 m KC1, 2 mM MgCl,, 5% glycerol, 20 mM Dl", and 15 pg of tRNA. Purified 3CDP" and fractions from different purification steps of the host factor were pre- incubated 5 min at 30 "C in a total volume of 25 pl. Cloverleaf RNA uniformly labeled with ta-32PlUTP (20,000 cpm) was then added (to 1.1 nM) to the reaction, and incubation was continued for 10 min at 30 "C. Five pl of 50% glycerol, 0.1% xylene cyanol, and 0.1% bromphenol blue was added prior to loading 15 pl of the reaction on a 6% native polyac- rylamide gel (acrylamidehisacrylamide 40:l) containing 0.5 x TBE (45 mM Tris, 45 mM boric acid; 1 mM EDTA, pH 8.0). The gels were prerun 30 min at 20 mA, the binding reactions were then loaded, and electro- phoresis was continued at 20 mAfor 2-3 h. The gels were then dried and subjected to autoradiography.

Plasmids-pPN6, a generous gift from Raul Andino, was used for run off transcriptions of the cloverleaf RNA (16). This plasmid also was subjected to deletion analyses to generate plasmids expressing stem- loops IABC (nt 1-45 of the polioviral genome) and stem-loops IAB (nt 1-34 of the polioviral genome) termed pIABC and p M , respectively (Fig. 2 A ) . Transcription of stem-loop D was performed using a partially double-stranded synthetic template, the T7 promoter region being dou- ble-stranded and the complementary sequence of stem-loop D (nt 46-81 of the polioviral genome) being single-stranded. Cold RNAs used in competition experiments were mixed with the probe prior to the addi- tion to the binding reaction. 3"Terminal RNA starting from nt 7332 to 7440 plus the poly(A) tail was generated by run-off transcripts of the plasmid, p3' PK-poly(A) (3"terminal pseudoknot with the poly(A) tail), linearized with EcoRI.

viral Polypeptides-Proteinases 3CPm and 3CDPm were prepared ac- cording to procedures described previously (9, 17). 3CDP" used in all experiments here is a mutant protein where the P4 Thr residue of the type I1 (Sabin) 3C/3D cleavage site (...x Q I Q/G E I Q...) was mutated to a Lys residue (...K Q I Q/G E I Q...) to impair autocleavage (9). Polypeptide 3AB was purified as described previously (6). 3DW' RNA polymerase was a generous gift of Dr. Stephen Plotch. Purified EF-la was a generous gift from Dr. William C. Merrick.

RNA Footprinting-21 pmol of in vitro transcribed dephosphorylated cloverleaf RNA was end-labeled with [Y-~~P]ATP using T4 polynucle- otide kinase. 1 x lo6 cpm of the end-labeled RNA was used in RNA gel shift assays. After the 10-min binding reaction with the viral and/or cellular proteins, T1, T2, or V1 nucleases were added and incubation

continued at 30 "C for 10 min. The reactions were then loaded onto a 6% nondenaturing 0.5 x TBE polyacrylamide gel. After electrophoresis the gel was exposed to Kodak XAR film for 15 min to visualize the bound complexes from the unbound probe. Bound complexes and unbound probe were separately excised from the gel, diced into small fragments, the 32P-labeled RNA was passively eluted by adding 0.6 ml of RNA elution buffer (0.1% SDS, 0.5 M ammonium acetate, 1 mM Na,EDTA, and 10 m magnesium acetate), and incubating at room temperature over- night. The next day the solutions were separately transferred to new Eppendorf tubes, 0.5 ml of RNA elution buffer was added to the gel pieces, and elution continued for 6 hs after which the supernatants were combined. Potassium acetate was then added to the eluted material to a final concentration of 0.3 M and incubated on ice for 15 min. After removal of the precipitated potassium dodecyl sulfate (KDS) by cen- trifugation, 10 pg of carrier tRNA was added to the supernatant, and the RNA was isolated by ethanol precipitation 30 min at -80 "C. The RNA pellet was dissolved in 300 pl of 0.3 M K+-acetate and reprecipi- tated with 750 pl of cold ethanol for 30 min at -80 "C. After centrifu- gation the RNA pellet was washed with 80% ethanol and dissolved in 20 pl of 7 M urea containing 0.1% xylene cyanol and 0.1% bromphenol blue. The total counts present were determined by Cherenkov counts, and 2,000 counts of each sample were then added to a 15% polyacrylamide 6 M urea sequencing gel, electrophoresed, and the gel was subjected to autoradiography.

