Leader Protein of Encephalomyocarditis Virus Binds Zinc, Is Phosphorylated during Viral Infection, and Affects the Efficiency of Genome Translation

Virology 290, 261–271 (2001) doi:10.1006/viro.2001.1193, available online at http://www.idealibrary.com on

Leader Protein of Encephalomyocarditis Virus Binds Zinc, Is Phosphorylated during Viral Infection, and Affects the Efficiency of Genome Translation Cheryl M. T. Dvorak,* ,1 David J. Hall,† ,1,2 Marchel Hill,㛳 Michael Riddle,‡ Andrew Pranter,§ Johnathan Dillman,‡ Michael Deibel, ¶ and Ann C. Palmenberg㛳 *Department of Veterinary PathoBiology, University of Minnesota, Minneapolis, Minnesota 55455; †Department of Biomolecular Chemistry, University of Wisconsin—Madison, Madison, Wisconsin 53706; ‡Indiana University School of Medicine, Indianapolis, Indiana 46202; §Department of Chemistry, University of Pennsylvania, Philadelphia, Pennsylvania 19104; ¶ Department of Chemistry, Earlham College, Richmond, Indiana 47374; and 㛳Institute for Molecular Virology, University of Wisconsin—Madison, Madison, Wisconsin 53706 Received January 19, 2001; returned to author for revision July 20, 2001; accepted September 12, 2001 Encephalomyocarditis virus (EMCV) is the prototype member of the cardiovirus genus of picornaviruses. For cardioviruses and the related aphthoviruses, the first protein segment translated from the plus-strand RNA genome is the Leader protein. The aphthovirus Leader (173–201 amino acids) is an autocatalytic papain-like protease that cleaves translation factor eIF-4G to shut off cap-dependent host protein synthesis during infection. The less characterized cardioviral Leader is a shorter protein (67–76 amino acids) and does not contain recognizable proteolytic motifs. Instead, these Leaders have sequences consistent with N-terminal zinc-binding motifs, centrally located tyrosine kinase phosphorylation sites, and C-terminal, acid-rich domains. Deletion mutations, removing the zinc motif, the acid domain, or both domains, were engineered into EMCV cDNAs. In all cases, the mutations gave rise to viable viruses, but the plaque phenotypes in HeLa cells were significantly smaller than for wild-type virus. RNA transcripts containing the Leader deletions had reduced capacity to direct protein synthesis in cell-free extracts and the products with deletions in the acid-rich domains were less effective substrates at the L/P1 site, for viral proteinase 3C pro. Recombinant EMCV Leader (rL) was expressed in bacteria and purified to homogeneity. This protein bound zinc stoichiometrically, whereas protein with a deletion in the zinc motif was inactive. Polyclonal mouse sera, raised against rL, immunoprecipitated Leader-containing precursors from infected HeLa cell extracts, but did not detect significant pools of the mature Leader. However, additional reactions with antiphosphotyrosine antibodies show that the mature Leader, but not its precursors, is phosphorylated during viral infection. The data suggest the natural Leader may play a role in regulation of viral genome translation, perhaps through a triggering phosphorylation event. © 2001 Academic Press

Key Words: picornavirus; enzyme purification; cardiovirus; translation.

ditis virus (EMCV), a prototype member of the cardiovirus genus, the IRES is a self-contained, 450-base segment which lies immediately 5⬘ of the polyprotein initiation codon (Jang et al., 1988; Evstafieva et al., 1991; Duke et al., 1992; Howell et al., 1990; Jackson et al., 1990). During translation of picornavirus genomes, the encoded polyproteins are processed by virally encoded proteases in a defined series of co- and posttranslational reactions to produce all the mature viral proteins required for an infectious cycle. The polyprotein features and their associated mature proteins are named according to an L-4-3-4 convention that subdivides them by related function and cleavage sites (Rueckert and Wimmer, 1984). The four P1 proteins (1A, 1B, 1C, and 1D) are virion structural components that function collectively as a promoter unit during viral capsid assembly. The three P2 region proteins (2A, 2B, 2C) and four P3 region proteins (3A, 3B, 3C pro, 3D pol) comprise the nonstructural components of the viral RNA-dependent RNA synthesis complexes, or contribute otherwise to intracellular virus– host interactions (Rueckert and Wimmer, 1984).

INTRODUCTION The picornavirus family of nonenveloped, positivesense RNA viruses is taxonomically divided into six genera, the aphthoviruses, cardioviruses, enteroviruses, hepatoviruses, parechoviruses, and rhinoviruses (King et al., 2000). The infectious, single-stranded genomes have long 5⬘-untranslated regions (5⬘UTRs), single open reading frames (ORFs) encoding large polyproteins, and 3⬘untranslated regions (3⬘UTRs) ending with 3⬘-poly(A) tails (Rueckert, 1996). The 5⬘UTRs contain multiple genus-specific or strain-specific RNA structural motifs that contribute variously to viral infectivity. Among these, and characteristic of all picornaviruses, is an internal ribosomal entry site (IRES) that directs cap-independent translation of the viral polyprotein. For encephalomyocar-

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These authors contributed equally to this work. To whom correspondence and reprint requests should be addressed at Dept. of Biomolecular Chemistry, Room 571 MSC, 1300 University Avenue, Madison WI 53706. Fax: 608-262-5253. E-mail: [email protected]. 2

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0042-6822/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.

