Specific interactions of HeLa cell proteins with Coxsackievirus B3 RNA: La autoantigen binds differentially to multiple sites within the 5′ untranslated region

Virus Research 90 (2002) 23 /36 www.elsevier.com/locate/virusres

Specific interactions of HeLa cell proteins with Coxsackievirus B3 RNA: La autoantigen binds differentially to multiple sites within the 5? untranslated region Paul Cheung, Mary Zhang, Ji Yuan, David Chau, Bobby Yanagawa, Bruce McManus, Decheng Yang * Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, Canada McDonald Research Laboratories/The iCAPTUR4E Centre, St. Paul’s Hospital, Vancouver, Canada Received 29 March 2002; received in revised form 20 June 2002; accepted 20 June 2002

Abstract Translation initiation of the coxsackievirus B3 (CVB3) RNA occurs by internal ribosomal entry. The internal ribosomal entry site (IRES) of the virus has been mapped to the 5? untranslated region (5? UTR) of the genome. As well, the 5? UTR has been suggested to play roles in determining the tissue tropism and infectivity of the virus. In this study, we investigated interactions between HeLa cell protein extracts and radiolabeled RNA of CVB3 5? UTR by competitive UV cross-linking. We have observed a number of proteins that specifically interact with the three sub-cloned regions of the 5? UTR. In particular, the molecular weights of five of these proteins resemble those of the eukaryotic translation initiation factors 4A, 4B and 4G, as well as the La autoantigen and the polypyrimidine tract binding protein. Based on this data, we focused on the interaction of the 5? UTR with the La autoantigen, which was purified by the glutathioneS-transferase affinity method. We have confirmed the highly specific interaction of the La autoantigen with the 5? UTR sequence nt 210 /529. The core IRES (nt 530 /630) and nt 1 /209 also appear to bind to the La protein at moderate and weak affinities, respectively. A functional role of the La autoantigen in translation initiation is suggested. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Coxsackievirus B3; La autoantigen; Translation initiation; Untranslated sequence; Protein /RNA interaction; Internal ribosomal entry site

1. Introduction

* Corresponding author. Address: St. Paul’s Hospital, Rm 292-1081 Burrard Street, Vancouver, BC, Canada V6Z 1Y6. Tel.: /1-604-806-8346; fax: /1-604-806-8351 E-mail address: [email protected] (D. Yang).

Coxsackievirus B3 (CVB3) is an enterovirus of the family Picornaviridae . The Group B coxsackieviruses include six serotypes (B1 to B6) that cause a variety of human diseases including myocarditis, meningitis and diabetes (Cherry 1995). Among the group B coxsackieviruses, the

0168-1702/02/$ - see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 8 - 1 7 0 2 ( 0 2 ) 0 0 1 3 8 - 7

24

P. Cheung et al. / Virus Research 90 (2002) 23 /36

B3 strain is mostly studied for its cardiovirulence, and its ability to cause acute and persistent infections (Carthy et al., 1997; Gauntt et al., 1989; McManus et al., 1993). Evidence of direct viral injury is derived from histochemical, immunohistological and in situ hybridization studies (Kandolf et al., 1987; Klingel et al., 1996; McManus et al., 1993). Persistent CVB3 infection may elicit a chronic host inflammatory response, leading to dilated cardiomyopathy (Johnson and Palacios 1982; Keeling et al., 1994; Martino et al., 1994; Sole and Liu, 1993). Upon cellular entry, translation of the CVB3 genome by the host translational machinery produces a single poly-protein. Subsequent auto-lytic cleavages of the poly-protein generate various structural and non-strutural proteins that are responsible for the subsequent viral replication and pathogenesis. Thus, the efficiency of translation initiation of the viral genome is a critical determinant in the course of infection. Translation of the CVB3 genome occurs via a cap-independent ribosomal internal entry mechanism, a process that is also widely observed in other picornaviruses and is distinct from the cap-dependent ribosomal scanning translation of most host mRNA (Martinez-Salas et al., 2001). It is known that internal translation initiation requires sequence-specific determinants in the 5? untranslated regions (5? UTR) of the viral genome, and particularly the cis -acting sequence termed internal ribosomal entry site (IRES) (Yang et al., 1997). The primary sequences of the picornaviral IRESes are not significantly conserved, with the exception of the short poly-pyrimidine tract (Fig. 1) (Pestova et al., 1991). Further studies indicate that the organization of IRES into specific secondary structures is crucial for its activity (Robertson et al., 1999). In fact, picornaviruses may be divided into three main groups based on their IRES secondary structures: the entero-rhinoviruses, the cadioaphthoviruses and the heptaoviruses (Drew and Belsham 1994; Jackson and Kaminski 1995). The relationship between the translation initiation activity and the secondary structures of the 5? UTR strongly implies an importance of proteinIRES RNA interactions in translation initiation (Lopez and Martinez-Salas, 1997). In the case of

CVB3, our previous studies have identified the core IRES and other sequences in the 5? UTR that are important for translation initiation (Yang et al., 1997) as well as sequences that appear to determine the infectivity of the virus (Liu et al., 1999). Also, it has been suggested that nucleotide substitutions in the 5? UTR region can alter the cardiovirulence of CVB3 (Chapman et al., 1994; Dunn et al., 2000; Tu et al., 1995). In the closely related poliovirus, the 5? UTR contains determinants of neural-virulence (La Monica and Racaniello, 1989; Macadam et al., 1992). Although there has been no conclusive data to explain the relationship between the 5? UTR sequence and viral pathogenesis, findings in CVB3 are in line with experimental observations in other viruses: the maintenance of high-order RNA structures in the IRES by the primary 5? UTR sequence and the interaction of such high order structures with host proteins are critical for translation initiation activity (Martinez-Salas et al., 2001). In related picornavirus systems, several host cellular proteins have been demonstrated to interact with the untranslated regions and their interactions are important for the replication and translation of various viruses (Andino et al., 1993; Blyn et al., 1997; Gamarnik and Andino 1998; Rohll et al., 1994). These proteins include the glyceraldehyde 3-phosphate dehydrogenase (GADPH) (Schultz et al., 1996), eukaryotic initiation factor 4G (eIF4G) (Kolupaeva et al., 1998), eIF3 (Sizova et al., 1998), La autoantigen (Svitkin et al., 1994), and poly-pyrimidine-tract binding protein (PTB) (Ali and Siddiqui 1995; Kolupaeva et al., 1996). Of note, the synergistic effect of PTB and La in internal translation initiation of a bicistronic mRNA containing the encephalomyocarditis virus (EMCV) IRES element has been demonstrated (Kim and Jang 1999). In particular, the La autoantigen has been reported to bind a variety of viral RNAs and stimulate translation initiation of uncapped RNA molecules (Crosio et al., 2000; De et al., 1996; Gutierrez-Escolano et al., 2000; Isoyama et al., 1999). The expression of La autoantigen may be tissue specific and dependent on cellular conditions (Carter and Sarnow 2000). The availability of the La protein in CVB3 infected