Purification of the Host Factor-20 liters of HeLa cells (4 x IO6 cells/ ml) were harvested, suspended, and swollen in lysis buffer (10 mM HEPES, pH 7.9, 10 mM KC1, 1.5 m MgCl,, 0.5 m~ D'IT, 0.5 mM phe- nylmethylsulfonyl fluoride (PMSF), 2 pg/ml aprotinin, and 2 pg/ml leu- peptin and lysed as described previously (18). All manipulations with the extract were done at 4 "C. After lysis by Dounce homogenization, 25 ml of 50% glycerol was added to 120 ml of lysate to give a final concen- tration of 10% (Fraction 1). The lysate was then spun at 10,000 x g for 5 min at 4 "C, and KC1 was added to the supernatant (S10) to a final concentration of 0.1 M (Fraction 2A). The ,510 was then centrifuged at 100,000 x g in a Ti-TY60 rotor for 1 h at 4 "C. The ribosomal pellet (P100) was washed with 0.5 M KC1 for 1 h at 4 "C and spun for 1 h at 100,000 x g. The supernatant (S100) and the ribosomal salt wash (su- pernatant of the 100,000 x g spin) were pooled and dialyzed overnight

A 'I"

A A c c c 6 A c A G G G c G u A b . . - ..

U G G G G U C U C C C G

70 20

100 ~~

A V U A I I

/ U U A C V C C C U U C C C G U A A C U U A 6 3 1

G G A U u

\" c c .L u

2: : " - , 4 1 0

C " C G

i l R " - II "

FIG. 2. A, predicted secondary structure of the 5'-109 nucleotides of polio type 1 (Mahoney) genomic RNA. B, predicted secondary structure of the 3"terminal 90 nucleotides plus the poly(A) tail. 3"Pseudoknot adapted from Ref. 28.

27006

A Polioviral Polypeptide 3 C P 0

Frc. 3. RNA gel shift activity of HeLa cell proteins with [(u-"P]UTF" labeled cloverleaf. A, gel shifts with Hela cell extracts. Lane 1 , probe incu- bated in buffer alone. Lane 2, probe incu- bated with 10 pg of protein from a polio- virus-infected HeLa lysate. Lane 3, probe incubated with 10 pg of protein from a mock-infected HeLa lysate. Lane 4, probe incubated with 10 pg of protein from a mock-infected HeLa lvsate and 3CDPm (0.2 p~ final concentration). Lane 5, probe incubated with 10 ue of orotein from a FrtcRobed mock-infected HeLa 510. 'Lane 6, probe incubated with 10 pg of protein from a mock-infected HeLa SI0 and 3CDP" (0.2

h e # 1 2 3 4 5 6 7 8 p~ final concentration). Lane 7, probe in- cubated with 10 pg of protein from a mock-infected HeLa SIOO. Lane 8, probe incubated with 10 pg of protein from a mock-infected HeLa SI00 and 3CDP" (0.2 p~ final concentration). B, protein compo- nents of complex B in the RNA gel shift assay. 2 pg of Fraction 6 (host factor) was added per binding reaction with various purified viral protein(s), the final concen- tration(s) of which is indicated. The host factor-induced complex (Complex A ) and the host factor-3CDP" complex (Complex B ) are indicated. Complex B exhibited two electrophoretic isoforms, occasionally even three isoforms as shown here.

B

Complex B

Complex A

LaneNo. 1 2 3 4 5 6 7 8 9 10 1 1 12

Polioviral Polypeptide 3CP" 27007

A

"97kD -66kD - p50 -43 kD

p36 -31 kD

- 21.5 kD - 14.4 kD

L a n e w 1 2 3 4 5 6 7 8

B l10 2P 3P 4P 5P 6P 190

is0 130 y o y o 170

V 0 IT0 1go 1po 290

2$0 230 2?o 2f0 2?o

270 270 2fjo 2po 390

M G K E K T H I N I V V I G H V D S G K S T T T G H L I Y K C G G I D K R T I E K F E K E A A E M G 70

I 90 K G S F K Y A W V L D K L K A E R E R G I T I D I S L W K F E T T K Y Y I T I I D A P G H R D F I K

N M I T G T S Q A D C A V L I V A A G V G E F E A G I S K N G Q T R E H A L L A Y T L G V K Q L I V

G V N K M D S T E P A Y S E K R Y D E I V K E V S A Y I K K I G Y N P A T V P F V P I S G W H G D N

M L E P S P N M P W F K G W K V E . R K E G N A S G V S L L E A L D T 1 L P P T R P T D K P L R L P L

Q D V Y K I G G I G T V P V G R V E T G I L R P G ~ V V T F A P V N I T T E V K S V E ~ H H E A L S

3tO 3:O 3 j O 3f 0 370

370 310 3 T O 3 T O 490

4$0 430 4 j O 4fO 470

470

E A L P G D N V G F N V K N V S V K D I R R G N V C G D S K S D P P Q E A A Q F T S Q V I I L N H P

G Q I S A G Y S P V I D C H T A H I A C K F A E L K E K I D R R S G K K L E D N P K S L K S G D A A

I V E M V P G K P M C V E S F S Q Y P P L G R F A V R D M R Q T V A V G V I K N V E K K S G G A G K

V T K S A Q K A Q K A G K FIG. 4. Purification and identification of host factor. A, fractionation of HeLa cell proteins. 25 pg of total protein was loadedflane on a