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The polyproteins of cardioviruses and aphthoviruses are different from the other picornaviruses in that their ORFs encode an additional N-terminal Leader peptide, L, that precedes the P1 capsid region (Rueckert, 1996). In both genome types, the Leader is the first sequence to be translated, and it is subsequently released from its polyprotein precursor by virally encoded proteolytic events unique to each genus. For aphthoviruses, as typified by foot-and-mouth disease virus (FMDV), there are two forms of Leader protein, 201 or 173 amino acids in length, that arise from different in-frame AUG start sites near the IRES (Belsham, 1992). Both of these share carboxy-terminal sequences that encode a self-cleaving acid proteinase with sequence and structural similarities to papain (Piccone et al., 1995; Kleina and Grubman, 1992). Aphthovirus Leaders cleave themselves autocatalytically from the viral polyprotein, and after release, catalyze an additional cleavage of translation initiation factor eIF-4G, a component of the eukaryotic cap-binding complex, thus contributing to viral-induced shut-off of host protein synthesis during infection (Roberts and Belsham, 1995; Devaney et al., 1988). Cardiovirus Leaders have different organizations and presumed functions than the aphthoviruses (Mosenskis et al., 1985). The EMCV-like viruses (e.g., Mengo, CSK, ME) have short Leaders that are 67 amino acids long (Palmenberg et al., 1984; Duke et al., 1992). The related Theiler’s murine encephalitis viruses (TMEV) have Leaders that are slightly longer (76 amino acids), and they only share about 50% aligned amino acid identity with EMCV (Chen et al., 1995). Unlike the aphthoviruses, neither type of cardiovirus Leader has sequences consistent with an inherent proteolytic function (Robertson et al., 1985). Rather, these Leaders are released (from L-P1-2A precursors) by posttranslational trans reactions with viral protease 3C pro, encoded in the distal region of each genome (Palmenberg and Rueckert, 1982; Palmenberg, 1982). The mechanistic contribution of cardioviral Leaders to viral infectivity is still an open question, although clues do exist that suggest potential functions. The TMEV Leader proteins bind zinc ions in interactions that are dependent upon a conserved zinc-finger motif (CHCC type) near the NH 2-terminus of the protein (Chen et al., 1995). This sequence is common to all cardioviruses but atypical among the more canonical proteins that exhibit zinc fingers and usually distribute the Cys and His residues in CCHC or CCCC patterns (Berg, 1986). Bacteriophage gene32 protein and HIV nucleocapsid protein (NC) are other examples of this cardiovirus-like CHCC motif (Giedroc et al., 1986; Coleman, 1992). The cardiovirus zinc finger, as with other such motifs, is probably also a nucleic acid binding signal (Chen et al., 1995; Kong et al., 1994; Pieler and Bellefroid, 1994; Draper, 1995; Aitken, 1999; Kong et al., 1994). In 1995, Hoffman and Palmenburg demonstrated that point muta-

tions in the IRES of EMCV reduced the translational efficiency of the viral RNA (Hoffman and Palmenberg, 1995), but as a surprising consequence of these experiments, it was also noted that phenotypic reversions of these mutations mapped to protein changes in the zinc finger of nearby Leader (Hoffman and Palmenberg, 1996), suggesting the Leader may be involved in IRES function and may even interact with this region of the RNA. Although EMCV IRES-dependent translation of nonviral genes does not require coexpression of other viral sequences, including the Leader (Witherell et al., 1995; Duke et al., 1992; Jang et al., 1988, 1989; Kaminski et al., 1990), the participation of certain cellular translational factors is conditional on the presence of Leader (Kaminski and Jackson, 1998). The relative efficiency of IRESdirected translation in cell-free extracts, for example, is dependent upon mRNA interactions with the cellular pyrimidine tract binding protein, PTB, unless the Leader protein is one of the products synthesized by the reaction (Kaminski and Jackson, 1998; Kaminski et al., 1995). Additional experiments with TMEV have shown that engineered deletions in the Leader gene gave viruses with altered growth patterns in BHK-21 cells. Such viruses also had lower infectivity than L929 cells and were attenuated in mice (Zoll et al., 1996; Kong et al., 1994; Calenoff et al., 1995). More recently, Badshah et al. (2000) examined another virus with a TMEV Leader deletion and determined that RNA translation, replication, and virion assembly were all diminished relative to the parental virus. Given the differences, outside of the CHCC motif, between the Leader sequences of EMCV and TMEV, it is not yet clear whether all cardiovirus Leaders have synonymous functions during infection, but the current data are consistent with the idea that Leaders probably serve some sort of regulatory role, perhaps in concert with the IRES during viral polyprotein translation. We have begun to examine EMCV Leader with the intention of creating recombinant reagents and assays that will enable a more thorough characterization of its infectious contributions and its (putative) interactions with the EMCV IRES. Three deletion sequences are now described that removed large portions of the Leader gene. Recombinant genomes carrying these deletions were found to be less effective mRNAs than wild-type sequences in cells and cell-free assays, and resultant progeny viruses had slower growth rates in HeLa cells, confirming a vital role for Leader in the EMCV replication cycle. Recombinant EMCV Leader protein has been expressed and purified. Zinc assays demonstrate that as with TMEV, the EMCV Leader binds zinc in reactions that require the zinc finger motif near the N-terminus of the protein. Immunoblot experiments with anti-Leader polyclonal sera further suggest that Leader precursors predominate over the mature form of the protein in infected HeLa cells, and surprisingly, the mature protein that could be detected was in a phosphorylated state and