P. Cheung et al. / Virus Research 90 (2002) 23 /36

25

Fig. 1. Diagram of the CVB3 5? UTR secondary structures and the relative locations of sub-cloned sequences used for probes synthesis. The range of numbers indicates the nucleotides of the respective stem-loop structures which are labeled A through K. The IRES core, authentic initiation codon AUG and conserved polypyrimidine tract/AUG are indicated. The three shaded bars indicate sequences covering nt 1 /209, 210 /529 and 530 /630, respectively, which were cloned into individual T/A vectors. Stem loops are not drawn to scale.

cells may play roles in determining the expression of viral proteins and the subsequent cellular injury. The stem-loop structures in the CVB3 5? UTR has been predicted and studied in relation to translation initiation (Yang et al., 1997; Zuker 1989), but interactions of host proteins with the CVB3 5? UTR have not been characterized. Information on these protein-RNA interactions can lead to a better understanding of the molecular mechanism of CVB3 translation initiation and cardiovirulence. In this study, we performed mobility shift assays and competitive UV crosslinking assays to demonstrate the interactions of HeLa cell protein-CVB3 5? UTR RNA. A number of 5? UTR binding proteins were identified by their respective molecular weights (MWs) and then we focused on the verification of a 52 kDa proteinRNA complex, which may be the human La autoantigen. We expressed and purified recombinant La autoantigen by the GST-fusion protein system. Using the purified La protein, we have

observed differential affinities of the La protein towards multiple sites in the 5?UTR. Considering the functional roles of the La protein in other RNA viruses and its ability to interact with the 40S ribosomal subunit, we propose that the La autoantigen is also important in forming a host protein-CVB3 RNA pre-translation complex.

2. Materials and methods 2.1. Plasmid construction Considering the importance of RNA secondary structures (stem loops) in forming protein complexes, we created three CVB3 5?UTR sub-clones: clone p(1 /209) includes complete stem-loops A to D; clone p(210 /529) includes stem-loops E, F and the beginning region of G; and clone p(530 /630) contains the IRES core sequence spanning stemloops G and H. The sequence nucleotide (nt) 529 /

P. Cheung et al. / Virus Research 90 (2002) 23 /36

26

630 was previously identified as the IRES of CVB3 and hence proteins interacting with this region are potentially important for translation initiation. The nt 1 /209 was selected because it encompasses the first four stem loop structures containing the so called cloverleaf-like secondary structures, which is essential for viral replication (Rohll et al., 1994; Yang et al., 1997). The nt 210/529 region includes potential cardiovirulence determinants of the virus (Chapman et al., 1994; Dunn et al., 2000; Tu et al., 1995), thus protein interactions with this region may determine viral pathogenesis and tissue tropism. PCR reactions were performed to amplify DNA fragments corresponding to these regions, and the respective primers are listed in Table 1. The amplified DNA fragments were cloned directly into the dual promoter T/A vector (Invitrogen ) as per manufacturer’s instruction. Cloned sequences and their orientations with respect to the RNA polymerase promoter on the vector were verified by DNA sequencing. 2.2. In vitro transcription Prior to transcription, plasmid templates were linearized and purified by phenol/chloroform extraction and ethanol precipitation. RNAs of the three cloned sequences were synthesized by in vitro transcription using 1 mg of linearized plasmids and the appropriate RNA polymerase (Promega ) in the presence of [a-32P]UTP (3000 Ci/mmol, Amersham) as per manufacturer’s instruction. Nonradioactive labeled RNA was also synthesized, and served as specific competitors in the mobility Table 1 Primers used for construction of the CVB3 5? UTR clones Primers Sequence (5? to 3?)

locationa (nt)

P1-5? P1-3? P209-5? P209-3? P530-5? P530-3?

1 /11 199 /209 209 /219 519 /529 529 /539 620 /630

a

TTAAAACAGCC GTGAGCAGTCT CGCGGTTGAAG GAGTTGCCCGT CTGCAGCGGAA ATCCAATAGCT

PCR temperature (8C)

63 45 47

The range of numbers indicate the location of each primer on the genomic RNA of CVB3 (Klump et al., 1990).

shift and UV cross-linking experiments. RNA transcripts were purified by RNase-free DNase I digestion, phenol/chloroform extractions and ethanol precipitation at /80 8C. The integrity and purity of RNA were verified either on a 1.5% formaldehyde-containing agarose gel (for nonradioactive transcripts) or autoradiography with 6% SDS-polyacrylamide gel ([a-32P]UTP labeled transcripts). Non-labeled transcripts were quantified by spectrophotometry and radioactive transcripts were adjusted to a specific activity of 1 x 107 / 1 x 108 cpm/mg. 2.3. Preparation of HeLa cell extracts HeLa cells (ATCC) were grown in Eagle’s minimal essential medium supplemented with 10% fetal bovine serum. Cells were harvested mechanically at approximately 95% confluency, washed 2x with PBS and incubated with lysis buffer (50 mM Tris /HCl pH 7.4, 150mM NaCl, 1% Triton, 0.1% SDS, 0.5% Na Deoyxcholate, 10 mM EDTA) for 10 min. Cell lysates were dialyzed against buffer A [90 mM KOAc, 1.5 mM Mg(OAc)2, 1 mM dithiothreitol (DDT), 10 mM HEPES, pH 7.6] for 12 hrs at 4 8C. Protein concentration was determined by spectrophotometry using the Bradford protein assay kit (Bio / Rad). The concentration of the protein extracts was adjusted to 20 mg/ml. The extract was used directly for RNA-protein interaction assays or stored in aliquots at /80 8C until use. 2.4. RNA mobility shift assays All RNA-protein interactions were carried out in a total volume of 20 ml. Five ml of binding buffer (2.5 mM KCl, 10 mM HEPES, 2 mM MgCl2, 0.1 mM EDTA, 2 mM DTT, 5 mg heparin and 3.8% glycerol), 500 ng (5 x 106 cpm) of radioactive probe and 20 mg of HeLa cell extract were incubated for 15 min at 37 8C. In competitive assays, either an unlabeled form of the probe RNA (self competitor) or yeast tRNA was incubated with cell extracts for 5 min prior to adding radioactive probes. Unlabeled yeast tRNA (Sigma) was used as a non-specific competitor because it is not homologous to the viral RNA

