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Originally published In Press as doi:10.1074/jbc.M312391200 on January 22, 2004

J. Biol. Chem., Vol. 279, Issue 16, 16571-16580, April 16, 2004
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Proteolytic Processing and Translation Initiation

TWO INDEPENDENT MECHANISMS FOR THE EXPRESSION OF THE SENDAI VIRUS Y PROTEINS*

Sylvain de Breyne, Romaine Stalder Monney, and Joseph Curran{ddagger}

From the Department of Microbiology and Molecular Medicine, The University of Geneva Medical School (Centre Médicale Universitaire), 1 rue Michel Servet, CH-1211 Geneva 4, Switzerland

Received for publication, November 12, 2003 , and in revised form, January 8, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The four Sendai virus C-proteins (C', C, Y1, and Y2) represent an N-terminal nested set of non-structural proteins whose expression modulates both the readout of the viral genome and the host cell response. In particular, they modulate the innate immune response by perturbing the signaling of type 1 interferons. The initiation codons for the four C-proteins have been mapped in vitro, and it has been proposed that the Y proteins are initiated by ribosomal shunting. A number of mutations were reported that significantly enhanced Y expression, and this was attributed to increased shunt-mediated initiation. However, we demonstrate that this arises due to enhanced proteolytic processing of C', an event that requires its very N terminus. Curiously, although Y expression in vitro is mediated almost exclusively by initiation, Y proteins in vivo can arise both by translation initiation and processing of the C' protein. To our knowledge this is the first example of two apparently independent pathways leading to the expression of the same polypeptide chain. This dual pathway explains several features of Y expression.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The Sendai virus P/C mRNA has become a paradigm for expressional elasticity, expressing six different polypeptide chains by ribosomal choice (namely, C', P, C, Y1, Y2, and X) (1). The P protein initiates at the second start site (AUG104). It is an essential cofactor of the viral polymerase (2). The first start site is an unusual ACG initiation codon at position 81 (ACG81) that gives rise to the C' protein (3, 4). This is a member of an N-terminally nested set of four proteins referred to as the "C-proteins" whose open reading frame (ORF)1 overlaps that of P. The second member of this group, the C protein, initiates at AUG114 and is accessed by leaky ribosomal scanning (5). Its good Kozak consensus signal is consistent with its high expression levels in virally infected cells. It is also the major translation product when the P/C mRNA is expressed transiently in mammalian cells or in rabbit reticulocyte lysates (RRLs). The remaining members of this group are the Y proteins (Y1 and Y2). Their AUG start codons were mapped in vitro to positions 183 and 201 (6). The C-proteins impact on both the readout of the viral genome (7, 8) and the host cell response to viral infection. In particular, they function to disrupt type 1 interferon signaling pathways (9, 10).

Although initiation from the first three start sites (ACG81/C', AUG104/P, and AUG114/C) is readily explained by leaky scanning, results suggest that ribosomes can access the Y start codons by discontinuous scanning or shunting (11, 12). The seminal observation in this work was that changing the C' ACG codon to AUG (referred to as AUG81) ablated expression of the P and C proteins but not Y1/Y2. In discontinuous scanning, ribosomes are loaded via the 5' cap and then at a defined donor site translocate to an internal acceptor site located close to the start codon (13). The first description of a eukaryotic shunt was the SeV X protein (14). This is initiated from an AUG codon more than 1,500 nucleotides from the 5' end of the P/C mRNA in a manner that is cap-dependent. However, the most studied shunts are those on the cauliflower mosaic virus 35 S RNA (15, 16) and the adenovirus late mRNAs (17, 18).

We have been studying the expression of the Y proteins. A number of mutations were previously characterized that significantly enhanced Y levels in vivo, a result we attributed to increased initiation (19). However, in this report we demonstrate that this "up-phenotype" is actually the consequence of a product-precursor relationship between the Y and C' proteins. Curiously, although Y expression in vitro is mediated almost exclusively by initiation at the Y1183 and Y2203 AUG codons, Y proteins in vivo can arise both by translation initiation and processing of the C' protein.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
DNA Constructions—All the constructs were made in the AUG81 background starting from the pBS SK AUG81, pBS SK {Delta}9, or pBS SK {Delta}9Y1Y2UGU clones (19). Unless otherwise stated constructs carried the triple HA epitope tag fused to the C terminus of the C ORF. All changes were performed by PCR. The pEBS-PL episomal Epstein-Barr virus-based mammalian expression vector carries an SR{alpha} promoter and a hygromycin selection cassette (20). Clones were transferred from pBS KS to pEBS-PL as SacI/XbaI fragments.

The HCV IRES2b was removed as an NcoI/BspHI fragment from the pTZ18R plasmid clone (21) and introduced into the NcoI site of pBS SK AUG81, {Delta}9, and {Delta}9Y1Y2UGU. The triple HA tag was then fused to the C terminus by PCR. The cricket paralysis virus (CrPV) IRES was excised as an EcoRI/NcoI fragment from the clone pRdEMCV-CrPV-F and introduced into the NcoI site of pBS SK AUG81, {Delta}9Y1Y2UGU, and {Delta}9. This positioned the A site of the ribosome 18 nucleotides upstream of AUG81. To position the AUG81 in the ribosomal A site, we modified the IRES sequence, introducing an NcoI site at the translational start position and a compensating second site change that restored the critical pseudoknot of the IRES (22). This permitted fusion of the C' AUG81 via its NcoI site.