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FIG. 1. Leader protein sequences. (A) Nonredundant Leader protein sequences representing the TMEV (top) and EMCV (bottom) clades of cardioviruses are aligned for comparison. A complete alignment of cardioviral polyproteins is available on the web (www.bocklabs.wisc.edu/acp). Uppercase letters and the Consensus line mark residues that are identical, or at least very similar (e.g., D or E), among all viruses. The CHCC zinc finger motif, [C-X-H-X(6)-C-X(2)C], the putative Tyr phosphorylation site, [K-X(2)-E-X(2)-Y], and the acid-rich domain, (D ⫹ E ⫽ 44%), are highlighted. (B) The Leader protein sequences encoded by pEC 9 and the three deletion plasmids are illustrated. Dots (. . .) denote relative indels.

reactive with antibodies specific for phosphotyrosine residues. RESULTS Leader protein motifs Leader sequences from the TMEV and EMCV clades of the cardiovirus genus were aligned with the PILEUP program of the Wisconsin Software Package (Version 10.1, Genetics Computer Group, Inc.). Figure 1A (Consensus) highlights the 33 (of 67) residues in EMCV-R that are identical or nearly identical among all viruses. The aminoterminal CHCC zinc finger, C-X-H-X(6)-C-X(2)-C, and acidrich carboxyl portion of these proteins have been described (Chen et al., 1995). Comparison of the EMCV-R and Mengo-M sequences with the Prosite database (http://expasy.cbr.nrc.cs) identified an additional motif characteristic of Tyr kinase phosphorylation sites (Prosite: PDOC00007). This motif, [KR]-X(2,3)-[ED]-X(2,3)-Y, is present in all EMC-like viruses (Hagemann and Rapp, 1999), but not the TME-like viruses, and requires a lysine or arginine located six to seven residues N-terminal to the reactive tyrosine, with at least one acidic residue (D or E) centered between these sites (Aitken, 1999; Hagemann and Rapp, 1999). Leader affects virus growth Deletion mutations removing the zinc-binding motif, the acidic domain (inclusive of the putative Tyr phosphorylation site), or both segments were engineered into EMCV plasmid cDNAs (Fig. 1B). Each construct left intact at least nine amino acids at the N-terminus of the protein and eight amino acids at the C-terminus, since these regions are known to play roles in ribosome recognition

of the IRES (Hunt et al., 1993), and 3C pro recognition of the L/P1 proteolytic cleavage site, respectively. Genome transcripts from all of the deleted sequences were infectious to HeLa cells (Fig. 2A), but at 29 h post infection (not shown), their plaques were only “minute” in size (Martin et al., 1996) and barely visible. It required at least 48 h of incubation to detect any subtle difference in plaque sizes induced by RNAs from pEC 9⌬Lz (2.5 mm) relative to those from pEC 9⌬La (1.5 mm) or pEC 9⌬L (1.5 mm). Although the specific infectivity of all transcripts was similar to pEC 9, an equivalent quantity of the parental transcript typically induces very large plaques by 29 h posttransfection (Hahn and Palmenberg, 1995), and nearly every cell on the plates was lysed by 48 h posttransfection (Fig. 2A). To confirm the minute-plaque phenotypes of the deletion mutants were not those of revertant viruses, several well-separated plaques from samples similar to those in Fig. 2A were isolated, amplified in HeLa cells, and then used to infect new cell monolayers. In all cases, the relative growth characteristics and plaque sizes at 29 and 48 h p.i. were the same as for the original transfected sequences (not shown). These effects were better quantitated in single-step growth experiments, where the cells were infected synchronously with the amplified isolates and progeny titers were determined at defined intervals (Fig. 2B). The wild-type isolates gave typical EMCV growth characteristics, with eclipse periods of ⬃2.5 h and progeny yields of 2–3 log10 PFU/cell. The viruses with partial deletions or complete deletions of Leader showed a less pronounced decrease in infectivity during the eclipse phase than wild-type, although delays in the eclipse phase (2.5–3 h) were identical. By 12 h postinfection, all three Leader deletions had produced overall yields twofold lower per

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[ 35S]met incorporation (relative %) a

pEC 9 pEC 9⌬Lz pEC 9⌬La pEC 9⌬L

100 79 71 67

a T7-derived RNA transcripts were used to program cell-free translation extracts as described under Materials and Methods. The incorporation of [ 35S]met into acid-insoluble material relative to the wild-type (pEC 9) reactions was averaged for triplicate samples. The standard deviation per sample type was ⬍3.5%.

P1-2A, P1, 1ABC, and 1AB, which are the successive, expected products when endogenous 3C pro recognizes and processes the L/1AB junction of the largest, cotrans-

FIG. 2. (A) Deletion growth phenotypes. RNA transcripts from pEC 9 and the leader deletion plasmids were transfected into HeLa cells as described under Materials and Methods. After 29 h (not shown) or 48 h of incubation, plates were stained with crystal violet for plaque visualization. Representative plates are shown. Determination of plaque size and specific infectivity (PFU/␮g of RNA) are average values from at least two experiments and three plates each. (B) Single-step viral growth. Viruses from amplified plaques were used to infect HeLa cell cultures (multiplicity of infection of 10) as described under Materials and Methods. Each point represents the viral titer determined by serial dilution for a sample removed at that time.