10%-20% gradient SDS-polyacrylamide gel except for lunes 6 and 7 where 10 pg of total protein was loaded. The gel was stained with Coomassie

tryptic digests of p50. Stippled burs indicate sequences obtained by Edman degradation of peptides derived from tryptic digests ofp36. for identity Brilliant Blue R-250. B, amino acid sequence of EF-la. Solid burs indicate sequences obtained by Edman degradation of peptides derived from

of p50 and p36, see Fig. 3B.

against bufferA(l0 mM Tris-HC1, pH 8.0,1.5 mM MgCl,, 10 mM KCl, 0.5 mM Dl", 0.5 mM PMSF, and 10% glycerol) with one buffer change. The dialysate was spun a t 3,000 rpm for 5 min to remove any particulate matter before loading the supernatant (Fraction 3) onto a 60-ml DEAE- cellulose column equilibrated with buffer A. The flow-through and a two-column volume wash were pooled, and 0.5 M Na,EDTA was added to a final concentration of 5 mM prior to ammonium sulfate fractionation. Proteins precipitating in the 3540% ammonium sulfate fraction (Frac- tion 4) were then dissolved in 10 ml of buffer B (10 mM MES, pH 6.1, 1.5

mM MgCI,, 10 mM KCI, 1 mM Dm, 0.5 mM PMSF, and 10% glycerol) and dialyzed against 1 liter of buffer B for 2 h with a buffer change. Fraction 4 was then loaded onto a Mono-S HR 5/5 column equilibrated with buffer B, and after a 5-column volume wash bound proteins were then eluted by a segmented gradient. The first segment consisted of a 10- column volume linear gradient from 10 to 100 mM KC1 followed by a 5-column volume wash of buffer B containing 100 mM KCl. The second segment consisted of a 10-column volume linear gradient from 100 to 500 mM KC1 followed by a 5-column volume wash of buffer B containing

27008 Polioviral Polypeptide 3CDP"

E! Competitor RNAs

+-Cloverleaf Fragments +

o IO so 250 IO so 250 IO so 250 IO so 250 IO 50 250 MolarExass

FIG. 5. Competi t ion experiments to demonst ra te the specificity of clover- leaf binding of the host factor-3CDP" complex to the full-length cloverleaf. The concentration of the "'P-labeled probe in the bindine reaction was 1.1 nM as me- viously determined (10). Blue S&t denotes RNA transcribed from the ComplexB polylinker region of the Blue Script plas- mid (100 nth

Complex A

LamNo. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

FIG. 6. Characterization of the 3AB- 3CDPm-cloverleaf RNA complex. Puri- tied 3AB was present a t a final concentra- tion of 0.5 PM in reactions were indicated. The concentrations of 3CPm, 3Dw', and 3CDPm in the binding reactions where the purified proteins were added are indi- cated. The asterisk indicates an altered migration of the probe through the non- denaturing gel occasionally seen.

LancNo. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

500 mM KCl. Peak activity was found in the 100 mM to 500 m~ KC1 gradient eluate and pooled (Fraction 5). Fraction 5 was dialyzed against 1 liter of buffer C (10 mM HEPES, pH 7.4, 1.5 mM MgCl,, 10 mM KCl, 1 mM DTT, 0.5 mM PMSF, and 10% glycerol) overnight with a buffer change then loaded onto a 1-ml heparin-Sepharose column equilibrated with buffer C. After a 4-column volume wash, peak activity was found in the 300 mM KC1 step-elution. This fraction (Fraction 6) was used in all subsequent reactions and consisted of a BO-kDa protein and a 36-

kDa protein present at 90 and 5%, respectively, as judged by SDS- polyacrylamide gel electrophoresis and staining with Coomassie Bril- liant Blue R-250. Fraction 6 was stored a t -80 "C.

UV Cross-linking-RNA uniformly labeled with [(U-~~PIUTP (200,000 counts) was incubated with cellular and/or viral proteins in the binding reaction described above. The reactions were UV cross-linked using a UV Stratalinker 1800 from Stratagene for 30 min and then digested by RNases A, T1, T2, and V1 for another 30 min a t 37 "C. An equal volume

Polioviral Polypeptide 3CDp" 27009

FIG. 7. Competition experiments to demonstrate the specificity of bind- ing of 3AB-3CDPm to the full-length cloverleaf. The concentration of the probe in the binding reaction was 1.1 nM as previously determined (18). The aster- isk indicates the slower migration of the +ABCD/-ABCD duplex RNA.