The bicistronic constructs were built starting from a pGEM4 plasmid clone containing the SeV M gene (23). This clone contained a unique EcoRI site downstream of the M gene into which was inserted the HCV-AUG81 as an EcoRI fragment.

The GFP tag was amplified by PCR from pEBS-PL GFP (24) using oligonucleotides containing XbaI (5') and NotI (3') restriction sites. This was then used to replace the triple HA tag (which is fused to the end of the C ORF via an XbaI site) in cDNA clones expressing C' (AUG81) and C.

Cell Culture, Transient Transfection, and Metabolic Labeling—Cell culture, infections, transfections, and metabolic labeling were performed as previously described in de Breyne et al. (19). Cells were transfected with the pEBS-PL clones using FuGENE (Roche Applied Science) according to the manufacturer's instructions. Cells were infected with SeV (strain Z) at a multiplicity of infection of 4.

Antibodies and Indirect Immunofluorescence Assay—HeLa cells seeded on coverslips were transfected with pEBS-PL C'-GFP, pEBS-PL C-GFP, or both pEBS-PL C'-GFP and pEBS-PL C-HA as described in Ref. 25. After 24 h the coverslips were washed in 150 mM NaCl, 100 mM Tris, pH 7.5, and the cells were fixed and permeabilized in 3% formal-dehyde, PBS during 20 min and 0.05% saponin, PBS during 5 min. They were then washed three times in PBS and blocked for 30 min at room temperature with PBS containing 1% bovine serum albumin. The anti-HA and anti-galactosyltransferase mouse antibodies were used at dilutions of 1:100 and 1:1000 in PBS, respectively. Coverslips were incubated with the antibodies for 20 min at room temperature. After three PBS washes, the secondary antibody conjugated with Alexa-568 was added at a dilution of 1:200 for 20 min at room temperature. Microscopic analyses were performed on a confocal laser scan fluorescence inverted microscope (LSM 410, Zeiss). Each time, the two channels were recorded either together or independently to ensure the absence of interference between the channels.

In Vitro Transcription and Translation—Run-off capped transcripts were synthesized with T7 RNA polymerase in 800 µM ATP/CTP/UTP, 400 µM GTP, and 2 mM m7GpppG cap analogue (New England Biolabs). Capped mRNAs (50 µg/ml) were translated in a RRL (Promega) in the presence of 0.5 mM MgOAc, 75 mM KCl, a 20 µM concentration of each amino acid (except methionine), and 0.5 mCi/ml 35S Translabel. In the experiments using eIF4A, 2 µg of eIF4Awt, eIF4AR362Q, or elution buffer (150 mM imidazole, 110 mM KCl, 20 mM HEPES, pH 7.4) was added (26).

Purification of eIF4Awt and eIF4AR362Q—The eIF4Awt and eIF4AR362Q clones were amplified by PCR and cloned downstream of the His6 tag in the bacterial expression vector pT7-7 His6. Transformed Escherichia coli strain BL21 were induced for 2 h with 0.4 mM isopropyl-1-thio-{beta}-D-galactopyranoside when the culture reached an A600 of 0.5. Rifampicin (400 µg/ml) was added, and the incubation continued for a further 2 h. Cells were lysed in 300 mM NaCl, 20 mM HEPES, pH 7.4, 1% Nonidet P-40 by sonication, and the proteins were isolated on a Talon affinity column (Clontech) following the manufacturer's protocols.

Analysis of Preinitiation Complexes (Toeprinting)—Primer extension analysis to position the 48 S ribosomal complex was performed as outlined in Ref. 27. Briefly capped RNA (250 ng) was incubated with a 32P-labeled oligonucleotide corresponding to nucleotides 261–282 (2 x 106 cpm) in a final volume of 5 µl, heated to 50 °C, and then allowed to cool. The mixture was added to 8 µl of RRL containing either 1 mM GMP-PNP or 5 mM EDTA. This was incubated at 30 °C for 5 min and then transferred onto ice. The lysate containing the EDTA was adjusted to 5 mM MgCl2. Twenty microliters of toeprint buffer (50 mM Tris, pH 8.3, 75 mM KCl, 3 mM MgCl2,10mM dithiothreitol, 250 µM dNTPs, 1,000 units/ml Rnasin) and 100 units of Superscript II reverse transcriptase (Invitrogen) were then added, and the lysates were incubated at 37 °C for 30 min. Reactions were terminated by adding 30 µl of phenol-Tris/EDTA. Primer extension products were analyzed on an 8% polyacrylamide-urea gel alongside a sequence ladder.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Enhanced Expression of the Y Proteins in the {Delta}9 Background Requires Expression of C'—In our previous report we described mutants within the AUG81 background that significantly increased (~10-fold) the expression of the Y proteins, a pheno-type initially attributed to an increase in shunt-mediated translational initiation (19). One of these, referred to as {Delta}9, removed nucleotides 189–197 (three codons) between the Y1 and Y2 AUGs. This increased expression was essentially independent of the nature of the Y1/Y2 codons (e.g. {Delta}9Y1Y2UGU also gave high levels of Y expression). The earlier studies had used a vaccinia-T7 transient expression system, but these phenotypes were conserved with the mammalian expression vector pEBS-PL, precluding the possibility that the viral infection had impacted on the expression patterns observed (Fig. 1, A and B). It should be noted that in the {Delta}9 background the Y1/Y2 proteins generally co-migrate on SDS-polyacrylamide gels (19).