cell. Collectively, the infectivity and growth curve data suggest that each of the engineered Leader deletions had a measurably negative impact on overall EMCV growth. Cell-free translation reactions RNA transcripts from the deletion sequences were used to program cell-free translation reactions in rabbit reticulocyte lysates. At equivalent, saturating RNA concentrations, none of the deletion sequences was as effective as pEC 9 RNA at directing protein synthesis (Table 1). In repeated samples, the negative impact of each mutation correlated roughly with the size of its deleted segment, in that pEC 9⌬L (67%) and pEC 9⌬La (71%) RNAs were generally less effective mRNA templates than pEC 9⌬Lz RNAs (79%). The synthesized Leader-containing proteins in each reaction had sizes consistent for each deletion type (Fig. 3). The pEC 9 and pEC 9⌬Lz lanes (1 and 3), for example, showed bands of

FIG. 3. Protein synthesis in cell-free extracts. Transcript RNAs (800 ng) from the indicated plasmids were used to program [ 35S]met-labeled cell-free reactions in reticulocyte extracts as described under Materials and Methods. The relative migration of marker proteins (kDa) in parallel lanes (not shown) is indicated.

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lational L-P1-2A precursor. A mature Leader band is also evident near the bottom of the pEC 9 lane but not in the pEC 9⌬Lz lane. Migration of the (⌬)L-P1-2A precursor from pEC 9⌬Lz (1031 amino acids, lane 3) is only slightly faster than the wild-type protein (1044 amino acids, lane 1). Samples synthesized with deletions in the Leader acidic domain (pEC 9⌬La or pEC 9⌬L) also produced a spectrum of proteins (lanes 2 and 4). In both cases, the largest precursors (⌬L-P1-2A) migrated notably faster than the parental or ⌬Lz proteins. Not only are these proteins smaller because of their deletions, but removal of the respective acidic domains is predicted to increase the precursor pI values by at least 1 pH unit (e.g., 6.63 for ⌬Lz and 7.74 for ⌬La), accelerating their relative migration toward the anode. The cleaved P1 and 1ABC bands were also much weaker in these lanes, and replaced instead with ⌬L-P1 and ⌬L-1ABC bands, that indicated the 3C pro-dependent processing at the L/1AB junction was much less effective when the acidic domain was removed from the Leader. Recombinant leader protein The EMCV Leader gene and derivative ⌬Lz fragment were cloned into the pET11a vectors and expressed in bacteria. After IPTG induction and lysis of cells, the insoluble pellets were treated with guanidinium HCl. The resulting supernatant was purified using a C18 column and reverse-phase HPLC, and then samples (rL and r⌬Lz) were lyophilized and analyzed for purity by atomic weight determination using mass spectroscopy. This method indicated a purity ⬎98% for each protein (not shown) with a yield of ⬃300 ␮g/L. The purified Leader (rL) was also used to raise murine polyclonal antibodies that were specific to Leader and no bacterial proteins (data not shown). ICP AES analysis of bound zinc Recombinant Leader protein from TMEV when affixed to nitrocellulose membranes has been shown to bind 65 ZnCl 2 in a manner dependent upon the presence of the N-terminal zinc-binding CHCC sequence motif (Chen et al., 1995). To determine whether EMCV proteins behaved similarly and to quantitate the specific molarity of bound zinc, HPLC-purified rL and r⌬L fractions in acetonitrile column mixture were dialyzed against 1 mM zinc acetate (Tris) buffer and then dialyzed extensively against buffer A (pH 7.2 without EDTA) to remove all metal that was not part of the protein. The molar absorbance coefficient of the EMCV Leader was calculated using the method of von Hippel (Gill and von Hippel, 1989), and subsequent protein concentrations were determined by UV spectroscopy. The samples were analyzed by inductively coupled plasma atomic absorption spectroscopy (ICP-AES). This particular method detects the presence of various metals with a detection limit in the parts per billion range (ng/

265 TABLE 2 ICP Analysis of Recombinant Leader

Protein rL

r⌬Lz

Moles of protein (⫻10 ⫺7)

Moles of zinc (⫻10 ⫺7) a

Ratio

2.29 ⫾ 0.10 1.87 ⫾ 0.20 9.02 ⫾ 0.40 3.57 ⫾ 0.50 8.21 ⫾ 0.20 4.32 ⫾ 0.40

2.06 ⫾ 0.02 1.79 ⫾ 0.05 9.57 ⫾ 0.10 ⬃0 b 0.10 ⫾ 0.020 ⬃0 b

1:1 1:1 1:1 NC c 80:1 NC c

a

Inductively coupled plasma atomic absorption spectroscopy (ICPAES) was performed to determine the zinc ion concentration in Leader protein samples. Each sample was assayed in triplicate. b Values too low to be measured. c NC, not calculated.

ml). The zinc ion concentrations for wild-type (rL) and r⌬Lz Leader proteins were determined in triplicate for each of three separate protein preparations (Table 2). The r⌬Lz contained zinc levels equivalent to background (i.e., no zinc bound the protein), while the wild-type Leader (rL) was found to bind zinc in stoichiometric amounts. That is, one ion per molecule of protein. Leader protein during virus infection Cell-free processing experiments as in Fig. 2 suggest that Leader is easily removed from precursors by the presence of 3C pro (Hall and Palmenberg, 1996; Parks et al., 1986; Palmenberg, 1990). To assess the Leader format in infected cells, the polyclonal mouse serum raised to rL was reacted with cell lysates to immunoprecipitate soluble Leader-containing proteins (Fig. 4). Extracts from uninfected cells were not reactive with this serum (lane 1), but extracts from infected cells gave strong signals for L-P1 and L-1ABC proteins (lane 2), both of which could be immunoprecipitated, before fractionation and detection by Western analyses (lane 3). The infected samples also contained reactive bands of ⬃30 and ⬃27 kDa, which are inconsistent with the known processing profiles for this region of EMCV. Surprisingly, however, the free form of mature Leader protein (⬃8 kDa) was virtually undetectable on these gels and could be observed only after overexposure of the blots. Leader is phosphorylated The EMCV Leader sequence has a centrally located tyrosine phosphorylation motif (KYDEEWY) of the type recognized by tyrosine kinases (Guarne´ et al., 1998; Cooper et al., 1984). HeLa cells were incubated with [ 32P]␥ATP for 30 min to allow a synchronous, pulsed release of 32 Pi (Lazarowski et al., 2000; Kubler et al., 1980) and subsequently infected with EMCV for 4 or 6 h. Extracts from infected and uninfected HeLa cells (4 or 6 h p.i.) were then immunoprecipitated with anti-Leader poly-