LaneNo. 1 2 3 4

of 2 x protein sample buffer (125 mM Tris-HC1, pH 6.8, 4% SDS, 50% glycerol, 1.3 M P-mercaptoethanol, and a trace amount of bromphenol blue) was added to the reactions and loaded onto a 13% SDS-polyacryl- amide gel electrophoresis gel. Gels were dried and subjected to autora- diography.

Sequence Determination of p5O and p36"Fraction 6 was separated on a SDS 1620% gradient polyacrylamide gel and electroblotted onto Problot membrane (Applied Biosystems). p50 and p36 were excised and submitted to in situ digestion with trypsin (19). A Vydac C18 2.1 mm x 150 mm high performance liquid chromatography reverse-phase col- umn was used to isolate tryptic peptides for sequencing via an auto- mated Edman degradation Applied Biosystems 477A protein sequencer. Further details of protein microsequencing have been previously described (20).

RESULTS

Purification and Identification of the Host Factor-Comput- er-aided folding (211, phylogenetic comparisons, and biochemi- cal analyses (10, 11, 22) have identified a highly structured element formed at the 5'-end of poliovirus RNA that is referred to as a cloverleaf (Fig. 2A) . Transcripts consisting of 109 5'- terminal nucleotides of poliovirus have been described recently to bind 3CDP" in the presence of a 36-kDa cellular protein (HF (11,12). We have used [a-32PlUTP-labeled cloverleaf RNAin gel shifts assays to monitor the purification of HF from extracts of uninfected HeLa cells. In the assay, constant amounts of probe and purified 3CDP" were incubated with cellular fractions, and the mixtures were subjected to "native" polyacrylamide gel electrophoresis as described under "Materials and Methods." As can be seen in Fig. 3A, cellular protein(s) were able to produce a shift of the probe (complex A ) (lane 3), while an additional shift (complex B ) was observed in the presence of purified 3CDP'O (lane 4 ). This shift is identical to the shift seen when the probe is incubated with a poliovirus-infected HeLa extract (lane 2). Moreover, all of the complex B-forming activity

*

5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

was found in the SlOO supernatant of a HeLa cell cytoplasmic extract (lane 8).

Purification of the gel shifting activity contained in 20 liter of HeLa cells (4 x lo" cells/ml) yielded two proteins that co-puri- fied in all steps of the procedure (Fig. 4A). Reproducibly, one of the proteins, referred to as p50, was in large excess over the smaller protein, referred to as p36 (lane 7). Tryptic peptides of polypeptides p50 and p36 were microsequenced according to a previously described procedure (19, 20) and a search in the Genbank (23) of the peptide sequences of p5O revealed that p50 was the eukaryotic elongation factor la (EF-la; Fig. 4B, solid bars). Surprisingly, p36 yielded identical peptide sequences originating from the NH,-terminal two-thirds of EF-la (Fig. 4 B , stippled bars). Two possibilities may explain this phenom- enon; either p36 is a naturally occurring product in HeLa cells with a specific function distinct from that of EF-la, or i t is the result of proteolytic degradation of EF-la that took place dur- ing preparation of the cell extract. Splice variants of EF-la, yielding a product of 36-kDa protein, are not known.3 On the other hand, some proteolytic degradation of the abundant elon- gation factor (-4% of total protein in HeLa cells) is likely even though we employed a battery of protease inhibitors during the initial stages of cell fractionation (see "Materials and Meth- ods").

Protein Components of Complex B and Specificity of Clover- leaf Binding-Only purified 3CDP" was used in the gel shift assay with p36. As can be seen in Fig. 3B, neither 3CP" nor 3DV' mimicked the shifting activity of 3CDPm even when the former proteins were present in the binding reaction a t a 10 molar excess relative to 3CDPm. Purified EF-la was observed to shift the cloverleaf, but it did not give rise to complex B when it was

W. C. Merrick, personal communication.

27010 Polioviral Polypeptide 3CDP"

200- 97- 68 kD-

43 kD-

31 kD-

14 kD-

Laae No. 1 2 3 4 5 6 FIG. 8. UV cross-linking of S2P-labeled cloverleaf RNA to host

factor 3AB and 3CDPm. 32P-Labeled cloverleaf RNA(200,OOO cpm) was incubated with HF, 3AB, or 3CDPm in various combinations. After U V cross-linking and digestion with nucleases, the reactions were loaded onto a 1 3 1 SDS-polyacrylamide gel. Lane I , I4C-labeled molecular

3, probe incubated with host factor (2 pg) and 3CDPm (0.2 mM final weight markers. Lane 2, probe incubated with host factor (2 pg). Lane

concentration). Lane 4, probe incubated with 3AB (0.25 PM final con- centration). Lane 5, probe incubated with 3CDPm (0.2 p~ final concen- tration). Lane 6, probe incubated with 3AB and 3CDPm (0.25 and 0.2 PM final concentration, respectively). Arrows indicate 3CDPm and its cis cleavage products, P 3 4 a and 3Dp'. The asterisk indicates undigested cloverleaf RNA.

incubated with 3CDP" and the cloverleaf (data not shown). To determine the specificity of HF-3CDP" complex binding to

the cloverleaf (complex B formation), specific and nonspecific cold competitor RNAs were added to the binding reaction con- comitantly with the labeled cloverleaf (Fig. 5). The different stem-loops of the cloverleaf were designated A, B, C, and D (see "Materials and Methods," and Fig. 2A 1. It is apparent that the full-length cloverleaf was the only molecule able to compete for complex B formation.