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  		FIG. 1.
The {Delta}9 phenotype. A, a schematic representation of the C ORF indicating the initiation sites for the four C-proteins and the site at which nucleotides were inserted. The HA tag refers to the triple HA epitope, and {Delta}9 is a clone in which nucleotides 189–197 have been deleted. B, HeLa cells were transfected with pEBS-PL clones as indicated above the panel. Two independent clones of each construction were examined. Cells were metabolically labeled with 35S Translabel at 18–20 h post-transfection. Cytoplasmic extracts were immunoprecipitated with an anti-HA monoclonal antibody. The immunoselected proteins were resolved on a 15% SDS-polyacrylamide gel and visualized by fluorography. C, HeLa cells infected with vaccinia-T7 were transfected with the clones as indicated above the panel. In the mock control, cells were transfected with the empty vector. Cells were metabolically labeled with 35S Translabel at 18–20 h postinfection. Cytoplasmic extracts were immunoprecipitated with an anti-HA monoclonal antibody. Immunoselected proteins were resolved on a 15% SDS-polyacrylamide gel and visualized by fluorography.

 
With the objective of ensuring that the only products of the C ORF would be the Y proteins, we introduced a nucleotide down-stream of the initiation codon for C' (at position +5 relative to the AUG81, i.e. nucleotide 85) within the {Delta}9 background, thereby creating a frameshift (Fig. 1A). We anticipated that such a construct would only express Y1/Y2 and would retain the {Delta}9 up-phenotype with regard to Y levels. Indeed, when expressed in mammalian cells, C' expression was totally lost (Fig. 1C, lanes 4 and 5). However, Y protein levels dropped dramatically, approaching those observed in the AUG81 background. In addition, a second band was observed that co-migrated with the C protein (initiated from AUG114), a result that was somewhat surprising since the good Kozak context of the AUG81 start site had not been changed. Confirmation that this was the C protein came from mutating the AUG114 to GCG (Fig. 1C, lanes 6 and 7). The addition of 2 nucleotides also destroyed the phenotype characteristic of {Delta}9 (data not shown). However, addition of 3 nucleotides restored the C ORF and generated a slightly slower migrating C' protein (Fig. 1C, lanes 8 and 9). There was no obvious expression of C, and Y protein levels were once again enhanced, i.e. the {Delta}9 phenotype had been restored.

A Product-Precursor Relationship between Y and C'—Pulsechase experiments performed on virally infected cells had previously failed to provide unambiguous evidence for a product-precursor relationship between C'/C and the Y proteins.2 However, the results outlined above demonstrated that the enhanced expression of the Y proteins observed in the {Delta}9 background was coupled to the expression of C'. We therefore performed a series of pulse-chase experiments in cells expressing the AUG81 and {Delta}9 clones (Fig. 2) and quantitated the proteins using a PhosphorImager. In the AUG81-transfected cells (Fig. 2A), the C' levels decayed during the 5-h chase (t1/2 = 180 min), and there was a small but reproducible rise in the Y proteins. This effect was more pronounced in the {Delta}9 transfection in which the {Delta}9C' protein turned over more rapidly (t1/2 = 69 min), whereas the Y proteins accumulated during the 1st h of the chase and then decayed with kinetics very similar to {Delta}9C' (Fig. 2B). The presence of a product-precursor relationship between Y and {Delta}9C' is most clearly demonstrated during a shorter chase period (Fig. 2C). In this latter experiment the Y1 and Y2 proteins were clearly discernable, and both accumulated at similar rates during the 60-min chase. The quantification of the gels also indicated that not all the {Delta}9C' protein turned over to generate Y proteins.



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  		FIG. 2.
A product-precursor relationship between Y and C' in vivo. Vaccinia-T7-infected HeLa cells were transfected with clones expressing AUG81 (A) and {Delta}9 (B and C). Cells were also infected with SeV (D). All were metabolically labeled with 35S Translabel for1hat 18 h postinfection and then chased for the times indicated above each panel. Proteins were immunoprecipitated with either an anti-HA monoclonal antibody (A, B, and C) or an anti-C polyclonal antiserum (D) and resolved on a 15% SDS-polyacrylamide gel. Bands were quantified on a PhosphorImager. The results of this latter analysis are depicted graphically below each gel.

 
The results above indicate a product-precursor relationship between C' and Y. However, is this the case in SeV-infected cells? Pulse-chase experiments were reperformed in virally infected cells, and the products of the C ORF were quantitated on a PhosphorImager (Fig. 2D). The C' protein decayed with a half-life of 90 min, whereas the Y proteins accumulated slowly throughout the 5-h chase.

