Cleavage of Poly(A)-Binding Protein by Coxsackievirus 2A Protease In Vitro and In Vivo: Another Mechanism for Host Protein Synthesis Shutoff?

Materials.

[³H]Leu (60 Ci/mmol) was purchased from DuPont-NEN, and [³⁵S]Met (>1,000 Ci/mmol) was purchased from both DuPont-NEN and Amersham. Horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G (IgG) and alkaline phosphatase-conjugated horse anti-mouse IgG were obtained from Vector Laboratories, Burlingame, Calif. Immobilon P was purchased from Millipore, Bedford, Mass. Glutathione-Sepharose 4B and protein A-agarose were obtained from Pharmacia and Pierce, respectively. Female frogs (Xenopus laevis) were purchased from Xenopus I, Madison, Wis. Cyanogen bromide-activated Sepharose 4B and poly(A) were purchased from Sigma.

Cells and cell culture.

HeLa cells (a gift from S. A. Huber, University of Vermont, Burlington, Vt.) and COS-1 cells (American Type Culture Collection, Rockville, Md.) were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco BRL, Gaithersburg, Md.) with high glucose (4.5 g/liter) supplemented with 10% heat-inactivated fetal bovine serum, 1 mM sodium pyruvate, 2 mM l-glutamine, 100 μg of streptomycin per ml, and 100 U of penicillin G per ml all from Irvine Scientific, Santa Ana, Calif.) at 37°C under 5% CO₂.

Virus and viral assays.

A plasmid containing the full-length, infectious cDNA of coxsackievirus B3 (37) was transfected into COS-1 cells. At 72 h posttransfection, the supernatant was transferred to HeLa cells. When the cytopathic effect was complete, cells were lysed and the virus was passaged once in HeLa cells for amplification. The lysate was subjected to three cycles of freeze-thawing for release of intracellular virus. The supernatant was then subjected to titer determination by a plaque-forming assay with HeLa cells as the titering cell line (29).

Infection of HeLa cells with coxsackievirus and pulse-labeling.

HeLa cells (2 × 10⁶) were plated on a 90-mm dish and allowed to adhere overnight. Cells were infected with coxsackievirus B3 at a multiplicity of infection of 2 in 1 ml of DMEM with 2% heat-inactivated fetal bovine serum. After 30 min of incubation, new medium (containing l-Met) was added to the cells. At 30 min prior to harvest, the medium was removed and the cells were washed three times with Met-deficient DMEM. Then Met-deficient medium was added to each plate, followed by 50 μCi of [³⁵S]Met, and the cells were returned to the incubator for 30 min. At 0 h (mock infected) and at various times postinfection, the cells were washed once with phosphate-buffered saline, trypsinized, and pelleted by centrifugation at 300 × g for 10 min. The cells were then resuspended in 100 ml of lysis buffer with 1 mM dithiothreitol (DTT; Clontech, Palo Alto, Calif.) and allowed to stand for 15 min at 4°C. After removal of insoluble material, the supernatant was stored in aliquots at −20°C.

Preparation of 2A protease.

Plasmid pET8c/CVB4 2A, a gift from Tim Skern, University of Vienna, encodes a fusion protein consisting of 13 amino acids of the T7 gene 10 protein, 34 amino acids of coxsackievirus B4 VP1, and 150 amino acids of coxsackievirus B4 2A protease. This plasmid was used to produce recombinant 2A protease in Escherichia coli (44). The enzyme was purified and stored at 4°C at a concentration of 2.9 mg/ml in buffer A (50 mM Tris-HCl [pH 8.0], 50 mM NaCl, 1 mM EDTA, 5 mM DTT, 50% glycerol).

Preparation of PABP.

Poly(A)-Sepharose was prepared by immobilization of poly(A) on cyanogen bromide-activated Sepharose 4B (53). For the preparation of rabbit PABP (rPABP), 40 ml of the postribosomal supernatant of RRL (15) was made 100 mM with respect to KCl and passed through 10 ml of poly(A)-Sepharose in a column as described previously (19). The bound PABP was eluted with buffer B (50 mM Tris-HCl [pH 7.6], 2 M LiCl). Fractions containing PABP (typically 0.5 ml) were identified by the Bradford assay (10), confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (10% polyacrylamide) with Coomassie blue staining, dialyzed overnight against 10 mM Tris-HCl (pH 7.6)–50 mM KCl at 4°C, and concentrated fourfold in a Microcon-30 apparatus (Amicon). This procedure typically yielded 50 μg of PABP.