3AB-3CW" Interaction with the Cloverleaf and Specificity of Binding-Although the interaction between HF-3CDPm and clo- verleaf produced a specific shift in gel electrophoresis, we en- tertained the possibility that the viral protein 3AB, the precur- sor for the genome-linked protein VPg (24,25) could replace HF as the natural partner of 3CDP'" in the cloverleaf binding reac- tion. This was based on the observations that 3AB binds to 3CDP" or 3DP1 in the absence of RNA (41.'

When incubated alone with cloverleaf RNA, in the presence of an excess tRNA, neither 3AB, 3CDPm, 3CPr0, nor 3Dp' could bind to the cloverleaf (Fig. 6, lanes 2-5). However, when 3AB and 3CDP" were combined together efficient binding was ob- served (lane 6). Genetic evidence suggests that the RNA-bind- ing domain of 3CDPm resides in the 3C moiety (10, 11). We were not surprised, therefore, to observe that 3AB-30" also pro- duced a gel shift albeit a t a proteinase concentration 10 times the molar concentration of 3CDP" (lanes 7-9). This suggested

that a portion of 3AB has binding affinity to 3CPr0 and that this complex interacts with the cloverleaf, a result contrasting bind- ing experiments with HF + 3CPm (Fig. 3B). Interestingly, puri- fied polymerase 3DP', when incubated with 3AB, did not react with the cloverleaf (Fig. 8, lanes 10-12). However, 3DP" has binding affinity to 3AB because, in a mixture of 3DP"', 3CPrn, and 3AB, 3Dw' prevents 3AB-3CP" from binding to the probe. In fact, the presence of 3DP"' inhibits the interaction of 3AB-3Cp" with the probe even when 3CP" is present in the reaction a t a 20 molar excess relative to 3DW1 (lanes 13-19). These data are suggestive of a high affinity interaction between 3AB and 3DP', a result that was proven to be the case in separate studies (6; see "Discussion").'

Competition experiments with the cold RNAs indicated that the formation of the 3AB-3CDPm-RNA complex requires the entire cloverleaf (Fig. 7, lanes 8-10). Interestingly, it appeared as if the minus strand cloverleaf (-ABCD) which is predicted to form a structure complementary to the plus strand cloverleaf could compete at 250 molar excess (lane 22). However, it is apparent that much of the unbound plus strand probe migrated slightly slower than in other lanes (lane 22, asterisk). This could be interpreted to mean that the plus and minus probes hybridized during incubation thereby removing free plus strand probe from the binding reaction. Some weak interaction between 3AB-3CDP" and -ABCD, however, cannot be excluded at the present time.

UV Cross-linking Using Labeled Cloverleaf RNA-Upon in- cubation of [~r-~~P]UTP-labeled cloverleaf RNA (200,000 cpm) with Fraction 6, U V cross-linking, and RNase digestion, EF-la (p50) and AEF-la (p36) were labeled (Fig. 8, lane 2). However, when purified 3CDPr0 was added to this mixture, p36 and 3CDP" were labeled but, significantly, p50 was not (compare lanes 2 with lane 3). 3AB alone did not cross-link, and 3CDP" alone demonstrated very weak cross-linking to the cloverleaf (lanes 4 and 5). In sharp contrast, the 3AB-3CDPm complex led to ex- tensive labeling of 3CDP" (and of 3DPl and P3-4a, two cleavage products of 3CDP"), but very little if any labeling of 3AB (lane 6). 3CDPm was present at a final concentration of 0.2 p~ in all incubations, and the concentration of HF and 3AB were also comparable.