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antibody or label with 32P indicates that these forms of the protein are probably not phosphorylated. DISCUSSION Our interest in the EMCV Leader protein stems from observations that reversion mutations in the putative zinc binding domain can partially compensate for translational deficiencies caused by primary mutations in the viral IRES (Hoffman and Palmenberg, 1996). Not all IRES defects can be reverted with Leader mutations (C. M. T.

FIG. 4. Protein immunoprecipitation. (A) Extracts from HeLa cells infected with vEC 24 virus were treated with anti-rL polyclonal mouse serum and fractionated by SDS–PAGE, as described under Materials and Methods. Lane 1, uninfected cell extract; Lane 2, unfractionated, infected cell extract; Lane 3, immunoprecipitated proteins from infected cell extract. Protein visualization was by Western analysis using the polyclonal anti-rL serum. (B) Longer exposure of A for the region between 2 and 20 kDa detects the presence of mature Leader.

clonal antibodies. The immunoreactive proteins were fractionated by SDS–PAGE, and then the gel was subjected to autoradiography (Fig. 5A). The uninfected HeLa cell lysate sample (lane 1) did not have any detectable radioactivity (lane 1). However, infected extracts harvested at 4 or 6 h p.i. (lanes 2 and 3, respectively) gave clear positive bands detectable by phosphorimaging at a molecular weight of about 8 kDa, signaling that the mature Leader protein in these samples contained phosphate. The larger Leader-containing precursor proteins, such as those seen in Fig. 4, were not labeled with 32P during infection. To determine if the mature form of Leader was phosphorylated at a tyrosine residue, the immunoprecipitation experiment was repeated and then followed by Western analysis (Fig. 5B) with a monoclonal antibody specific for phosphotyrosine moieties (Shaw et al., 2000; Dreschers et al., 2001). Consistent with Fig. 5A, the mature (8 kDa) Leader protein isolated at 4 or 6 h p.i. (lanes 2 and 3, respectively) was also immunoreactive with the tyrosine-phosphate antibody, indicating that Leader is phosphorylated at a tyrosine. That the precursor forms of Leader did not react (even weakly) with this

FIG. 5. (A) Leader phosphorylation. HeLa cell monolayers were incubated with [ 32P]␥-ATP for 30 min and subsequently infected with vEC 9 at an m.o.i. of 10. At 4 h (lane 2) or 6 h (lane 3) p.i., the cells were harvested and lysed. Leader protein (and precursors) were collected by immunoprecipitation and then fractionated by SDS–PAGE as described under Materials and Methods. Lane 1 is from an uninfected cell sample treated the same way. The proteins were visualized by phosphorimaging. (B) Tyrosine phosphorylation of Leader. HeLa cell monolayers were infected with vEC 9 at an m.o.i. of 10. At 4 h (lane 2) or 6 h (lane 3) p.i., the cells were harvested and lysed. Leader protein (and precursors) were collected by immunoprecipitation and then fractionated by SDS– PAGE as described under Materials and Methods. Lane 1 is from an uninfected cell sample treated the same way. The proteins were visualized by chemiluminescence after Western analyses with an antiphosphotyrosine monoclonal antibody.

EMCV LEADER PROTEIN

Dvorak and C. Palmenberg, unpublished observations), but when combined with experiments that link altered EMCV translational requirements (i.e., PTB) with the presence or absence of Leader (Kaminski and Jackson, 1998), the idea of a regulatory role for this protein in viral protein synthesis becomes compelling. To that end, we expressed and purified Leader to homogeneity and established that the zinc binding capacity of rL was precisely quantitated by plasma emission spectroscopy at one ion per protein. Recombinant protein r⌬L did not bind zinc, establishing as with TMEV (Chen et al., 1995) that the CHCC motif, though an atypical form of a zinc-binding sequence, was required for this activity, and the motif actually functioned in the full-length EMCV protein. Murine polyclonal antibodies to Leader detected L-P1 and L-1ABC precursors, and two additional infection-specific proteins of ⬃30 and ⬃27 kDa from infected HeLa cell extracts. Yet, most surprising about these experiments was not so much the unanticipated bands as that the majority of detected Leader sequences in these cells was apparently in precursor form (e.g., L-P1 or L-1ABC), with only a tiny fraction as mature protein. EMCV-infected HeLa cells typically lyse at 5–6 h p.i., releasing progeny assembled between 4–6 h p.i. (Hahn and Palmenberg, 1995). By 4 h p.i., one expects robust concentrations of capsid precursors without Leader attached. Though it is not yet clear whether these new species represent precursor degradation products, nondissociable multimers of Leader, or previously unrecognized cleavage products, we intend to sequence them and probe with other viral antibodies to determine their origins. Mature Leader is usually detected after cell-free translation reactions with viral sequences (Fig. 2) (Parks and Palmenberg, 1987). The absence of mature Leader in infected cells could mean that (a) the mature Leader protein might have a short half-life in cells, (b) the mature Leader might be entirely membrane-bound or rendered insoluble when the cells were lysed, or (c) our polyclonal serum was only poorly effective at recognizing the mature Leader protein in its natural format in infected cells. Subsequent database searches with the EMCV sequence suggested the surprising presence of a modifying tyrosine phosphorylation site. Identification of this motif was unexpected, not only because it is not conserved among the TME viruses, but because few picornaviral proteins of any kind are documented to be phosphorylated (Neufeld et al., 1991). Yet, highly specific, recombinant monoclonal antibodies against phosphotyrosine residues clearly reacted with an 8-kDa protein from vEC 9-infected cells, after an initial immunoprecipitation with the anti-Leader polyclonal serum. Recombinant rL, uninfected cell extracts, and the slate of Leadercontaining capsid precursor proteins were unreactive with this MAb. The results suggest that the active form of the Leader during infection may be as a zinc-binding