Y Protein Expression in Vitro Is Exclusively via Initiation— The Y1/Y2 initiation codons were initially mapped on the P/C gene by mutating AUG186 and AUG201 and expressing the mRNAs in RRLs (6). Such changes ablated all Y expression. Furthermore toeprinting analysis, which maps the position of the 48 S preinitiation complex on the mRNA, demonstrated the presence of ribosomes at the Y2 AUG (generally the major Y protein expressed in RRLs (11) (Fig. 3A)). In vitro translation of the P/C and AUG81 mRNAs yielded protein profiles similar to those observed in mammalian cells (11). However, in vitro translation of the {Delta}9 mRNA did not reproduce the enhanced expression of the Y proteins observed in vivo (compare Fig. 1B, lanes 3–6, with Fig. 3B, lanes 2 and 3), and Y expression remained AUG codon-dependent even when S10 HeLa extracts were added to the translation mixture (data not shown), suggesting that the RRL did not simply lack soluble cellular factors. Since Y expression was driven by translational initiation we decided to examine to what extent ribosome scanning was required. For this we obtained clones expressing eIF4A and a dominant negative form of eIF4A carrying the mutation R362Q (26). eIF4A is an RNA helicase that forms part of the eIF4F complex (28–30). It functions to unwind the RNA during scanning. The eIF4AR362Q mutant effects in a dominant negative fashion initiation mediated via the 5' cap but not that mediated via the HCV IRES (31). Both wt and mutant proteins were His-tagged and purified from E. coli. Capped transcripts from the bicistronic clone M-IRESHCV-AUG81 (expression of the M protein is 5' cap-dependent, whereas C' is driven by the HCV IRES; Fig. 3C) were added to a RRL supplemented with 2 µg of either HiseIF4Awt or HiseIF4AR362Q (26). As a control, an equivalent volume of buffer was added. Addition of HiseIF4Awt to a RRL programmed with M-IRESHCV-AUG81 mRNA had virtually no effect on the expression of M, C', Y1, and Y2 (Fig. 3C). However, addition of HiseIF4AR362Q dramatically reduced expression from the first cistron (M) while only marginally effecting expression from the second (C', Y1, and Y2), confirming that ribosome recruitment by the HCV IRES does not require functional eIF4A (31). To facilitate interpretation the image was quantitated on a PhosphorImager (Fig. 3C). The addition of HiseIF4AR362Q reduced both the M/C' and M/Y protein ratios to a similar extent relative to the control (between 5- and 6-fold), whereas the C'/Y ratio was unchanged. We conclude that both ribosomal loading and subsequent initiation at the Y start codons are largely independent of eIF4A.



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  		FIG. 3.
Y expression in vitro is mediated exclusively by translation initiation. A, toeprinting analysis of the preinitiation complexes that assemble on the P/C wt and AUG81 mRNAs in RRLs. The left-hand four lanes contain a sequencing ladder derived from the AUG81 plasmid clone using the same oligonucleotide utilized in the primer extension analysis. Toeprints corresponding to the C',P, C, and Y2 start sites are highlighted with stars. B, the {Delta}9 and {Delta}9Y1Y2UGU constructs were expressed in HeLa cells (lanes 5 and 6), and capped mRNA transcripts from AUG81 and {Delta}9 were translated in a RRL (lanes 2 and 3). Proteins were immunoselected with the anti-HA antibody and resolved on a 15% SDS-poly-acrylamide gel. As controls, cells were transfected with an empty plasmid vector (lane 4), or RRLs were programmed with buffer alone (lane 1). C, starting from the bicistronic M-IRESHCV-AUG81 plasmid clone (upper panel), 5' capped transcripts were made in vitro. Approximately 100 ng of transcript was added to 10 µl of RRL containing 2 µg of either His-tagged eIF4Awt (HiseIF4Awt) or His-tagged eIF4AR362Q (HiseIF4AR362Q) or an equivalent volume of buffer (Control). The lysates were immunoprecipitated with a combination of anti-M and anti-C poly-clonal antisera. Proteins were resolved on a 17.5% polyacrylamide-SDS gel and visualized by fluorography (lower left-hand panel). The region of the gel containing the Y proteins has been exposed longer and is therefore depicted as a separate panel. The protein bands were quantitated on a PhosphorImager, and the relative protein ratios are depicted in the lower right-hand panel.