RNA Footprinting-To determine contact points between the polypeptides and the cloverleaf, footprinting experiments were performed. 5'-End-labeled cloverleaf RNA was added to bind- ing reactions that contained either HF alone, HF-3CDPro, or 3AB-3CDPm. After the 10-min binding period at 30 "C, ribo- nucleases T1 or T2 (single strand-specific) or cobra venom nuclease V1 (double strand-specific) were added, and incuba- tion was continued for an additional 10 min. Bound RNA.protein complexes and free probe were then processed as described under "Materials and Methods," and equal counts from each complex were analyzed by polyacrylamide gel elec- trophoresis. As shown in Fig. 9, binding of HF alone appears to cause considerable structural changes in the cloverleaf as it renders the probe more sensitive to nucleases T1 (see, for ex- ample, Ga, lane 4 ) and T2 (Gd6 and C,, lane 9). The most sensitive bases in the unprotected probe to cleavage by T2 are U,, and 4, (lanes 8 and 12). In general, the HF-3CDP" and 3AB-3CDPr0 complexes protected with similar efficiency against the pronounced cleavages by single strand-specific nucleases. An exception is cleavage at G,, which is clearly enhanced in the complex with either HF-3CDP" or 3AB-3CDP'" (compare lanes 5 and 6 with 7). Comparison of V1 cleavages led us to conclude the HF-3CDP" and 3AB-3CDP" complexes bind preferentially to single-stranded loop regions (lanes 13 to 17).

Interaction of 3AB and 3CP" with the 3'-Non-translated Region-Whereas complex formation between 3AB-3CDP" and

FIG. 9. RNAfootprint of cellular and viral proteins bound to 5"end-la- beled cloverleaf RNA using T1, T2, and V1 nucleases. Lanes 1 and 18, alka- line ladder of the cloverleaf; the number- ing does not include the first 2 G residues derived from the T7 promoter. Lane 2,

Lanes 3 and 7, probe incubated with T1 probe incubated in binding buffer alone.

nuclease. Lane 4, probe incubated with host factor and T1 nuclease. Lane 5, probe incubated with host factor, 3CDPm, and T1 nuclease. Lane 6, probe incubated with 3AB, 3CDPm, and T1 nuclease. Lanes 8 and 12, probe incubated with T2 nuclease. Lane 9, probe incubated with host factor and T2 nuclease. Lane IO, probe incu- bated with host factor, 3CDP", and T2 nuclease. Lane 11, probe incubated with 3AB, 3CDPm, and T2 nuclease. Lanes 13 and 17, probe incubated with V1 nucle- ase. Lane 14, probe incubated with host factor and V1 nuclease. Lane 15, probe incubated with host factor, 3CDPm, and V1 nuclease. Lane 16, probe incubated with 3AB, 3CDP'" and V1. Stem-loop domains are indicated on the sides.

Polioviral Polypeptide 3CDp"

I T1 T2 t 27011

&

LancNo. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

I" 114 f

[ 1 -

the cloverleaf may be crucial for the initiation of RNA plus strand synthesis (see "Discussion"), we have also explored the possibility that a similar complex could form at the 3'-end of the poliovirus genome to facilitate initiation of minus strand RNA synthesis. An RNA probe consisting of the 3'-terminal 108 nucleotides plus poly(A) (Fig. 2 B ) was prepared by run-off tran- scription. This probe contains a segment of the genome that is conserved among several enteroviruses (26) with the propen- sity to form a pseudoknot (27, 28). Based on genetic and bio- chemical data, this structure may be involved in the initiation of minus strand RNA synthesis (28, 29).

Recently, i t has been observed that incubation of either

poiy(A)/primer' or the 3'-PK-poly(A) probe4 with 3AB produces a gel shift of the labeled RNA. We have incubated either 3AB or 3CDPm alone or 3AB and 3CDP" together with [ C ~ - ~ ~ P ] U T P - ~ ~ - beled 3' PK probe and U V cross-linked as described above. Surprisingly, 3AB binds to the probe (Fig. 10, lane 31, a result that contrasts binding experiments with the 5"terminal clover- leaf. 3CDP" has no apparent affinity to the 3"probe under these conditions (lane 4 ) whereas this protein is efficiently cross- linked when presented to the RNAin the presence of 3AB (lane 5). Interestingly, two cleavage products of 3CDPm, 3Dw' and

A. V. Paul and E. Wimmer, unpublished observations,

27012 Polioviral Polypeptide 3 C P 0

mkD-

WkD- 68kD-

43 kD-

31 kD-

14kD-

-3cD

r3b-p

-x

-3AB

LaneNo. 1 2 3 4 5 6 FIG. 10. Binding of 3AB and 3CDP" to the 3'-pseudoknot.

32P-Labeled 3"PK (200,000 cpm) was incubated with 3AB or 3CDPm separately and together. After UV cross-linking and digestion with nucleases, the reactions were loaded onto a 107~20% SDS-polyacrylam- ide gel. Lane 1, 'IC molecular weight marker. Lane 2, probe incubated alone. Lane 3, probe incubated with 3AB (0.25 PM final concentration). Lane 4, probe incubated with 3CDPm (0.2 PM final concentration). Lane 5, probe incubated with 3AB and 3CDPm (final concentrations 0.25 and 0.2 PM, respectively). Lane 6 , ["%]Met-labeled poliovirus-infected HeLa cell extract. Asterisk indicates undigested probe. Arrows indicate 3CDPm cis cleavage products, P 3 4 a , and 3DP"'.