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phosphoprotein that probably is not easily recognized by the polyclonal serum raised to (unphosphorylated) rL. As soon as fresh preparations of rL are in hand, we intend to assay for cellular kinases that may carry out this phosphorylation reaction and perhaps raise new antibodies that can recognize the modified Leader, as the (true?) mature form of the protein. A requirement for phosphorylation could easily explain why preliminary IRES–Leader mixing experiments (with rL) were not initially successful (not shown). The EMCV Leader segment is clearly important for viral function. Deletion of the zinc domain, the acidic domain, or both domains allowed viral infectivity, but the progeny had severely restricted plaque phenotypes and growth curves. Removal of these regions also affected the ability of transcript RNAs to direct protein synthesis in cell-free extracts and (for the acidic domain) diminished the proteolytic processing efficiency of 3C pro at the L/P1 junction. The combination of zinc-binding domain, phosphorylation site, and notably charged (acidic) segments are highly suggestive of cellular or viral RNA binding proteins that typically mediate and regulate RNA–protein interactions by serving as functional adaptors (Oehler and Angel, 1992; Kouzarides, 2000). The T4 gp32 protein autoregulates its own translation (Shamoo et al., 1991), and the HIV NC protein nucleates RNAdependent virion assembly (Fu et al., 1985; Coleman, 1992). These, and other auto regulatory transcription factors that bind zinc (Oehler and Angel, 1992), are obvious models for putative Leader–IRES interactions that should be further explored. MATERIALS AND METHODS Genome cDNAs Standard recombinant methods were used (Sambrook et al., 1989; Ausubel et al., 1993). By convention, the nucleotide and protein numbering scheme is according to EMCV-R, a genome with a 5⬘ poly(C) tract of C 125UCUC 3UC 10 (Genbank Accession No. M81861) (Hahn and Palmenberg, 1995; Duke et al., 1992). Plasmid pEC 9 (5⬘ poly(C) tract of C 9) has been described (Hahn and Palmenberg, 1995). To facilitate Leader region cloning, a pEC 9 subclone, pEC 9L1AB, was constructed by digestion with NdeI and SalI enzymes (New England Biolabs), treatment with Klenow polymerase, and then ligation. Deletion mutagenesis with dual asymmetric PCR (Martin and Palmenberg, 1996; Sandhu et al., 1992; Jayaraman and Puccini, 1992) was used to delete viral bases encoding Leader amino acids 10–22 (⌬Lz), 37–59 (⌬La), or 10–59 (⌬L) from pEC 9 cDNAs. Amplicons with these deletions were used to replace the BstXI to BstBI fragment of pEC 9L1AB, and the identity of the resultant plasmids was carefully confirmed by restriction analyses and sequencing. Fragments of cDNAs containing the deletions (NheI to SphI) were then transferred back into a

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pEC 9 plasmid that had been similarly digested. The resultant genome-length plasmids, pEC 9⌬Lz, pEC 9⌬La, and pEC 9⌬L, were again characterized by comprehensive restriction analyses and sequenced (IRES and Leader regions). Plasmid transcription Genome-containing cDNAs were linearized with SalI and used to program standard T7 RNA polymerase runoff transcription reactions. When required, [␣- 32P]UTP (13 ␮Ci/ml) was added as a radiolabel. Reactions were terminated by DNase I addition, extracted with phenolchloroform, and the RNA collected after precipitation with ethanol. Radiolabeled reaction efficiencies were monitored by scintillation counting after adsorption to Whatman DE81 filters, as described (Hoffman and Palmenberg, 1995). Agarose gel electrophoresis was used to assay the size and character of the transcripts. Cell-free translation reactions RNA transcripts (400 ng) were used to program cellfree translation reactions (15 ␮l) in rabbit reticulocyte lysates containing [ 35S]methionine (1.67 ␮Ci/␮l), as described (Pelham and Jackson, 1976; Duke et al., 1992; Hoffman and Palmenberg, 1995). After 1 h of incubation at 30°C, the reactions were terminated by addition of cyclohexamide and RNase A (to 0.6 ␮g/␮l each). Samples were then incubated an additional hour at 30°C to allow for viral processing reactions. Incorporation of [ 35S]methionine into acid-insoluble products was performed as described (Palmenberg and Rueckert, 1982). Proteins were fractionated (typically, 1 ␮l containing 400,000 [ 35S]cpm) by standard SDS–PAGE (5–20% gradient Laemmli gels) and autoradiography. HeLa cell infectivity Confluent monolayers of H1-HeLa cells (cell line ATCC CRL 1958) in 60-mm plates were transfected with viral RNA transcripts (5 or 50 ng RNA, incorporated into liposomes) (Rose et al., 1991), or infected with virus, overlaid with noble agar, and incubated at 37°C under 5% CO 2, as described (Hoffman and Palmenberg, 1995; Hahn and Palmenberg, 1995). After appropriate times (29–48 h) postinfection, the agar was removed and the monolayers were stained with crystal violet to visualize plaques. Plaque size was measured with a micrometer, and the values averaged for each plate. Specific infectivities were assigned after dividing the number of plaques per plate by the RNA amount added to that plate (Martin et al., 1996; Martin and Palmenberg, 1996). HeLa cell growth curves Virus was clonally propagated by picking an isolated plaque and inoculating a fresh cell monolayer (60-mm-