P. Cheung et al. / Virus Research 90 (2002) 23 /36

sequence but contains similar stem-loop structures. Protein-RNA mixes were analyzed by electrophoresis through a 4% non-denaturing polyacrylamide gel. After drying the gel, radioactivity was visualized by autoradiography. 2.5. UV cross-linking experiments To determine the MW of the RNA-binding proteins, UV cross-linking experiments were conducted. Reactions were carried out in a total volume of 20 ml containing 5 ml of binding buffer. In each reaction, 500 ng of labeled transcripts (5 x 106 cpm) was incubated with 10 mg of HeLa cell extracts or 2.0 mg purified La protein at 37 8C for 15 min. Specific competitors (self RNA competitors), were added, five min before adding radioactive probes, at specified molar excess ratios. In the UV cross-linking reactions between the La protein and the 5? UTR probes, competitors were added at 2x molar excess ratios over that of the probe. The reaction mixtures were kept on ice and UV cross-linked (Strategene 2400 ) for 25 min at a distance of 3 cm from the UV bulb. Total UV energy applied was 22.5kJ. After UV-irradiation, all samples were treated with 200 ng of RNase A at 37 8C for 20 min. As control, a reaction not treated with UV irradiation was digested with RNase A. Protein-probe complexes were then analyzed under reducing conditions by 9% SDSPAGE. The gels were fixed in a solution containing 15% methanol and 20% acetic acid for 10 min and dried prior to autoradiography. MWs of probe-protein complexes were determined by comparing against simultaneously loaded protein markers. 2.6. Protein purification GST-La fusion protein was produced in E. coli (BL21 DE3) cells (Novagen ) transformed with the pGEX-La plasmid (a gift from Dr. Y. K., Kim/S. K. Jang, Pohang University of Science and Technology, Korea). In brief, the E. coli (BL21 DE3) cell was induced at a mid-log phase by Isopropylb-D-thiogalactopyranoside (IPTG, final concentration 0.1mM) and allowed to grow at 26 8C for 20 hrs. Cells were lysed by freeze-thaw cycles and

27

sonication. The lysate was treated with glutathione sepharose beads (Pharmacia Biotech) at room temperature for 1 hr. The mixture was centrifuged and the pelleted beads were washed 5 times with 10 x bead volume of PBS buffer. Five units of thrombin were added to the washed beads together with 100 ml PBS per 100 ml of beads. The supernatant was removed and tested for protein concentration and purity by the Bradford assay and 10% SDS-PAGE, respectively. The final concentration of the protein was adjusted to 0.5 mg/ml.

3. Results 3.1. Specific interactions of the HeLa cell proteins with the 5? UTR sequences To determine whether cellular proteins bind the CVB3 5? UTR in a specific manner, we measured mobility shift of the 32P-labeled 5? UTR transcripts in the presence of HeLa cell proteins and various competitor RNAs. The locations of these probes in the CVB3 5? UTR sequence are indicated in Fig. 1. Data for the mobility shift assays are presented in Fig. 2a /c. In each of these figures, the integrity and migration distance of the free probes (nonprotein-bound 32P-labeled probes) were demonstrated in lane 1. Changes in the electrophoretic mobility of the radioactive probes are observed in lane 2, indicating the formation of protein-probe complexes (Co). The smear-like patterns (lane 2) suggest the presence of multiple RNA-protein complexes over a range of MWs. Specificity of the protein-probe interaction was demonstrated when self competitor RNAs were added to the respective reactions (lane 3 /5). Unlabeled RNA of the same sequence specifically competed against the 32P-labeled probes for protein-interactions, allowing free 32P-labeled probes to migrate at near normal mobility. In the case of sequence nt 210/529, the mobility of the probe under competition appeared slightly faster than that of the free probe (lane 1 compared to lane 5). This may be due to changes in the reaction conditions (pH, salt), resulting from the addition of protein extracts. In the case of nt 530/630 (Fig. 2c), addition of specific competitors resulted in the

28

P. Cheung et al. / Virus Research 90 (2002) 23 /36

detection of two major bands of protein-complexes (lane 5). This suggests two possible processes: the presence of multiple host proteins that bound the probes at differential affinities and quantities; or the cooperative binding of multiple copies of the same protein towards the probe. Nonetheless, formation of the protein-probe complexes was reduced by the addition of increasing amounts of unlabeled self RNA (lane 3 to 5). This demonstrates the specificity of the HeLa cell protein interactions. In contrast, formation of complexes between HeLa cell proteins and yeast tRNA was minimal, as the addition of tRNA did not present significant competition effects (Fig. 2a/c, lane 6 /8). 3.2. Determination of MWs of the UTR-binding proteins

Fig. 2. Competitive mobility shift assays to demonstrate specific interactions between HeLa cell proteins and the 5? UTR probes. [a/32P]UTP labeled probes (5 x 105 cpm, /50 ng) were loaded in lane 1 as a control (P). In lane 2 /8, labeled probes (5 x 106 cpm, /500 ng) were added to HeLa cell total protein extracts. Specific competitors (unlabeled RNA of the probe sequence) were added in lane 3 /5 at the indicated molar excess ratios. Yeast tRNA was added in lane 6 /8 at the indicated quantities, and served as a non-specific competitor. Protein-RNA complexes (Co) were analyzed by 4% nondenaturing PAGE. Note that the increase in specific competitor (lane 3 /5) resulted in the corresponding decrease in the proteinprobe complex formation. However, this was not observed for the non-specific competitor (lane 6 /8), indicating the specific nature of protein binding to the three probes.

To identify the proteins that bind to the 5? UTR probes, we performed competitive UV cross-linking experiments. After UV cross-linking, RNase A was added to digest the non-interacting regions of the labeled probes, and thus allows realistic MW estimates (Fig. 3a/c). Lane 1 in each assay illustrates probe-protein complexes without competitor. Lane 2 is a negative control which includes samples that were not treated with UV radiation. Self competitor of the corresponding probe sequence was added in lanes 3, 4, and 5 at the indicated molar ratios relative to the probe. As in the mobility shift experiments, specific interactions were concluded when the increase in band intensity corresponds to the decrease in competitor ratios. Fig. 3a demonstrates a total of six proteins specifically interacting with the probe nt 1/209. The MWs of these proteins are 200, 80, 62, 52, 45 and 39 kDa. Similarly, a total of eight proteins were observed to bind the nt 210/529 sequence (Fig. 3b). These proteins have MWs of 200, 100, 85, 65, 57, 52, 45 and 39 kDa. These protein-RNA complexes were differently susceptible to competitions. For example, the 200 kDa protein band was restored only at 1/50x molar excess ratio (lane 5) while the 45 kDa band was detected at 1x molar excess ratio (lane 4). The differences in susceptibility to competition can be a consequence of the binding specificities of the respective proteins

P. Cheung et al. / Virus Research 90 (2002) 23 /36

29

of these proteins are 52, 57, 95, and 170 kDa. All these proteins appeared to specifically interact with the core IRES sequence as they were all susceptible to competitions. Overall, the different patterns of protein-RNA interactions among these three 5? UTR probes are indicative of the presence of various HeLa proteins that specifically bind to the different locations of the CVB3 5? UTR.