 
The Y Proteins in Vivo: Protein Processing Versus Translation Initiation—The Y proteins in vivo can arise by processing of the C' protein, but can they also be expressed via translation initiation as observed in vitro? To address this question we exploited the frameshift approach outlined earlier in combination with ribosome recruitment via the HCV IRES. The former assures that no C' protein is expressed (i.e. no cleavage precursor), whereas the IRES eliminates the possibility that the Y start sites are accessed by leaky scanning (31, 32). The IRES was positioned at the C' AUG81 codon in a {Delta}9 background. A single nucleotide was inserted at position 103 (referred to as +1*, Fig. 4A) altering the ORF of the IRES-mediated translation product. This insertion was downstream of that presented earlier because the +5 position relative to the AUG has been reported to influence initiation at least that which is 5' cap-dependent (33, 34). The protein products expressed from this construct (HCV-C'+1*{Delta}9) were identical to those observed when the translation was 5' cap-mediated (compare Fig. 4B, lanes 5 and 6 with lane 4). We were particularly surprised to observe a C protein given that the HCV IRES does not permit leaky scanning (31). This suggests that either leaky scanning did occur in this particular context or that on this mRNA C was initiated by shunting. The Y proteins expressed in the HCV-C'+1*{Delta}9 background were lost when the Y1/Y2 codons were changed (Fig. 4B, lanes 7 and 8). Similar results were obtained when the experiment was performed in the AUG81 background (Fig. 4B, lanes 9–14), demonstrating that Y expression in vivo can be mediated via translation initiation in an AUG-dependent manner. This, in turn, indicates that the Y proteins observed in vivo from constructs in which the AUG183 and AUG201 have been mutated are generated exclusively via the processing of C'. Curiously the shorter C protein does not give rise to Y proteins when these AUG codons are changed (even in the {Delta}9 background; Fig. 4B, lanes 7 and 8 and lanes 11 and 12), indicating that although C turns over rapidly (t1/2 = 140 min, Fig. 2D), it is not processed to generate Y proteins.



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  		FIG. 4.
Y proteins can arise by initiation in vivo. A, the HCV IRES was fused to the C' AUG start codon as illustrated schematically. The P ORF is depicted below the line, and the C ORF is depicted above the line. The site of the nucleotide insertions is indicated: +1 and +1* refer to positions 85 and 103, respectively. B, vaccinia-T7-infected HeLa cells were transfected with the clones indicated above the panel and analyzed as in Fig. 1.

 
The in vivo and in vitro studies outlined above leave us with an apparent paradox since both propose different models to explain the expression of the same proteins. As an initial step at trying to resolve this we examined to what extent the proteins generated by processing of the C' in vivo actually corresponded to the Y proteins initiated at AUG183 and AUG201, i.e. did they share a common N terminus. The approach we used took advantage of the fact that there is normally only one cysteine codon in the C ORF, and it is just downstream of Y2. {Delta}9Nsi is a {Delta}9 background in which the CG nucleotide pair just upstream of the Y2 AUG has been inverted thereby generating an NsiI restriction site (19). This change also introduced a new cysteine codon just upstream of Y2 (Fig. 5A; the reason for its selection is indicated below). The cysteine codon downstream of Y2 was changed to tyrosine in a background in which the Y1/Y2 start codons were either AUG ({Delta}9*NsiMet) or UGU ({Delta}9*NsiCys). Transfected cells were metabolically labeled with either 35S Translabel or [35S]cysteine. Upon labeling with the former, both {Delta}9*NsiMet and {Delta}9*NsiCys gave the high Y expression characteristic of {Delta}9 (Fig. 5B, lanes 2–5). In the {Delta}9*NsiMet transfection, the Y proteins co-migrated (lanes 2 and 3), whereas with {Delta}9*NsiCys the two proteins were clearly separated (lanes 4 and 5). This change in Y protein mobility (in this particular case Y1) is probably the result of alterations in the primary sequence (arising from the methionine/cysteine mutations), changes that have also affected the mobility of the {Delta}9C' proteins (compare lanes 2 and 3 with lanes 4 and 5). Although the {Delta}9Nsi background introduced a second cysteine codon upstream of Y2 it was selected for this experiment because in the {Delta}9*NsiCys clone the Y1 and Y2 proteins were clearly resolved (this is unfortunately not the case in the normal {Delta}9 construct). This was critical for the interpretation of the labeling patterns (see below). When labeled with [35S]cysteine, bands corresponding to C' and Y1 were observed in the {Delta}9*NsiMet-transfected cells (Fig. 5B, lanes 7 and 8). The Y2 protein would not be detected since it contains no cysteine. In cells transfected with {Delta}9*NsiCys, the C', Y1, and Y2 proteins were clearly visible (Fig. 5B, lanes 9 and 10). The radiolabeling of the faster migrating Y2 protein with [35S]cysteine positions its N terminus at either the precise site of the Y2 initiation codon or just 1 amino acid upstream (since the amino acid immediately N-terminal is now also a cysteine; Fig. 5A).



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  		FIG. 5.
Mapping the N terminus of the Y2 protein. A, the nucleotide and amino acid sequences flanking the Y start sites in the {Delta}9Nsi background are depicted in the upper part of the figure. The UGU cysteine codon downstream of Y2 was changed to a UAU tyrosine codon (dotted vertical line). This mutated construct is indicated as {Delta}9*NsiMet. The Y1 and Y2 initiation codons were then changed to UGU ({Delta}9*NsiCys). The scissors indicate the sites at which processing of C' must occur to give rise to the Y2 protein based upon the results below. B, vaccinia-T7-infected HeLa cells were transfected with the pBS {Delta}9*NsiMet or {Delta}9*NsiCys clones as indicated above the panel. Mock cells (lanes 1 and 6) were transfected with an empty plasmid vector. Cells were metabolically labeled with 35S Translabel (lanes 1–5) or [35S]cysteine (lanes 6–10) at 18–20 h postinfection and analyzed as in Fig. 1.