diameter plates). After the development of cytopathic effect, infected cells were lysed by three cycles of freezing and thawing, the cell debris was removed by centrifugation for 10 min at 2000 g, and the supernatant was used to infect another monolayer (100-mm-diameter plates) for virus amplification. Subsequent cell lysis and virus purification by sedimentation through sucrose cushions have been described (Hoffman and Palmenberg, 1995; Hahn and Palmenberg, 1995). Prior to infection for single-step growth assays, the HeLa cells from suspension cultures were collected, washed once in phosphate-buffered saline (PBS), gently pelleted, and then resuspended at a concentration of 4 ⫻ 10 7 cells/ml. Virus samples were added at a multiplicity of infection of 10 PFU per cell, and attachment was carried out for 30 min at room temperature under constant rotation. After another gentle pelleting, unattached virus was removed from the cells by washing with PBS (10 ml). The infected cultures were resuspended to a concentration of 4 ⫻ 10 6 cells/ml in medium A (Hahn and Palmenberg, 1995) and incubated at 37°C in a shaking water bath. Aliquots (0.5 ml) were removed at the required times (0–12 h), buffered with 15 ␮l of 1.25 M HEPES, pH 7.4, and then frozen in an ethanol–dry ice bath before storage at ⫺80°C. After two subsequent freeze–thaw cycles, virus titers (PFU per cell) were determined by standard plaque assays at 37°C for 30 h. Bacterial expression plasmids The pEC 9 and pEC 9⌬L Leader coding regions were amplified by PCR using primers (A) 5⬘-CCGGCATATGGGTCCAAATCCGACTAT-3⬘ and (B) 5⬘-CCGCGGGATCCACTACTGGGGCTCGAAGGCCT-3⬘. Primer A contained an engineered NdeI site linked to 17 nucleotides complementary to the 5⬘ end of the pEC 9 Leader region. Primer B contained 17 nucleotides complementary to the 3⬘ end of the pEC 9 Leader, and also the complement to a stop codon (CTA) and a BamHI restriction sequence. Each amplicon was subcloned into pET11a (Novagen Inc.) using the NdeI and BamHI sites. The resultant plasmids, pL and p⌬Lz, were transformed into Escherichia coli (strain MV1190), amplified, and sequenced throughout their inserted fragments. Protein expression Colonies of E. coli strain BL21(DE3)pLysS (Novagen Inc.) transformed with plasmids pL or p⌬Lz were isolated and amplified (1 L) at 37°C to an absorbance of 0.7 (600 nm) in 2XYT broth supplemented with M9 salts, 100 ␮g/ml ampicillin, and 30 ␮g/ml chloramphenicol (Studier et al., 1990). Isopropyl ␤-D-thiogalactoside (IPTG) was added to 1 mM final concentration and the incubation continued for 5 h. Cells were pelleted by centrifugation and then resuspended in 1/10 vol of buffer A (50 mM Tris acetate, pH 8.5, 1 mM EDTA, 1 mM DTT, 10% glycerol).

EMCV LEADER PROTEIN

The suspension was lysed by freezing and thawing; DNase I was added (25 U per 10 ml suspension) and the samples incubated for 20 min at 37°C. The lysate was clarified (27,000 g, 15 min) and the resulting pellet washed two times with 5 ml of 1% deoxycholate in buffer A. The inclusion bodies were repelleted (12,000 g, 5 min) and then resuspended in 10 ml of buffer B (50 mM Tris acetate, pH 8.5, 1 mM EDTA, 1 mM DTT, 8 M guanidinium HCl). After removal of insoluble material (12,000 g, 5 min), the supernatant was applied to a HPLC C-18 column (5 cm, Beckman) preequilibrated in water with 0.1% TFA. Samples were fractionated by reverse-phase HPLC (Beckman C18 cartridge, 5 ␮m particle size, 4.6 mm ⫻ 25 cm) using a 0–70% acetonitrile gradient in 0.1% TFA (flow rate 1 ml/min, Beckman System Gold HPLC). The peak area and retention time were recorded and quantitated by Beckman Gold software. The appropriate peak was collected and then dialyzed 12–15 h against buffer A (without DTT, 10% glycerol, or 1 mM zinc acetate) at 4°C. Precipitate material was removed by centrifugation (12,000 g, 5 min), and the soluble fraction (rL or r⌬Lz) concentrated (centricon-3, Amicon), before DTT was added to 1 mM final concentration. The protein in the final preparation was quantitated by the Bradford method (Bio-Rad, Hercules, CA) and stored at ⫺70°C. To refolded the protein in the presence of zinc, rL and r⌬L HPLC fractions in the acetonitrile column mixture were dialyzed against a 1 mM zinc acetate (Tris) buffer and then dialyzed against buffer A to remove all ions except those which were part of the protein. The molar absorbance coefficient of the EMCV Leader was calculated using the method of von Hippel (Gill and von Hippel, 1989). Subsequent protein concentrations were determined by UV spectroscopy. Antibodies Purified, rL protein (50 ␮g), emulsified in complete Freund’s adjuvant (Sigma), was injected intraperitoneally into female BALB/c mice. Booster injections of a similar dose and route were on day 14, 28, and 42. On day 45, animals were sacrificed and bled out by cardiac puncture. The plasma was isolated from blood after centrifugation at 14,000 g for 5 min. Recombinant monoclonal antibody RC20:HRPO against phosphotyrosine was purchased from Transduction Laboratories (Catalog No. E120H). This highly specific, high-affinity reagent was altered through mutation to remove the IgG domain and is directly linked to horseradish peroxidase for use in Western blotting experiments. Western analyses SDS–PAGE gels (Mini-PROTEAN II, Bio-Rad) contained a 5% polyacrylamide stacking gel and a 15% polyacrylamide resolving gel. Samples were fractionated (210 V