3.3. Interaction of the La autoantigen with the 5? UTR

Fig. 3. UV cross-linking to determine the MW of RNAbinding proteins. [a/32P]UTP-labeled probes (5 x 106 cpm, /500 ng) were added to HeLa cell total protein extracts and cross-linked by UV light exposure, followed by RNase A treatment. Proteins that cross-linked to radioactive probes were detected by the 9% SDS-PAGE and subsequent autoradiography. Lane 1 demonstrates protein-probe interactions without competitor. Lane 2 is a control without UV-crosslinking. Lanes 3 /5 demonstrate the specificity of interactions by adding the indicated amount of specific competitors to the reaction. The MW of each complex was determined by comparing to a concurrently loaded MW marker. Data for probe nt 1 /209, 210 /529, and 530 /630 are shown in Fig. 3a, b, and c, respectively.

towards the probe sequence. Four major proteins are observed to bind the IRES (Fig. 3c). The MWs

As shown in Fig. 3, a 52 kDa protein appears to interact with all three of the UTR probes. As mentioned earlier, the La autoantigen (which also has a MW of 52 kDa) binds to other RNA viruses closely related to CVB3. To confirm whether the 52 kDa can specifically interact with different regions of the CVB3 5? UTR, we performed UV cross-linking experiments using purified recombinant La protein. The purity of the La protein was verified by Commassie blue staining of 10% SDSPAGE (Fig. 4). Both the GST-La fusion protein (/80 kDa) and the La autoantigen (52 kDa) were prepared. Two mg of the proteins were UV crosslinked to the three labeled probes at the same conditions as described above. RNA probes of the nt 1/209, 210 /529 and 530 /630 were tested for protein interaction in Fig. 5a, b and c, respectively, with the addition of different 5? UTR competitors or non-homologous competitor yeast tRNA. RNase A digestion was performed prior to analyzing the protein-RNA complex. The observation of bands thus indicates the specific interaction of the La protein with the respective RNA probe. The use of GST-La serves as a control to indicate that the observed bands were protein-specific. In each of the probes (Fig. 5a /c), both the GST-La fusion protein and La protein can form a protein-probe complex (band) of different intensities at the expected MW locations, suggesting that the La protein can specifically interact with all three 32Plabeled probes at different affinities. Interaction of the GST-La fusion protein with probes were detected at significantly lower intensities (comparing lanes 1 and 3). This is probably because the uncleaved GST domain of the fusion protein

30

P. Cheung et al. / Virus Research 90 (2002) 23 /36

3.4. Relative affinities of La autoantigen to the 5? UTR sequences In order to determine the relative affinities of the La autoantigen towards each of the three regions of the CVB3 5? UTR, we introduced unlabeled RNAs of the 5? UTR sequences as competitors against labeled probes as shown in Fig. 5(a /c). In each experiment, the protein-probe complex in lane 2 was subjected to competition from unlabeled self RNA. The intensity of the

Fig. 4. Expression and purification of the recombinant La autoantigen. E. coli (BL21 DE3) transformed with pGEX-La (Materials and Methods ) were induced by IPTG to produce the GST-fused La autoantigen ( /80 kDa). Total lysate was prepared from the bacterial culture (lane 4). Affinity purification with glutathione sepharose beads produced the GST-fused La autoantigen (lane 2). Cleavage of the GST-fused La autoantigen by thrombin on the affinity column produced the 52 kDa La autoantigen (lane 1). Negative control (lane 3) was affinity column wash without the use of thrombin. The protein preparation was then used in subsequent UV cross-linking experiments.

constituted unfavorable conformations for interaction with the probe.

Fig. 5. Specific interaction between the La autoantigen and the 5? UTR probes. The experiments for competitive UV crosslinking were performed as described in Fig. 3. Lane 1 in each panel shows that the GST-La protein interacts with the probes but at significantly reduced intensity as compared to that with the pure La protein (lanes 2 /6). In each competitive interaction assays, competitor RNAs were added at 3-fold molar excess over that of the probe. The use of self competitor results in competition (lane 2) and the effect of such was compared to the use of competitor from other sequences (lane 4 /5) (see Results). Lane 3 demonstrates the uncompleted interaction between the La autoantigen and the respective probes. Yeast tRNA did not compete against the probe for interaction (lane 6). Data for probe nt 1 /209, nt 210 /529, and nt 530 /630 is shown in Fig. 5a, 5b, and 5c, respectively. For the sequence nt 1 /209, preheating of the probe to 95 8C for 2 min prior to reaction at 37 8C was required to detect the bands.

Fig. 5

P. Cheung et al. / Virus Research 90 (2002) 23 /36

probe-protein complex in the presence of self competitor is a standard for comparison to the effect of competition from other UTR RNAs. An RNA competitor with a stronger affinity for the La protein would result in more significant reduction in band intensity than a competitor with weaker affinity. The excess free probe is then digested by the RNase A treatment, and thus by comparing the intensities of La protein-probe complexes in each gel we can deduce the relative affinities of the UTR sequences towards the La autoantigen. Fig. 5a demonstrates the UV cross-linking of nt 1-209 to the La autoantigen. It is important to note that protein-probe complex could only be observed when the labeled probes of nt 1 /209 were preheated to 95 8C for 2 min prior to reaction at 37 8C. Hence, we believe that complex formation between the La protein and the nt 1 /209 was suboptimal at 37 8C. Intensity of the La proteinprobe complex in lane 4, in which nt 210/529 was added as a competitor, is much weaker than that in lane 2, hence competition from nt 210/529 is considered to be much stronger than that of the self-competitor (Fig. 5a, lane 2). Similarly, competitor of the IRES core sequence (lane 5) also resulted in the reduction of band intensity and this RNA sequence also appears to have a stronger competition effect than the self competitor (lane 2). This observation is in line with the relative order of affinity that was deduced in the experiments using nt 210/529 and the IRES core sequence (Fig. 5b and c) as probe. In Fig. 5b, the intensity of the probe-protein complex in the presence of self competitor (lane 2) is much weaker than in lane 4 and 5. The band intensity in lane 5 is in turn weaker than that in lane 4. In other words, the La protein binds most strongly to the nt 210/ 529 (lane 2), less so to the 530/630 region (Fig. 5b, lane 5), and the weakest to the nt 1 /209 region (Fig. 5b, lane 4). This hierarchy of affinity is also consistent with observation in the experiment using sequence nt 529 /630 as probe (Fig. 5c). The band intensity in lane 5, in which nt 210/529 was used as a competitor, is weaker than that of lane 2, in which the self competitor was added. This indicates that the competitor of sequence nt 210 /529 presented a stronger La-binding activity