 
The N Terminus of C' Is Required for Processing to Generate Y1/Y2—We introduced the CrPV IRES just upstream of C' in the {Delta}9 background. Remarkably this IRES directs protein synthesis on non-AUG codons in a manner that is methionyl-tRNA -independent, i.e. they initiate using the cognate charged tRNA (22, 35). Ribosomes recruited via this IRES would start translation on a GCU codon 6 amino acids before the start site of C' (Fig. 6A). In cells this construct produced a slightly slower migrating form of {Delta}9C' (Fig. 6B, lanes 3 and 4, {Delta}9C'*) consistent with IRES-driven expression. However, no Y proteins could be detected. The 6-amino acid addition did not change the stability of the C' (Fig. 6C), suggesting that it had altered the processing pathway that generated the Y proteins. To rule out any effect of the IRES itself, we first modified it so that the critical pseudoknot that occupies the ribosomal A site is formed with AUG81 instead of GCU (22). This removes the N-terminal extension and ensures that the IRES-mediated product is identical to that expressed from the 5' cap-driven {Delta}9. This modification restored the {Delta}9 expression phenotype (Fig. 6B, lanes 5 and 6). We next introduced the 6-amino acid extension immediately downstream of the C' AUG such that C'* would be expressed in a 5' cap-dependent manner. This destroyed the {Delta}9 phenotype (Fig. 6B, lanes 7 and 8).



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  		FIG. 6.
A 6-amino acid extension on the N-terminus of C' prevents Y expression via processing. A, schematic representation of the CrPV C ORF constructs. The IRES was initially positioned so that translation would commence on its normal GCU codon. This added 6 amino acids to the N-terminus of C' (referred to as C'*) as indicated below the line. The IRES was then modified such that translation would start on the AUG81 of C' (as indicated above the line). Below the panel the primary sequence between the C'* and C proteins is indicated. B, vaccinia-T7-infected HeLa cells were transfected with the clones as indicated above the panel. In the mock control, cells were transfected with an empty plasmid. Transfections were analyzed as in Fig. 1. C, pulse-chase experiments with the {Delta}9 and CrPV {Delta}9 clones (for further details see Fig. 2). aa, amino acids.

 
Mutation of Arg34 of the C' Protein Severely Perturbs Processing—While performing these studies we changed the arginine codon immediately upstream of the Y1 AUG (Arg34) to either alanine or cysteine in the background {Delta}9 (Fig. 7A). These changes severely reduced Y expression in cells (Fig. 7B). Therefore, a single change in the primary sequence of the C' protein just upstream from Y1 can ablate the {Delta}9 up-phenotype.



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  		FIG. 7.
The R34A mutation perturbs dramatically the {Delta}9 phenotype. A, the primary protein sequence flanking the Y start sites in the {Delta}9 and {Delta}9 Y1Y2UGU backgrounds is depicted as well as the position of the R34A change. B, vaccinia-T7-infected HeLa cells were transfected with the clones indicated above the panel. In the mock control, cells were transfected with an empty plasmid. Transfections were analyzed as in Fig. 1.

 
The N-terminal Region of C' May Serve as a Targeting Signal—The failure to process either the C'* or C protein suggests that the very N-terminal region of C' itself plays a pivotal role in this event. The C' and C protein differ by 11 amino acids (Fig. 6A), and assays have yet to discern a clear functional difference between the two. Both inhibit viral genome expression (7, 8) and bind Stat1 to induce its turnover (10, 36). However, we reasoned that the additional amino acids on C' may serve as a targeting signal that could direct at least a fraction of the protein to a particular cellular location. It would be at this site that processing would occur. As an initial means to test this hypothesis we examined the cellular distribution of the C' and C proteins. For this, both were C-terminally tagged with GFP and expressed in HeLa cells. At low expression levels, C'-GFP showed mainly a punctate cytoplasmic distribution (Fig. 8A, upper panels). However, we frequently observed accumulation at a condensed patch whose tight perinuclear location was reminiscent of the Golgi stack. Indeed double labeling with a marker for the trans-Golgi (galactosyltransferase) confirmed that this patch was in the vicinity of the Golgi, although the extent of co-localization was limited (Fig. 8A, upper panels). As C'-GFP accumulated, this patch became the dominant feature (Fig. 8A, lower panels). Interestingly these cells generally showed a more rounded morphology, and the trans-Golgi was now undetectable, suggesting that the accumulation of C'-GFP had induced its disruption. Fragmentation of the Golgi apparatus is associated with apoptosis (37). This is mediated via caspase-induced cleavage of a number of Golgin proteins that play a key role in organelle architecture (38–40). Sendai virus infection has been reported to induce apoptosis through activation of caspases (41). The images observed with the C-GFP construct were different. It tended to be more distributed in the cytoplasm (Fig. 8B). No condensed patches were observed, and there was no disruption of the trans-Golgi. We extended these studies by co-expressing C'-GFP with a C-terminally HA-tagged version of C (C-HA). This showed a co-localization of the two proteins at the plasma membrane. However, there was a more limited overlap in the cytoplasm (Fig. 8C). Curiously the viral C-proteins have been reported to have both apoptotic (42) and antiapoptotic (43) properties. The system of expression that we used permitted selection of transformants (24). Although clones expressing both C'-GFP and C-GFP could be observed, they did not divide. However, we could readily select cell lines expressing Y-GFP (data not shown). The Y-GFP showed a diffuse cytoplasmic distribution very similar to GFP alone.