A plasmid encoding recombinant human PABP fused at the N terminus to glutathione S-transferase (GST-hPABP) in the pGEX-2T vector (Pharmacia Biotech) was provided by Jnanankur Bag (University of Guelph, Canada) (4). The GST-hPABP was expressed in E. coli XL1-Blue cells (Stratagene) by induction with 0.1 mM isopropyl-β-d-thiogalactopyranoside and purified on glutathione-Sepharose (4). Bound GST-hPABP was eluted with 10 mM Tris-HCl (pH 8.0)–10 mM reduced glutathione.

hPABP was enriched from extracts (500 μl) of coxsackievirus-infected HeLa cells by the addition of 50 μl of a 50% (vol/vol) slurry of oligo(dT)-cellulose followed by 50 μl of a 50% (vol/vol) slurry of poly(A)-Sepharose. To the slurry was added 600 μl of 20 mM HEPES (pH 7.0)–100 mM KCl–250 mM NaCl–2 mM magnesium acetate–0.1 mM EDTA–0.5 mM DTT. The slurry was rotated at 4°C for 1 h and centrifuged at 500 × g. The supernatant was discarded, and the resin was washed once with the same buffer. The bound material was eluted by heating in a twice-concentrated Laemmli sample buffer and subjected to SDS-PAGE (39).

Preparation of protein fragments for sequencing.

For preparation of the 55-kDa N-terminal cleavage product of rabbit PABP (rPABP_N), 20 ml of the postribosomal supernatant from RRL was treated overnight with 110 μl of 2A protease (final concentration, 15 μg/ml) at room temperature. The solution was passed over a poly(A)-Sepharose column, and the bound material (rPABP_N) was eluted with buffer B, as described for purification of intact rPABP. The protein sample was concentrated in a Microcon-30 apparatus and electrophoretically transferred to a Teflon membrane at Kendrick Laboratories (Madison, Wis.), and the C-terminal amino acid was determined by sequencing on a Hewlett-Packard G1009A C-terminal sequencer at Argo BioAnalytica, Inc. (Morris Plains, N.J.).

For preparation of the 15-kDa C-terminal cleavage product of human PABP (hPABP_c), 658 μg of GST-hPABP was treated with 13 μl of 2A protease (final concentration, 50 μg/ml) in a total volume of 0.75 ml of buffer A (without glycerol) at 30°C for 4 h. A slurry of glutathione-Sepharose (50% [vol/vol]; 0.65 ml) was added, and the incubation was continued for 1 h at room temperature with constant rotation. The slurry was then poured into a column, and the flowthrough fraction, containing both hPABP_c and 2A protease, was collected. (GST-hPABP_N remained bound to the glutathione-Sepharose.) To resolve hPABP_c from 2A protease, an aliquot of the flowthrough fraction (∼5%) was applied to a 0.46- by 25-cm C₅ reverse-phase column (Phenomenex, Inc., Torrance, Calif.) equilibrated in 0.1% aqueous trifluoroacetic acid, and the column was developed with a linear gradient of acetonitrile (40). Fractions were analyzed by SDS-PAGE to identify hPABP_c. The pure hPABP_c (0.8 μg) was subjected to automated Edman degradation with an Applied Biosystems model 470A Sequenator at the University of Kentucky Macromolecular Structure Analysis Facility.

Expression and cleavage of PABP in Xenopus oocytes.