31

than the probe (IRES core sequence). Also, the band intensity in lane 4, in which nt 1 /209 was used as a competitor, is slightly higher than that in lane 2. However, band intensities in both lane 2 and 4 are lower than that of the non-competed interaction (lane 3). Comparing lane 2 to lane 3 in each panel of Fig. 5, we have observed differential susceptibilities of the protein-probe complexes to self competitors: i.e. the change in band intensities from the uncompeted interaction (lane 3) to the self competed (lane 2) is different for each probe. For example, in the experiment with probe nt 1 / 209 (Fig. 5a), the reduction in band intensity in the presence of self competitor (lane 2) is minimal as compared to that of probe nt 210/529 (Fig. 5b, lane 2). This is likely due to the differences in the higher-order structures and the kinetics of the protein-RNA interactions of the respective probe sequences, which will be discussed later. Nonetheless, considering the strong signal of the probeprotein complex (lane 3), and that the intensity of the probe-protein complex appears unaffected by the addition of the yeast tRNA (lane 6), we conclude that the La autoantigen binds to the three 5? UTR probes specifically but at differential affinities.

4. Discussion The work presented here is the first attempt to identify interactions between host proteins and the 5? UTR of CVB3. The HeLa cell line has been chosen because it is a well established system for studying viral replication and infectivity (Liu et al., 1999). In addition, the HeLa cell lysate contains host proteins that support internal translation initiation of the viral RNA (Villa-Komaroff et al., 1975). The high specificity of interactions between HeLa cell proteins and the three CVB3 5? UTR probes can be demonstrated by the contrast between competition effects of the specific RNA competitors and the yeast tRNA. The later competitor had negligible effect on protein-probe complex formation because it has no sequence homology to the CVB3 5? UTR. The mobility shift assays (Fig. 2a /c) were performed without RNase

32

P. Cheung et al. / Virus Research 90 (2002) 23 /36

A digestion and were under non-reducing conditions, thus the smear like patterns represent multiple protein-probe complexes which could not be distinguished as discrete bands. The addition of specific competitor pre-occupied the probe-binding sites on the proteins that were available to the formation of complexes, thus allowing labeled probes to be observed at their corresponding electrophoretic mobilities. As verified by the UV cross-linking in Fig. 3, there are many different complexes formed between the HeLa cell proteins and the probes. These complex formations had different interaction kinetics and thus were differentially affected by the competitor. Factors such as the relative abundance of the 5? UTR binding proteins in the HeLa cell extracts and the affinities of those proteins towards the probe would determine how the complexes were affected by the addition of competitors. Consequently we observed several band-like patterns of complexes at high molar excess of competitors, such as that observed in lane 5 of Fig. 2c. This suggests that multiple proteins interact with the same RNA molecule and that some of these proteins are in relative higher content than other specific binding proteins. In this study, we have demonstrated a number of proteins binding to the three different 5? UTR sequences. Some of these UTR-binding proteins may be identical proteins that have multiple binding sites in the 5? UTR; some of them may be different proteins with similar MWs. Since there is no previous report on protein-RNA interactions of the CVB3, we compare our findings to a close relative of CVB3, the poliovirus. Dildine and Semler (1992) reported that HeLa cell proteins that interact with the poliovirus 5? UTR can be cross-competed by the CVB3 5? UTR, suggesting a conservation of both the computer predicted stem loop structures as well as the basic function of the RNA-protein interaction in life cycles and perhaps IRES-directed translation initiation. Further work by other researchers have identified a number of unknown proteins (Haller and Semler, 1995) and known proteins such as the hnRNP, PTB, La autoantigen and PCBP2 that bind to the IRES, with MW 68, 57, 52 and 39 kDa, respectively (Andino et al., 1993; Blyn et al., 1997; Hellen et al.,

1993; Meerovitch et al., 1993). Of note, the PTB can work against another host protein GAPDH on IRES function (Yi et al., 2000), while GAPDH is also capable of IRES interaction (Schultz et al., 1996). This observation is contrasted to the synergistic effect between the La autoantigen and PTB in the IRES function of EMCV. Although a detailed mechanism of how these proteins contribute to the regulation of internal ribosomal entry on the viral 5? UTRs is unknown, it is clear that an intricate network of host proteins are involved in the general process of enteroviral translation initiation. Reflecting on these findings, we report that some of the CVB3 5? UTR-binding proteins have molecular weights that are identical to the above mentioned five polio-UTR interacting proteins, suggesting possible identities of those CVB3 5? UTR-binding proteins. It is also important to note there are similarities between the CVB3 5? UTR-binding proteins and host proteins that are pertinent to the translation process. These host proteins include the eIF4A (45 kDa), eIF4B (80 kDa), and eIF4G (170 /220kDa) (reviewed by Gingras et al., 1999). The three eIF4 proteins are constituent molecules of the 220kDa eIF4F that is critical in the translation initiation of eukaryotic mRNA. Although MW determinations were not definitive in our UV cross-linking assay, the protein complexes that we have detected appear to be within reasonable range of these abovementioned proteins. A large 170 kDa protein complex binding to the CVB3 IRES sequence is similar to the MW of the well-characterized eIF4G. The eIF4G is sometimes detected as a 220 kDa protein when it combines with other eIF factors to form the eIF4F complex (Thomas et al., 1992). The integral eIF4F is crucial for host capdependent mRNA translation initiation (Merrick 1992). Incidentally, there is a /200 kDa complex detected for probes nt 1 /209 and 210/529. The normal cellular functions of the eIF4A and eIF4B are to resolve complex secondary structures of the mRNA prior to binding of eIF4G and ribosome, followed by translation initiation (Gingras et al., 1999). The binding of these factors to the nt 1 /209 and nt 210/529 of the CVB3 5? UTR would not be surprising since extensive stem-loop structures have been predicted within these sequences (Fig.