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  		FIG. 8.
The intracellular localization of the C' and C proteins. HeLa cells transfected with pEBS-PL C'-GFP (A), pEBS-PL C-GFP (B), or pEBS PL C'-GFP plus pEBS-PL C-HA (C) were fixed with formaldehyde. GFP (green), HA (red), and galactosyltransferase (red) were detected by indirect immunofluorescence. Images were recorded by confocal microscopy. The left panels correspond to images obtained with the red filter, and the central panels correspond to those with the green filter. The right panels represent merged images. The overlap of red and green signals results in a yellow color.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Y Protein Expression: Initiation Versus Proteolytic Processing—The SeV P/C mRNA has become a paradigm for expressional elasticity since it expresses no less than eight different polypeptides (1). Studies performed in vivo and in vitro indicated that six of these products arose via independent translational initiation events. In particular, the C' protein was initiated from an ACG codon (3, 4), and the Y1, Y2, and X proteins appeared to be the product of ribosomal shunting (12, 14). The AUG initiation sites for Y1/Y2 were mapped in vitro by mutagenesis (6). However, work performed in vivo mounted two curiosities associated with Y expression. First, it was largely independent of the nature of the start codons (12), and second, deletion mutations around Y1/Y2 (referred to as {Delta}8 and {Delta}9) increased expression by as much as 10-fold in a manner that was once again largely codon-independent (19). Curiously these expression phenotypes were not observed in vitro.

The studies presented in this report offer an explanation for these observations. Whereas Y expression in vitro is mediated by de novo initiation at the Y1183 and Y2203 AUG codons, Y proteins in vivo can arise both by translation initiation and processing of a protein precursor, the C' protein. To our knowledge this is the first example of two apparently independent pathways leading to the expression of the same polypeptide chain. This conclusion is based upon a number of experimental observations. First, the increased Y expression observed in the {Delta}9 mutant can be traced to a direct product-precursor relationship with C'. This processing of C' is insensitive to the nature of the Y1 and Y2 codons and explains the apparent codon degeneracy of Y expression, an observation that was difficult to reconcile with independent initiation events. The fact that Y protein expression in vitro is strictly AUG codon-dependent indicates that C' processing does not occur in these extracts. The absence of the {Delta}9 up-phenotype in the RRL is also consistent with this conclusion.

The exact nature of the C' processing event that leads to Y expression is unclear. All four C-proteins are relatively unstable in animal cells, and in the AUG81 background only a small fraction of C' is actually processed to Y proteins. Clearly the very N-terminal region plays a key role since if it is removed (as in the C protein) or displaced internally by the N-terminal addition of 6 amino acids processing is severely diminished. In addition, a single mutation that changes the amino acid immediately upstream of Y1 (namely R34A) also seriously perturbs Y expression. Although both these events have similar effects on Y, we suspect that they disrupt different steps in the processing pathway. The importance of the very N-terminal region is reminiscent of the signal sequences that target proteins to different intracellular compartments. Indeed at least a fraction of the C' protein appears to be located in regions distinct from C. The coupling of intracellular targeting and cleavage could also explain the failure to observe processing in RRLs even upon addition of HeLa cell extracts. Being distal from the C' N terminus, it is unlikely that Arg34 would form part of this targeting signal. It seems more likely that this change is perturbing the processing event itself. Curiously deletion mutations that flank Arg34, namely {Delta}8 (removal of amino acids 27–33) and {Delta}9 (removal of amino acids 38–40), actually enhance cleavage (19), indicating that the information that directs this event falls in this region. However, no consensus sequence for cellular proteases, in particular caspases (see below), can be found flanking Y1/Y2. In addition, the exact nature of the amino acid C-terminal to the scissile bond is only of marginal importance, a result that explains the continued high expression of Y proteins in the {Delta}9 background even when the Y codons are changed (19).

Shunting as a Means of Expanding the Decoding Complexity— In vitro studies demonstrate that preinitiation complexes can assemble at the Y start sites and that the proteins can be expressed via independent initiation events that are AUG-dependent. Likewise results in vivo support the notion that at least a fraction of the Y proteins are initiated de novo in a codon-dependent manner. Since most of these studies were performed in backgrounds that limit leaky scanning past the AUG81 C' codon (namely an excellent Kozak consensus or a HCV IRES), it is likely that the Y1/Y2 AUG codons can be accessed by discontinuous scanning or shunting as postulated earlier (12, 19). This would also be consistent with the in vitro observation that Y expression is largely insensitive to a dominant negative form of eIF4A. Although most examples of shunting are viral, it has been proposed that an AUG initiation codon in human cysteine disulfurase (NifS) mRNA may be selected by ribosomal shunting (44). In this example, initiation from the first AUG produces a form of NifS that is targeted to the mitochondria, whereas shunting permits initiation at the second in-frame AUG that produces a form of the protein that is cytosolic/nuclear. Interestingly the relative initiation frequency from the first and second start sites is regulated in a pH-dependent fashion, suggesting that this shunt may be modulated by the physiological state of the cell. Clearly several aspects of this system can be extrapolated to the C'/Y model presented in this work.