Capped, polyadenylated RNA encoding Xenopus PABP with an N-terminal Myc epitope tag (Myc-xPABP) was synthesized in vitro (47) from plasmid pSDM (provided by Peter Good, Louisiana State University Medical Center) by using SP6 polymerase (Promega Biotech). An ovary was surgically removed from female Xenopus laevis and rinsed with modified Barth’s saline. Stage VI oocytes were isolated manually, sorted, and injected with RNA (34). The oocytes were cultured for 12 h at room temperature, injected with recombinant 2A protease, cultured further for 2.5 h, and frozen in groups of 17. The oocytes were homogenized at 4°C in 0.5 ml of 30 mM HEPES (pH 7.5)–70 mM NaCl–7 mM 2-mercaptoethanol–0.1% Triton X-100–0.01% SDS–10 μg of RNase A per ml–0.5 μg of leupeptin per ml–1 mM phenylmethylsulfonyl fluoride and centrifuged at 25,000 × g for 5 min. Supernatants were diluted to 1 ml with the same buffer, and Myc-xPABP was immunoprecipitated with 1 μl of anti-Myc tag monoclonal 9E10 antibody (provided by Kelly Tatchell, Louisiana State University Medical Center). Incubation was carried out for 1 h at 4°C with gentle agitation. Immune complexes were immobilized on protein A-agarose and washed three times with the same buffer at room temperature. Protein was eluted from the beads with twice-concentrated Laemmli sample buffer at 100°C for 5 min. The immunoprecipitates were subjected to immunoblotting as described below.

Immunoblotting.

eIF4G, eIF4E and PABP were separated by SDS-PAGE and electrophoretically transferred to a polyvinylidene difluoride (PVDF) membrane with a Bio-Rad Mini Trans-Blot cell. The membranes were incubated with antibodies against either eIF4E, eIF4G, PABP, or Myc tag. Anti-PABP antibody was provided by Dan Schoenberg (Ohio State University, Columbus, Ohio). Immunoblotting for eIF4G was performed with antiserum which recognizes amino acid residues 327 to 342 in cp_N (76). Myc-xPABP was detected with the anti-Myc tag antibody described above. eIF4E was purified from human erythrocytes by affinity chromatography on m⁷GTP-Sepharose (71) and reverse-phase high-pressure liquid chromatography (HPLC) on a C₄ column (33) and used as a source of antigen for production of eIF4E antiserum in New Zealand rabbits. Immunoreactive species were visualized with goat anti-mouse secondary antibody conjugated to alkaline phosphatase (for Myc) or goat anti-rabbit secondary antibody conjugated to either horseradish peroxidase or alkaline phosphatase.

In vitro translation.

In vitro protein synthesis rates in a micrococcal nuclease-treated RRL translation system were measured by incorporating [³H]Leu into newly synthesized globin as described previously (13), except that the potassium acetate concentration was 100 mM instead of 150 mM. Translation reaction mixtures of 25 μl were programmed with total rabbit globin mRNA (10 μg/ml) and incubated at 30°C for the times indicated in the figures. Aliquots of 4 μl were withdrawn and precipitated in 10% trichloroacetic acid, and the radioactivity was determined by scintillation spectrometry.

Cleavage of PABP by 2A protease in RRL.

A number of published studies have used recombinant 2A protease (from rhinovirus or coxsackievirus) or L protease (from foot-and-mouth disease virus) to study the effects of cleaving eIF4G on cap-dependent and cap-independent translation in vitro (7, 8, 42, 44, 51, 52). Under conditions that are typically used for eIF4G cleavage in an RRL translation system, PABP was also cleaved (Fig. 1A). The 70-kDa PABP remained intact for 10 min of incubation in the control translation reaction mixtures (lane C) or in reaction mixtures to which only buffer was added (lane B) but was cleaved to a ∼55-kDa product by 2A protease addition (lane 2A). eIF4G also remained intact in the control and buffer-containing translation reactions but was cleaved to cp_N by 2A protease (Fig. 1B). (The antigen used to produce the anti-eIF4G antiserum was a peptide located in the N-terminal portion; hence, only full-length eIF4G and cp_N are reactive.) The eIF4E in these lysates remained intact under all three conditions (Fig. 1C).

The reduction in size of the 70-kDa PABP to ∼55 kDa suggests that an ∼15-kDa fragment was removed. Such a fragment, however, was not detected in immunoblots, due to the absence of an antigenic site. (The antigen used for the production of the anti-PABP antiserum was a peptide derived from the central proline-rich region of PABP.) To test for its presence, we synthesized [³⁵S]Met-labeled Myc-xPABP in an RRL translation system. The reaction mixture was incubated with 100 μg of 2A protease per ml overnight, and the cleavage products were subjected to SDS-PAGE on 15% polyacrylamide gels followed by autoradiography. Radioactive products of ∼55 and ∼15 kDa were present in the samples treated with 2A protease but not in control samples incubated with buffer A alone (data not shown).