P3-4a (a polypeptide consisting of a COOH-terminal segment of 3C"" and all of 3DP'') (301, also cross-link to the probe. This observation suggests that, in the presence of 3AB, the binding of 3CDP'O to the 3"PK probe occurs via contact points in 3DW'. Competition experiments to determine the specificity of 3"PK binding by 3AB and 3CDP" are underway as well as RNA foot- printing to determine the site of the 3'-PK- 3AB/3CDPm inter- action.

DISCUSSION

All mechanisms that have been proposed to function in the initiation of poliovirus RNA synthesis must take into consider- ation that newly synthesized RNA strands are VPg-linked (31). The polypeptide that may deliver VPg to the sites of initiation is 3AB, a protein found to be membrane-associated in infected HeLa cells (25, 32). Indeed, genetic and biochemical evidence supports the notion that 3AB is involved in viral RNA synthesis (6,33,34). We have proposed a mechanism of initiation of RNA synthesis by which the membrane-associated 3AB is uridyly- lated, then cleaved to release VPg-pU. The peptidyl nucleotide, in turn, will act as a primer for 3DW' (13, 14,24). Moreover, we have recently suggested that poliovirus double-stranded RNA (RF), is a replication intermediate and not a by-product of RNA synthesis (1).

The question of how the assembly of the membrane-associ- ated initiation complex proceeds remains unanswered. Data reported by Andino et al. (10-12) suggested that, dependent

upon a host cell polypeptide (HF), the poliovirus proteinase 3CDPm has the ability to bind to the 5'-end of the viral RNA. I t was speculated that the complex formation at the cloverleaf serves to bring the precursor of the polymerase to the site of initiation of plus strand synthesis (12). Based on the data re- ported here, we believe that the viral protein 3AB, rather than the host factor HF, is the partner for 3CDP" to bind to the cloverleaf of the genome.

In our studies a purified derivative of 3CDPm was used that is impaired in self-cleavage but has retained all other properties known for this protein (hence we refer to it as 3CDP"; Ref. 9). The cellular activity capable of complex B formation with 3CDP'" was contained in both the ribosomal salt wash and in the SlOO cytoplasmic fraction for which reason we have com- bined these fractions in the isolation procedure. Of the two cellular polypeptides (p50 and p36) that stubbornly copurified in all steps, only p36, when incubated with 3CDPr0, gave rise to complex B as described before (12). Yet we were surprised to note that p36 was not a bona fide host cellular protein. Instead, our data suggest that HF is a NH,-terminal proteolytic frag- ment of p50, the latter being the eukaryotic elongation factor EF-la. EF-la, one of the most abundant proteins in the host cytoplasm, is an essential component of the translational ma- chinery (35). Its identification as a possible partner in poliovi- rus genome replication was exciting because the prokaryotic elongation factors EF Tu and EF Ts are essential components of phage Qp replicase (36). Disappointingly, however, purified EF-la when incubated with 3CDPr", did not give rise to a com- plex, corresponding to complex B (data not shown). I t is, on the other hand, quite perplexing that an NH,-terminal proteolytic fragment (AEF-la) of EF-la would be involved in poliovirus replication. I t is possible that the p36 fragment originated from nonspecific degradation during the preparation of the cell ex- tracts and that it is, therefore, an artificially produced factor. However, we cannot exclude the possibility that the p36 de- scribed previously (12) and AEF-la described here may be dif- ferent proteins.

Several observations concerning the properties of the polio- virus polypeptides 3CDPT0, 3D"l, and 3AB prompted us to test whether 3AB can replace a host factor in the gel supershift assay. 3AB and 3CDPm form a complex that leads to the accel- erated autoproteolysis of 3CDP" (4). 3CDP'" is the proteinase that cleaves membrane-bound 3AB to VPg and 3A (6), and 3AB is a stimulating factor of 3D"' polymerase activity (61.' These reactions can be summarized as follows: 1) 3AB + 3CDP" -+

3D]"""'P"l; 3) [membrane-3AB] + 3CDP'" .--f VPg + 3A + 3CDP". The observation that the 3AB-3CDPm complex binds strongly

to the 5"terminal cloverleaf fits a model of the assembly of a replication complex at the left end of poliovirus RF. However, we consider it also significant that 3AB-3CDP'" has the propen- sity to bind to a 3"terminal fragment of the poliovirus genome (Fig. lo).* The binding parameters of the formation of the com- plex on either end of the genome, however, are quite different, as would be expected. Whereas sequences in the 3C moiety of 3CDPm participate in binding to the cloverleaf (Fig. 8 , the bind- ing to the 3'-terminal fragment involves sequences of the 3D moiety (Fig. 101.'