P. Cheung et al. / Virus Research 90 (2002) 23 /36

1) and the eIF4A and eIF4B could resolve the secondary structures prior to internal ribosomal entry and translation. Pertinent to viral pathogenesis, eIF4G alone has been reported to bind the IRES of the EMCV (Kolupaeva et al., 1998); the eIF4E binding domain of the eIF4G is cleaved by CVB3 protease 2A during an active infection (Etchison et al., 1982; Lamphear et al., 1995), leading to the inhibition of host mRNA translation while viral uncapped RNA remains unaffected. Reflecting on these findings, we propose that eIF4G may also bind the IRES of CVB3 and play important roles in the translation initiation of the viral RNA, while eIF4A and eIF4B can be involved in the resolution of secondary structures in the 5? UTR and facilitate the binding of eIF4G and ribosomes. La autoantigen is another host protein that appears to play a role in the translation initiation of various viral RNA (Isoyama et al., 1999; Kim and Jang 1999). In normal cellular environments, La has also been reported to interact with the Urich 5? regions of ribosomal protein mRNAs in vitro and stimulate their translation in vivo in a Xenopus tissue culture system (Crosio et al., 2000; Gottlieb and Steitz 1989b). A similar U-rich region is also present in the CVB3 IRES sequence (Fig. 1). Interestingly, poliovirus protease 3C is capable of cleaving this protein and may be responsible for the translocation of La from nucleus to cytoplasm during picornaviral infection, indicating a potential functional role of the modified protein in the IRES-mediated translation initiation (Meerovitch et al., 1993). La autoantigen was initially identified in patients suffering from autoimmune disorders systemic lupus erythematosus and Sjogren’s syndrome (Alspaugh and Tan 1975; Mattioli and Reichlin 1974). It is now understood that the La protein mainly functions in the synthesis, termination and release of polymerase III transcripts (Gottlieb and Steitz 1989a). La can also process tRNA molecules and form snRNP complexes prior to ribosomal binding (Gottlieb and Steitz 1989b). In our experiments, by comparing crosscompetition from the 5? UTR sequences, the La autoantigen appears to bind most strongly to the sequence nt 210 /529. The presence of a major tRNA-like stem loop in this region (nt 210 /481,

33

Fig. 1) echoes with the tRNA processing function of the protein. This sequence, although not as critical as the core IRES is in the translation initiation of the virus (Yang et al., 1997), may thus serve as an important landmark for recognition of the CVB3 RNA by the La autoantigen. As mentioned in the Introduction, determinants of the CVB3 cardiovirulence have been mapped to nucleotides in this region (Chapman et al., 1994; Dunn et al., 2000; Tu et al., 1995) The binding of other yet unknown proteins as detected by our UV cross-linking with the HeLa cell extract may also participate in the process of translation initiation and, eventually form a protein-RNA complex that has a high affinity towards the 40S ribosome. Although the La autoantigen appears to bind to all three probes of the 5? UTR (Fig. 5), we observe no specifically conserved primary sequences within the three regions that could be responsible for the potential La protein binding. Thus, we suggest that the interaction of La with the CVB3 5? UTR occurs mostly via recognition of high-order structures of the 5? UTR RNA. This postulate is also supported by the observation that pre-heating the sequence nt 1 /209 was required for the probe-La complex formation. Heating the probe at 95 8C prior to experiment denatures high-order structures, while the addition of the probes to the reaction allows the probes to assume an intermediate conformation that can be readily recognized by the La protein. Nonetheless, this observation suggests that the La protein alone does not bind the nt 1/209 very efficiently at 37 8C and probably requires chaperon proteins that resolve the stem-loop structures prior to its interaction. Under normal cellular conditions, RNA stem-loop structures can be resolved by proteins such as eIF4A and eIF4B (Gingras et al., 1999), thus possibly optimizing the RNA structures for further protein-RNA interactions. Hence, the apparent relative affinities of the La autoantigen towards the three 5? UTR probes that we have tested could be different once other cellular proteins are present. In other words, additional host proteins could serve as scaffolding or chaperone molecules that enhance the complex formation of 5? UTR and the La protein. Reflecting on the finding that the La protein can associate

34

P. Cheung et al. / Virus Research 90 (2002) 23 /36

with the 40S ribosomal subunits (Peek et al., 1996; Pellizzoni et al., 1996), our data suggests that the La autoantigen is critical to the internal translation initiation of the CVB3 genome by forming a pre-translation initiation protein-RNA complex. This postulate is also compatible with the observations that the La autoantigen binds to the IRES of EMCV and stimulates translation initiation (Kim and Jang 1999), and that La protein interacts with multiple sites on the hepatitis C virus RNA (Ali and Siddiqui 1997). Our study with the La autoantigen is the first step towards the identification of host proteins involved in the internal translation initiation of the CVB3 genomic RNA. Verification of other protein-RNA interactions (such as the above mentioned eukaryotic translation initiation factors) and identification of other unknown proteins would greatly facilitate the understanding of the pre-initiation complex formation and possibly the mechanisms of 5? UTR-related tissue tropism and cardiovirulence.

Acknowledgements We would like to thank Dr. Sung Key Jang, Pohang University of Science and Technology, Korea, for generously providing the La-GST recombinant plasmid. This work is supported by grants from the Canadian Institutes of Health Research, and the Heart and Stroke Foundation of B.C. and Yukon (HSFBCY) (Drs. D Yang and B McManus) and a studentship from the HSFBCY (P. Cheung and B. Yanagawa).

References Ali, N., Siddiqui, A., 1995. Interaction of polypyrimidine tractbinding protein with the 5’ noncoding region of the hepatitis C virus RNA genome and its functional requirement in internal initiation of translation. J. Virol. 69, 6367 /6375. Ali, N., Siddiqui, A., 1997. The La antigen binds 5’ noncoding region of the hepatitis C virus RNA in the context of the initiator AUG codon and stimulates internal ribosome entry site-mediated translation. Proc. Natl. Acad. Sci. USA 94, 2249 /2254. Alspaugh, M.A., Tan, E.M., 1975. Antibodies to cellular antigens in Sjogren’s syndrome. J. Clin. Invest. 55, 1067 / 1073.