Several studies have provided evidence that a latent cellular protease becomes activated by 2A protease and is responsible for the cleavage of eIF4G during poliovirus infection (reviewed in reference 73). Other studies have indicated that eIF4G is a substrate for cellular calcium-dependent proteases (74) and requires initiation factor eIF3 for efficient 2A protease-induced cleavage (75). It was therefore of interest to test whether PABP was a direct substrate for 2A protease. rPABP was purified to homogeneity from RRL (Fig. 1D, lane C) and incubated with the homogeneous, recombinant 2A protease. Appearance of the 55-kDa cleavage product (lane 2A) indicates that neither secondary proteases nor ancillary proteins are required.

Cleavage of Myc-xPABP by 2A protease in Xenopus oocytes.

The cleavage of PABP by 2A protease was also studied in Xenopus oocytes. An in vitro-synthesized mRNA encoding Xenopus PABP containing an N-terminal Myc epitope tag was microinjected into oocytes, and the Myc-xPABP was allowed to accumulate. Doses of 2 and 10 ng of 2A protease were subsequently microinjected into the oocytes, and the incubation was continued. Myc-xPABP was immunoprecipitated from oocyte extracts with an anti-Myc tag antibody, and the products were analyzed by immunoblotting with the same antibody (Fig. 2A). PABP was partially cleaved at 2 ng of protease per oocyte and almost completely cleaved at 10 ng. The same extracts were also analyzed for the cleavage of endogenous eIF4G (Fig. 2B). The eIF4G was completely cleaved at both 2 and 10 ng of protease per oocyte, indicating that eIF4G is more sensitive to cleavage by 2A protease than is PABP. Also, the detection of the 55-kDa band by the anti-Myc antibody identifies it as the N-terminal cleavage product of PABP (henceforth referred to as PABP_N) and establishes that the C-terminal ∼20% of the molecule (PABP_c) is removed by 2A protease. Finally, these results indicate that PABP from evolutionarily distant organisms (rabbit and frog) are similarly cleaved.

Cleavage of PABP in coxsackievirus-infected HeLa cells.

The foregoing experiments establish that PABP can be cleaved in vitro (Fig. 1) and in vivo (Fig. 2) by 2A protease, but they do not indicate whether the levels of 2A protease achieved during picornavirus infection of mammalian cells are sufficient for PABP cleavage. This was investigated by analyzing PABP and eIF4G at various times after infection of HeLa cells with coxsackievirus B3 (Fig. 3). PABP cleavage was more than 50% complete by 6 h postinfection and was essentially complete by 9 h (Fig. 3A). In addition to the 55-kDa PABP_N, a faster-migrating, secondary cleavage product, PABP_N*, appeared by 9 h postinfection, which could indicate the presence of a second cleavage site for the 2A protease on PABP. However, since secondary cleavage was not observed in reactions with only purified PABP and 2A protease (Fig. 1D), it is more likely that secondary cleavage in HeLa cells was due to another protease. Cleavage of eIF4G in infected HeLa cells was extensive by 3 h and was complete by 6 h (Fig. 3B).

To more precisely determine the kinetics of PABP cleavage relative to those of eIF4G cleavage and also to correlate these cleavages with the progress of viral infection and host shutoff, another experiment was performed in which more frequent time points were used and total protein synthesis was measured by pulse-labeling with [³⁵S]Met for 30 min before each time point. Host protein synthesis continued for 3 h, after which viral protein synthesis predominated until 7.5 h (Fig. 4A). Cleavage of eIF4G was ∼75% complete by 3 h and was ∼100% complete by 4.5 h (Fig. 4B). PABP cleavage was detectable by 4.5 h, ∼75% complete by 6 h, and essentially complete by 9 h (Fig. 4C). PABP cleavage therefore lagged behind eIF4G cleavage by ∼3 h, suggesting that PABP is less sensitive to 2A protease than is eIF4G. This is consistent with the dose-response results obtained with Xenopus oocytes (Fig. 2).