Recent genetic and biochemical analyses of the FG loop of poliovirus 3Cpr0 have identified mutations conferring a null phenotype to poliovirus without interfering with proteolytic activity (37). We suggest that these mutations impair RNA binding of 3CDPm andor the interaction of 3CDPm with 3AB. Porter and his colleagues (38, 39) have recently proposed that 3CP'" of human rhinovirus 14 (HRV14) binds to the 5'-NTR of HRV14 RNA, and 3DW' of encephalomyocarditis virus (EMCV)

[3AB-3CDProl -+ 3AB + 3DW' + 3CP'"; 2) 3AB + 3D"' + [3AB-

Polioviral Polypeptide 3CP" 27013

ApA Plus strand Upup@ Minus strand

Proteolytic Processing

J.

Plus strand - Synthesis

t l

Production of VPg-linked plus strand genomic RNAs

( 5 ) Replicative Intermediate (RI)

FIG. 11. Model of initiation of polioviral RNA replication. Step 1, initiation of minus strand synthesis mediated by a 3'-NTR poly(A)-3AB- 3CDPm ribonucleoprotein. Step 2, minus strand synthesis and cleavage of VPg from the 3AB precursor generating the replicative form (RF). Step 3, recognition of cloverleaf structure by the 3AB-3CDPm complex; helicase activity (2CP) unwinds the double-stranded RNA duplex at the B'-end of the plus strand allowing the cloverleaf structure to form. The 3AB-3CDPm complex then recognizes the cloverleaf structure. Step 4, initiation of plus strand synthesis is followed by proteolytic processing of 3CDPm in cis to activate 3DP'. Step 5, formation of replicative intermediate (RZ).

binds to the 3'-NTR-poly(A) fragment of EMCV RNA. Both plex (Step 3). Initiation then occurs on the free 3' terminus of HRV14 and EMCV belong to the same virus family (Picorna- the minus strand RNA by [3AB-3DlsU~"PO' (Steps 4 and 5). viridae) as poliovirus. Although no second proteins were found It is apparent that a major step of poliovirus RNA synthesis to be necessary for binding of HRV14 3CP'" or EMCV 3DP0' to has yet to be elucidated: the covalent linkage of VPg to the their cognate RNA fragments, a general pattern of affinity of nascent RNA strands. We have shown previously that synthe- picornavirus 3C- and 3D-related sequences to, respectively, 5'- sis of VPg-pU occurs in membrane-bound replication complexes and 3"terminal sequences appears to emerge. However, these data do not exclude the possibility that other viral proteins may participate in RNA binding. HRV14 3Cpr0 is needed at a final concentration of 17 p~ in order to achieve efficient binding (381, whereas poliovirus 3Cp'" can efficiently bind the cloverleaf at a final concentration of 2 p~ if 3AB is present at 0.25 p~ (Fig. 6). A detailed analysis of the contact points between poliovirus proteins and the pseudoknot-poly(A) of the 3"terminal poliovi- rus RNA fragment is underway.

A model combining these considerations is shown in Fig. 11, Initiation of plus strand RNA synthesis involves formation of a 3'4erminal ribonucleoprotein complex followed by proteolysis of 3CDPm to activate the polymerase activity (Step 1). 3AB and 3DP"' then form the enzyme [3AB-3DlSu"P"'P"' that may use uri- dylylated VPg (released from membrane-bound 3AB by 3CDP") to synthesize minus strands leading to the formation of RF molecules (Step 2). A hypothetical helicase, possibly poliovirus polypeptide 2C (see Ref. 5)' may then melt the left end of RF thereby allowing formation of the 3AB-3CDPr0-cloverleaf com-

isolated from infected HeLa cells and that the peptidyl nucle- otide is elongated to longer VPg-RNA molecules (14, 15). At- tempts to uridylylate purified 3AB or VPg in vitro have failed.5 Clearly, an essential component(s) for this reaction is still miss- ing. It is possible, and even likely, that host cellular proteins are involved in any of the processes described here. EF-la remains a candidate as a cofactor in one of the reactions leading to poliovirus genome replication.

Bernhardt, Nick Delihas, and Matthew Schmidt for technical advice Acknozdedgments-We are grateful to Patrick Hearing, Giinter

and stimulating discussions. We are particularly indebted to William C. Memck for suggestions and the gift of purified EF-la and to Stephen J. Plotch for the gift of purified poliovirus 3DP'. We offer special thanks to J o h n Mugavero and Renee Robinson for excellent technical assis- tance, Peter Kissel for oligonucleotide synthesis, and Chris Helmke for photography.

K. S. Harris, W. Xiang, L. Alexander, W. S. Lane, A. V. Paul, E. Wimmer, unpublished observations.

27014 Polioviral Polypeptide 3 C P o

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