Andino, R., Rieckhof, G.E., Achacoso, P.L., Baltimore, D., 1993. Poliovirus RNA synthesis utilizes an RNP complex formed around the 5’-end of viral RNA. EMBO J. 12, 3587 /3598. Blyn, L.B., Towner, J.S., Semler, B.L., Ehrenfeld, E., 1997. Requirement of poly(rC) binding protein 2 for translation of poliovirus RNA. J. Virol. 71, 6243 /6246. Carter, M.S., Sarnow, P., 2000. Distinct mRNAs that encode La autoantigen are differentially expressed and contain internal ribosome entry sites. J. Biol. Chem. 275, 28301 / 28307. Carthy, C.M., Yang, D., Anderson, D.R., Wilson, J.E., McManus, B.M., 1997. Myocarditis as systemic disease: new perspectives on pathogenesis. Clin. Exp. Pharmacol. Physiol. 24, 997 /1003. Chapman, N.M., Tu, Z., Tracy, S., Gauntt, C.J., 1994. An infectious cDNA copy of the genome of a non-cardiovirulent coxsackievirus B3 strain: its complete sequence analysis and comparison to the genomes of cardiovirulent coxsackieviruses. Arch. Virol. 135, 115 /130. Cherry, J.D., 1995. Enteroviruses. Remington, J.S., Klein, K.O., (Eds.), Infectious diseases of the fetus and newborn infant, 4th ed. Saunders, W.H., Philadelphia, pp. 404 /446. Crosio, C., Boyl, P.P., Loreni, F., Pierandrei-Amaldi, P., Amaldi, F., 2000. La protein has a positive effect on the translation of top mRNAs in vivo. Nucleic Acids Res. 28, 2927 /2934. De, B.P., Gupta, S., Zhao, H., Drazba, J.A., Banerjee, A.K., 1996. Specific interaction in vitro and in vivo of glyceraldehyde-3-phosphate dehydrogenase and LA protein with cisacting RNAs of human parainfluenza virus type 3. J. Biol. Chem. 271, 24728 /24735. Dildine, S.L., Semler, B.L., 1992. Conservation of RNAprotein interactions among picornaviruses. J.Virol. 66, 4364 /4376. Drew, J., Belsham, G.J., 1994. Trans complementation by RNA of defective foot-and-mouth disease virus internal ribosome entry site elements. J. Virol. 68, 697 /703. Dunn, J.J., Chapman, N.M., Tracy, S., Romero, J.R., 2000. Genomic determinants of cardiovirulence in coxsackievirus B3 clinical isolates: localization to the 5’ nontranslated region. J. Virol. 74, 4787 /4794. Etchison, D., Milburn, S.C., Edery, I., Sonenberg, N., Hershey, J.W., 1982. Inhibition of HeLa cell protein synthesis following poliovirus infection correlates with the proteolysis of a 220,000-dalton polypeptide associated with eucaryotic initiation factor 3 and a cap binding protein complex. J. Biol. Chem. 257, 14806 /14810. Gamarnik, A.V., Andino, R., 1998. Switch from translation to RNA replication in a positive-stranded RNA virus. Genes Dev. 12, 2293 /2304. Gauntt, C., Godney, E., Lutton, C., Arizipe, H., Chapman, N., Tracy, S., Revtyak, G., Valente, A., Rozek, M., 1989. Mechanisms of coxsackievirus induced acute myocarditis in the mouse. In: De la Maza, L., Peterson, E. (Eds.), Medical Virology (8th). Lawrence Erlbaum Associates, Hellsdale, N.J, pp. 161 /182.

P. Cheung et al. / Virus Research 90 (2002) 23 /36 Gingras, A.C., Raught, B., Sonenberg, N., 1999. eIF4 initiation factors: effectors of mRNA recruitment to ribosomes and regulators of translation. Annu. Rev. Biochem. 68, 913 / 963. Gottlieb, E., Steitz, J.A., 1989a. Function of the mammalian La protein: evidence for its action in transcription termination by RNA polymerase III. EMBO J. 8, 851 /861. Gottlieb, E., Steitz, J.A., 1989b. The RNA binding protein La influences both the accuracy and the efficiency of RNA polymerase III transcription in vitro. EMBO J. 8, 841 /850. Gutierrez-Escolano, A.L., Brito, Z.U., del Angel, R.M., Jiang, X., 2000. Interaction of cellular proteins with the 5’ end of Norwalk virus genomic RNA. J. Virol. 74, 8558 /8562. Haller, A.A., Semler, B.L., 1995. Stem-loop structure synergy in binding cellular proteins to the 5’ non-coding region of poliovirus RNA. Virology. 206, 923 /934. Hellen, C.U., Witherell, G.W., Schmid, M., Shin, S.H., Pestova, T.V., Gil, A., Wimmer, E., 1993. A cytoplasmic 57-kDa protein that is required for translation of picornavirus RNA by internal ribosomal entry is identical to the nuclear pyrimidine tract-binding protein. Proc. Natl. Acad. Sci. USA 90, 7642 /7646. Isoyama, T., Kamoshita, N., Yasui, K., Iwai, A., Shiroki, K., Toyoda, H., Yamada, A., Takasaki, Y., Nomoto, A., 1999. Lower concentration of La protein required for internal ribosome entry on hepatitis C virus RNA than on poliovirus RNA. J. Gen. Virol. 80 ((Pt 9)), 2319 /2327. Jackson, R.J., Kaminski, A., 1995. Internal initiation of translation in eukaryotes: the picornavirus paradigm and beyond. RNA 1, 985 /1000. Johnson, R.A., Palacios, I., 1982. Dilated cardiomyopathies of the adult (first of two parts). N. Engl. J. Med. 307, 1051 / 1058. Kandolf, R., Ameis, D., Kirschner, P., Canu, A., Hofschneider, P.H., 1987. In situ detection of enteroviral genomes in myocardial cells by nucleic acid hybridization: an approach to the diagnosis of viral heart disease. Proc. Natl. Acad. Sci. USA 84, 6272 /6276. Keeling, P.J., Lukaszyk, A., Poloniecki, J., Caforio, A.L., Davies, M.J., Booth, J.C., McKenna, W.J., 1994. A prospective case-control study of antibodies to coxsackie B virus in idiopathic dilated cardiomyopathy. J. Am. Coll. Cardiol. 23, 593 /598. Kim, Y.K., Jang, S.K., 1999. La protein is required for efficient translation driven by encephalomyocarditis virus internal ribosomal entry site. J. Gen. Virol. 80 ((Pt 12)), 3159 /3166. Klingel, K., Stephan, S., Sauter, M., Zell, R., McManus, B.M., Bultmann, B., Kandolf, R., 1996. Pathogenesis of murine enterovirus myocarditis: virus dissemination and immune cell targets. J. Virol. 70, 8888 /8895. Kolupaeva, V.G., Hellen, C.U., Shatsky, I.N., 1996. Structural analysis of the interaction of the pyrimidine tract-binding protein with the internal ribosomal entry site of encephalomyocarditis virus and foot-and-mouth disease virus RNAs. RNA 2, 1199 /1212. Kolupaeva, V.G., Pestova, T.V., Hellen, C.U., Shatsky, I.N., 1998. Translation eukaryotic initiation factor 4G recognizes