All four parameters are quantified in Fig. 4D. Interestingly, at 3 h, host protein synthesis had decreased only ∼20% and viral protein synthesis had just become detectable at a time when eIF4G cleavage was ∼75% complete, indicating a lack of correlation between the translational switch and eIF4G cleavage. At 4.5 and 6 h, by contrast, PABP was partially cleaved, host protein synthesis had been almost completely shut off, and viral protein synthesis was at maximal levels. The subsequent decline in viral synthesis correlated with a further decline in the amount of intact PABP.

Determination of the cleavage site in PABP.

We next determined the cleavage site of 2A protease in PABP. Recombinant GST-hPABP was digested with 2A protease in vitro, and hPABP_c was separated from GST-hPABP_N on glutathione-Sepharose. hPABP_c was further purified by reverse-phase HPLC on a C₅ column (Fig. 5), and fractions were analyzed by SDS-PAGE (inset). The hPABP_c peak was subjected to automated Edman degradation for determination of its N-terminal sequence (Fig. 6A). Ten residues were unambiguously assigned, and their sequence exactly matched that of hPABP beginning at Gly-491. These results indicate that the cleavage by 2A protease occurs at ⁴⁹⁰M↓G⁴⁹¹ in hPABP. The sequences of hPABP and xPABP are identical in this region (Fig. 6A). The sequence of rPABP does not appear in public databases. However, amino acid sequences derived from human and mouse cDNAs are 99.5% identical, and those derived from human and Xenopus cDNAs are 93.7% identical. Thus, it is likely that PABP from all four species is cleaved at the same site. This is consistent with the fact that the apparent molecular masses of cleavage products, derived from electrophoretic mobility, are the same for rPABP, xPABP, and hPABP (after correction for N-terminal tags) (Fig. 1–4).

Cleavage by 2A protease is highly dependent on the amino acid sequence of the substrate (64, 65), but it was possible that it cleaved PABP at two nearby sites, thus removing a short peptide between PABP_N and PABP_c. To verify that a single cleavage occurs, we prepared rPABP_N and subjected it to C-terminal sequencing (see Materials and Methods). Since C-terminal sequencing is much less processive than N-terminal sequencing, it was possible to assign only the C-terminal amino acid residue with confidence. The assignment of Met matched the sequence of both hPABP and xPABP upstream of Gly-491 (Fig. 6A), supporting the existence of only a single cleavage site. Further evidence comes from the observation that the molecular masses for hPABP_N and hPABP_c estimated from electrophoretic mobility (55 and 15 kDa, respectively) are in excellent agreement with the calculated masses for cleavage at ⁴⁹⁰M↓G⁴⁹¹ (55.2 and 15.5 kDa, respectively).

Cleavage of PABP by 2A protease diminishes its ability to stimulate translation in vitro.

The effect of PABP cleavage on in vitro translation was determined in an RRL translation system programmed with exogenous rabbit globin mRNA. The system was preincubated for 5 min at room temperature with the cap analog m⁷GTP and with low levels of poly(A) to make it less dependent on cap-mediated translation (69) and more dependent on exogenous PABP (23), respectively. The addition of both m⁷GTP and poly(A) inhibited translation synergistically: 100 μM m⁷GTP alone caused 15% inhibition, 8 μg of poly(A) per ml alone caused 58% inhibition, but the combination of 100 μM m⁷GTP and 8 μg of poly(A) per ml caused 95% inhibition (data not shown). Inhibition by m⁷GTP was relatively weak because of the low potassium acetate concentration (14). These results are consistent with earlier published studies showing that the cap and poly(A) are functionally redundant for mRNA recruitment (21, 68), although this has not previously been demonstrated in RRL.

Equimolar amounts (0.6 nM) of either GST-hPABP or GST-hPABP_N were added to this PABP-dependent system to measure restoration of translation (Fig. 7). GST-hPABP was more effective than GST-hPABP_N in restoring translation (28% restoration of mRNA-stimulated protein synthesis after 60 min compared with 9% for PABP_N). At 0.4 and 0.2 nM GST-hPABP and GST-hPABP_N, the extent of restoration was lower, but GST-hPABP was consistently more effective than GST-hPABP_N (data not shown). These results indicate that cleavage of PABP impairs poly(A)-dependent translation in vitro.