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for 45 min), and the bands visualized by silver staining. Alternatively, gel-fractionated samples were transferred to polyvinylidene fluoride membranes (Immobilon PVDF, Millipore) in transfer buffer (0.025 M Tris, pH 8.0, 0.19 M glycine, 20% methanol) at 30 V for 2 h (Genie blotter, Owl Scientific). The membranes were treated with Blotto buffer (5% w/v nonfat dry milk, 0.05% w/v Tween-20 in PBS, 0.1 M phosphate, 100 mM NaCl, pH 7.2) for 30 min at room temperature and then rinsed twice in wash buffer (0.05% w/v Tween-20 in PBS). A 1:5000 dilution of appropriate anti-Leader antibody in Blotto buffer was incubated with the membrane for 1 h at 22°C, and then the membrane was rinsed twice in wash buffer. Membranes were subsequently incubated with horseradish peroxidase conjugated, anti-mouse antibodies (1:2000 dilution, Amersham Pharmacia Biotech, Inc.) in Blotto buffer for 1 h, rinsed twice in wash buffer, and once in distilled water, and the bands were visualized by chemiluminescence (ECL Kit, Amersham Pharmacia Biotech, Inc.). Protein immunoprecipitation Confluent HeLa cell monolayers on 60-mm plates were infected with vEC 24 virus (Palmenberg and Osorio, 1994) at a multiplicity of infection of 20 plaque-forming units (PFU) (Hoffman and Palmenberg, 1995; Hahn and Palmenberg, 1995). For 32P-labeling, the cells were incubated with 250 ␮Ci [ 32P]␥-ATP (3000 mmol/mCi) for 30 min prior to infection to enable the breakdown of ATP and the uptake of the resulting 32Pi (Kubler et al., 1980; Lazarowski et al., 2000). At 4 or 6 h postinfection, the medium was removed and the monolayers washed in PBS. Cells were lysed on ice by the addition of 0.5% NP-40 and 1 mM ZnCl 2 in 50 mM Tris–Cl, pH 7.5 (200 ␮l). After clarification (14,000 g, 10 min, 4°C), the supernatants were stored at ⫺70°C. Anti-Leader polyclonal antibody (1 ␮g) was added to an aliquot (10 ␮l) of thawed lysate and reacted at 4°C for 2 h. Goat anti-mouse IgG antibodies coupled to magnetic beads (Advanced Magnetics) were added (5 ␮l of suspension) and incubation continued for 1 h at 4°C. The beads were isolated with a magnetic stand (Advanced Magnetics), washed three times with NP-40 buffer (25 mM Tris–Cl, pH 7.5, 5 mM MgCl 2, 1 mM EDTA, 1 mM DTT, 0.1% NP-40), three times with NP-40 buffer containing 1 M LiCl, and then three times again with NP-40 buffer alone. The immunocomplexes were solubilized with Laemmli gel sample buffer (Cleveland et al., 1977), fractionated by electrophoresis on 7–18% gradient SDS–PAGE, and visualized by chemiluminescence (ECL Kit, Amersham Pharmacia Biotech, Inc.) or phosphorimaging (Molecular Dynamics). Images were captured electronically and processed with Adobe Photoshop software.

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Plasma emission spectroscopy The analysis of zinc (Zn) was performed using a Perkin–Elmer ICP/6000 inductively coupled plasma atomic emissions spectrometer equipped with a CETAC U5000-AT ultrasonic nebulizer. The 213.856-nm line of Zn was used for analysis. A calibration curve was established using a set of Zn standards directly prior to analysis of the samples. The Zn standards were prepared by serial dilution of a 1000 g/mL Zn standard obtained from High Purity Standards. All dilutions used 16 M cm ⫺1 water (Millipore). In all operations, care was taken to avoid exogenous contamination. Plastic and glassware were soaked in 5% HNO 3 for at least 48 h and then rinsed with copious amounts of 16 M cm ⫺1 water prior to use. ACKNOWLEDGMENTS This work was supported by National Institutes of Health Grant AI-17331 to A.C.P. and predoctoral training Grant T32-GM07215 to C.M.T.D. The authors thank the Holcomb Research Institute and Butler University for providing support and facilities that aided in this work and Joe Binder for critical reading of the manuscript.

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