Why Two Mechanisms to Produce the Same Polypeptide Chain?—C', C, Y1, and Y2 form an N-terminal nested set of proteins that impinge on both the readout of the viral genome and the host cell response to viral infection (7, 8, 36). In particular, all four have been shown to interact with Stat1, an interaction that blocks type 1 interferon signaling to reporter genes carrying interferon-stimulated response elements. However, whereas binding to either C' or C leads to proteasome-mediated Stat1 turnover and hence a subsequent block on the interferon-induced antiviral state, binding to the Y proteins does not destabilize Stat1, and the antiviral state can be observed (36, 45). Our own work suggests that both C' and C expression but not Y is detrimental to cell viability. The fragmentation of the Golgi observed in C'-expressing cells is consistent with apoptosis (37). Intriguingly the C protein did not produce the same Golgi disruption, a result that hints at possible differences in the apoptotic pathways induced by each protein.

The C-proteins therefore play a critical role in modulating the cellular response to viral infection. In this regard, ribosomal shunting may be a means to ensure continued access to the Y start sites under conditions in which linear scanning is limited. Indeed the translational machinery is a major target for cellular regulation during periods of stress (46, 47), and a number of initiation factors are cleaved during apoptosis (48). Expression via processing of C' is also not completely unconnected to translational control since the latter is initiated from an unusual ACG codon. ACG codons may be regulated in a manner different from AUG start codons (49). It is also conceivable that there is interplay between scanning and shunting modes as a response to changes in cell physiology, an interplay that ensures continued expression of Y proteins throughout the viral infection (e.g. scanning produces the C' protein, which serves as the precursor for Y via processing, whereas shunting ensures Y expression via de novo initiation). Switching between linear scanning and shunting modes in response to changes in the cell has already been observed in the adenovirus late mRNAs (in this example the switch is the phosphorylation status of eIF4E) (17, 18). Protein processing could offer other levels of regulation. It may also be a means of inactivating part of the C' function at a particular intracellular location while conserving those activities associated with the Y module. Indeed a number of important functions map to the N-terminal extension of C and C' that are not present in the Y proteins (8, 10, 36). Alternatively the small N-terminal cleavage product itself (34 amino acids at Y1 and 40 amino acids at Y2) may exercise a specific and unique function in the cell. This type of modification of a protein function by N-terminal proteolytic processing was recently described in the bacterium Myxococcus xanthus. This organism expresses a p25 protein homologous to alcohol dehydrogenases. Processing at the outer membrane generates a p17 protein that has lost the NAD+ binding module and now functions as an extracellular morphogen (50). Cell-mediated processing of viral proteins is also frequent, although they tend to be structural components. However, it was recently reported that the caspase-mediated cleavage of the NS1 nonstructural protein of a parvovirus was actually required for a permissive infection, opening the possibility that the cleavage products of such proteins may have distinct and important functions in the cell (51). Intriguingly, with regard to the C-proteins, the NS1 is the primary protein responsible for cell cytotoxicity and may be directly responsible for the induction of apoptosis (52).

In conclusion, the current work resolves a number of out-standing issues associated with expression of the Y proteins. The apparent codon degeneracy observed in cells has been traced to a processing event from the larger C' protein. Nevertheless proteins are also expressed by translation initiation events at the Y1 and Y2 AUG codons in a manner that appears to involve non-linear scanning of the ribosome on the mRNA. The fact that the C-proteins are all expressed via translation initiation routes that are rather non-classical (C', a non-AUG codon; C, leaky scanning; and the Y proteins, discontinuous scanning) and that they all impinge upon the interferon signaling pathway, which in turn is known to modulate protein synthesis principally at the level of initiation, creates an intriguing biological loop. The molecular details of both the mechanisms that lead to Y expression and their interplay represent the focus of our on-going studies.


   FOOTNOTES
 
* This work was supported by Swiss Science Foundation Grants 31-57434.99 and 3100A0-100221. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed. Tel.: 41-22-3795655; E-mail: Joseph.Curran{at}medecine.unige.ch.

1 The abbreviations used are: ORF, open reading frame; RRL, rabbit reticulocyte lysate; SeV, Sendai virus; HA, hemagglutinin; HCV, hepatitis C virus; IRES, internal ribosome entry site; CrPV, cricket paralysis virus; GFP, green fluorescent protein; PBS, phosphate-buffered saline; eIF, eukaryotic initiation factor; GMP-PNP, guanosine 5'-({beta},{gamma}-imino)triphosphate; wt, wild type; Stat, signal transducers and activators of transcription. Back

2 J. Curran, unpublished. Back


   ACKNOWLEDGMENTS
 
We thank Nahum Sonenberg, Peter Sarnow, Michel Strubin, and Richard Elliott for kindly supplying reagents.



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 DISCUSSION
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