35

a specific structural element within the internal ribosome entry site of encephalomyocarditis virus RNA. J. Biol. Chem. 273, 18599 /18604. La Monica, N., Racaniello, V.R., 1989. Differences in replication of attenuated and neurovirulent polioviruses in human neuroblastoma cell line SH-SY5Y. J. Virol. 63, 2357 /2360. Lamphear, B.J., Kirchweger, R., Skern, T., Rhoads, R.E., 1995. Mapping of functional domains in eukaryotic protein synthesis initiation factor 4G (eIF4G) with picornaviral proteases. Implications for cap-dependent and cap-independent translational initiation. J. Biol. Chem. 270, 21975 / 21983. Liu, Z., Carthy, C.M., Cheung, P., Bohunek, L., Wilson, J.E., McManus, B.M., Yang, D., 1999. Structural and functional analysis of the 5’ untranslated region of coxsackievirus B3 RNA: In vivo translational and infectivity studies of fulllength mutants. Virology 265, 206 /217. Lopez, D.Q., Martinez-Salas, E., 1997. Conserved structural motifs located in distal loops of aphthovirus internal ribosome entry site domain 3 are required for internal initiation of translation. J. Virol. 71, 4171 /4175. Macadam, A.J., Ferguson, G., Burlison, J., Stone, D., Skuce, R., Almond, J.W., Minor, P.D., 1992. Correlation of RNA secondary structure and attenuation of Sabin vaccine strains of poliovirus in tissue culture. Virology 189, 415 /422. Martinez-Salas, E., Ramos, R., Lafuente, E., Lopez, D.Q., 2001. Functional interactions in internal translation initiation directed by viral and cellular IRES elements. J. Gen. Virol. 82, 973 /984. Martino, T.A., Liu, P., Sole, M.J., 1994. Viral infection and the pathogenesis of dilated cardiomyopathy. Circ. Res. 74, 182 /188. Mattioli, M., Reichlin, M., 1974. Heterogeneity of RNA protein antigens reactive with sera of patients with systemic lupus erythematosus. Description of a cytoplasmic nonribosomal antigen. Arthritis Rheum 17, 421 /429. McManus, B.M., Chow, L.H., Wilson, J.E., Anderson, D.R., Gulizia, J.M., Gauntt, C.J., Klingel, K.E., Beisel, K.W., Kandolf, R., 1993. Direct myocardial injury by enterovirus: a central role in the evolution of murine myocarditis. Clin. Immunol. Immunopathol. 68, 159 /169. Meerovitch, K., Svitkin, Y.V., Lee, H.S., Lejbkowicz, F., Kenan, D.J., Chan, E.K., Agol, V.I., Keene, J.D., Sonenberg, N., 1993. La autoantigen enhances and corrects aberrant translation of poliovirus RNA in reticulocyte lysate. J. Virol. 67, 3798 /3807. Merrick, W.C., 1992. Mechanism and regulation of eukaryotic protein synthesis. Microbiol. Rev. 56, 291 /315. Peek, R., Pruijn, G.J., Van Venrooij, W.J., 1996. Interaction of the La (SS-B) autoantigen with small ribosomal subunits. Eur. J. Biochem. 236, 649 /655. Pellizzoni, L., Cardinali, B., Lin-Marq, N., Mercanti, D., Pierandrei-Amaldi, P., 1996. A Xenopus laevis homologue of the La autoantigen binds the pyrimidine tract of the 5’ UTR of ribosomal protein mRNAs in vitro: implication of a protein factor in complex formation. J. Mol. Biol. 259, 904 / 915.

36

P. Cheung et al. / Virus Research 90 (2002) 23 /36

Pestova, T.V., Hellen, C.U., Wimmer, E., 1991. Translation of poliovirus RNA: role of an essential cis-acting oligopyrimidine element within the 5’ nontranslated region and involvement of a cellular 57-kilodalton protein. J. Virol. 65, 6194 /6204. Robertson, M.E., Seamons, R.A., Belsham, G.J., 1999. A selection system for functional internal ribosome entry site (IRES) elements: analysis of the requirement for a conserved GNRA tetraloop in the encephalomyocarditis virus IRES. RNA 5, 1167 /1179. Rohll, J.B., Percy, N., Ley, R., Evans, D.J., Almond, J.W., Barclay, W.S., 1994. The 5’-untranslated regions of picornavirus RNAs contain independent functional domains essential for RNA replication and translation. J. Virol. 68, 4384 /4391. Schultz, D.E., Hardin, C.C., Lemon, S.M., 1996. Specific interaction of glyceraldehyde 3-phosphate dehydrogenase with the 5’-nontranslated RNA of hepatitis A virus. J. Biol. Chem. 271, 14134 /14142. Sizova, D.V., Kolupaeva, V.G., Pestova, T.V., Shatsky, I.N., Hellen, C.U., 1998. Specific interaction of eukaryotic translation initiation factor 3 with the 5’ nontranslated regions of hepatitis C virus and classical swine fever virus RNAs. J. Virol. 72, 4775 /4782. Sole, M.J., Liu, P., 1993. Viral myocarditis: a paradigm for understanding the pathogenesis and treatment of dilated cardiomyopathy. J. Am. Coll. Cardiol. A22, 99A /105A. Svitkin, Y.V., Meerovitch, K., Lee, H.S., Dholakia, J.N., Kenan, D.J., Agol, V.I., Sonenberg, N., 1994. Internal translation initiation on poliovirus RNA: further character-

ization of La function in poliovirus translation in vitro. J. Virol. 68, 1544 /1550. Thomas, A.A., Scheper, G.C., Kleijn, M., De Boer, M., Voorma, H.O., 1992. Dependence of the adenovirus tripartite leader on the p220 subunit of eukaryotic initiation factor 4F during in vitro translation. Effect of p220 cleavage by foot-and-mouth-disease-virus L-protease on in vitro translation. Eur. J. Biochem. 207, 471 /477. Tu, Z., Chapman, N.M., Hufnagel, G., Tracy, S., Romero, J.R., Barry, W.H., Zhao, L., Currey, K., Shapiro, B., 1995. The cardiovirulent phenotype of coxsackievirus B3 is determined at a single site in the genomic 5’ nontranslated region. J. Virol. 69, 4607 /4618. Villa-Komaroff, L., Guttman, N., Baltimore, D., Lodishi, H.F., 1975. Complete translation of poliovirus RNA in a eukaryotic cell-free system. Proc. Natl. Acad. Sci. USA 72, 4157 / 4161. Yang, D., Wilson, J.E., Anderson, D.R., Bohunek, L., Cordeiro, C., Kandolf, R., McManus, B.M., 1997. In vitro mutational and inhibitory analysis of the cis-acting translational elements within the 5’ untranslated region of coxsackievirus B3: potential targets for antiviral action of antisense oligomers. Virology 228, 63 /73. Yi, M.K., Shultz, D.E., Lemon, S.M., 2000. Functional significance of the interaction of hepatitis A virus RNA with glyercaldehyde 3-phosphate dehydrogenase (GAPDH): opposing effects of GAPDH and polypyrimidine tract binding protein on internal ribosomal entry site function. J. Virol. 74, 6459 /6468. Zuker, M., 1989. On finding all suboptimal foldings of an RNA molecule. Science 244, 48 /52.