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Originally published In Press as doi:10.1074/jbc.M608948200 on January 9, 2007

J. Biol. Chem., Vol. 282, Issue 10, 7056-7065, March 9, 2007
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A Bipartite Sequence Motif Induces Translation Reinitiation in Feline Calicivirus RNA*

Christine Luttermann and Gregor Meyers1

From the Institut für Immunologie, Friedrich-Loeffler-Institut, D-72001 Tübingen, Germany

Received for publication, September 20, 2006 , and in revised form, December 18, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The mechanism leading to reinitiation of translation after termination of protein synthesis in eukaryotes has not yet been resolved in detail. One open question concerns the way the post-termination ribosome is tethered to the mRNA to allow binding of the necessary initiation factors. In caliciviruses, a family of positive strand RNA viruses, the capsid protein VP2 is translated via a termination/reinitiation process. VP2 of the feline calicivirus is encoded in the 3'-terminal open reading frame 3 (ORF3) that overlaps with the preceding ORF2 by four nucleotides. In transient expression studies, the efficiency of VP2 expression was 20 times lower than that of the ORF2 proteins. The close vicinity of the ORF2 termination signal and the ORF3 AUG codon was crucial, whereas the AUG could be replaced by alternative codons. Deletion mapping revealed that the 3'-terminal 69 nucleotides of ORF2 are crucial for VP2 expression. This sequence contains two essential sequence motifs. The first motif is conserved among caliciviruses and complementary to part of the 18 S rRNA. In conclusion, VP2 is expressed in a translation termination/reinitiation process that is special because it requires a sequence element that could prevent dissociation of post-termination ribosomes via hybridization with 18 S rRNA.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Translation of proteins is a key process in living cells and a prominent site of gene regulation that occurs at the step of translational initiation (reviewed in Refs. 1–3). For most eukaryotic mRNAs, initiation begins with recruitment of the translation machinery at a 5' m7GpppN cap structure and conforms to elements of the ribosomal scanning mechanism (4). Because the small ribosomal subunit starts scanning at the 5'-terminal cap structure of the RNA and then migrates linearly in the 3' direction, translation usually starts at the AUG codon closest to the 5'-end. In higher eukaryotes, the AUG has to reside in a favorable sequence context; otherwise at least some of the ribosomal subunits can pass by and initiate at an AUG further downstream, which can result in expression of proteins that differ from the relevant mRNA (5–10).

Several alternative mechanisms have been identified that lead to the start of translation. Many of these were first detected in viruses because viruses have developed a variety of strategies to ensure translation of their RNAs in competition with cellular RNAs (reviewed in Refs. 9, 11–13).

One possible alternative initiation mechanism is the transfer of an initiation complex assembled at the 5'-end to a downstream initiation site by a so-called shunt mechanism (14–16). Different types of these mechanisms have been identified that rely on special sequences in the RNA and in some cases on the presence of specific proteins that facilitate shunting (17, 18).

Translation can also be initiated without implication of the RNA 5'-end when an internal ribosomal entry site (IRES)2 is present. IRESs represent specialized RNA structures that are able to assemble initiation complexes at internal sites of the mRNA, a principle that is found in a variety of cellular and especially viral RNAs (reviewed in Refs. 19–21).

Furthermore, ribosomes can be recruited by downstream translational start sites after having terminated translation of a preceding ORF (reviewed in Refs. 9, 22, and 23). The biochemistry behind such a termination/reinitiation process is not fully understood. In two cases, the implication of upstream sequences has been reported although the function of these sequences is not known (24, 25).

Caliciviruses represent a group of rather poorly studied pathogens that are causative for epidemically occurring gastrointestinal diseases in humans and a variety of sometimes highly aggressive and fatal syndromes in animals (reviewed in Ref. 26). Members of the Caliciviridae are nonenveloped viruses with nonsegmented single-stranded RNA genomes of positive polarity. The caliciviral genomes have a length of about 7.5 kb and carry a viral protein VPg that is covalently linked to the RNA 5'-end. The viral genomic RNA contains 2 or 3 functional ORFs. Within the infected cells, genome-sized RNA and one species of subgenomic mRNA (sg mRNA) are found. The sg mRNA is 3'-coterminal with the viral genome and codes for a major capsid protein VP1 of about 60 kDa and a minor capsid protein VP2 with a size that varies among viruses from ~8 to more than 20 kDa. VP2 is expressed from a small 3'-terminal ORF that overlaps with the preceding VP1-encoding ORF.

Caliciviruses are special with regard to initiation of viral protein translation because they seem to use VPg as kind of a cap substitute that interacts with translation initiation factors (27–29). Moreover, the small 3'-terminal ORF of rabbit hemorrhagic disease virus (RHDV) was shown to be translated according to a novel termination/reinitiation mechanism, dependent on an RNA element upstream of the start/stop site (25).

The present report shows that the 3'-terminal ORF3 of the feline calicivirus is also initiated by a termination/reinitiation mechanism dependent on an upstream sequence element. Deletion analyses were conducted to identify crucial elements in this sequence, which were further characterized, so that a functional model could be proposed.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cells and Viruses—BHK-21 cells (kindly provided by T. Rümenapf) and CRFK cells (American Type Culture Collection) were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and nonessential amino acids. Vaccinia virus MVA-T7 (30) was kindly provided by B. Moss (NIH, Bethesda, MD) and the FCV vaccine strain 2024 by K. Danner, Hoechst Roussel Vet GmbH.

Construction of Recombinant Plasmids—Restriction and subcloning were done according to standard procedures (31). Restriction and modifying enzymes were purchased from New England Biolabs (Schwalbach, Germany) and Fermentas GmbH (Sankt Leon-Rot, Germany). Plasmid pCITE-2A containing a T7 RNA polymerase promoter followed by a picornavirus IRES was purchased from AGS (Heidelberg, Germany).

Plasmid pCH1 was established by inserting a fragment generated by PCR with pCL1 and primers Ol-FCV-NdeI-Nterm and Ol-FCVSphI-Cterm, cut with NdeI and SphI into pCITE2a. pCL1 was generated by cutting pCI (Promega) with BglII and EcoRI, end-filling with Klenow polymerase, and religation; the resulting plasmid was cut with SmaI and BamHI, end-filled with Klenow polymerase, and religated, followed by cutting with AccI, end-filling with Klenow polymerase, cut with KpnI, ligated with a PCR fragment containing ORF2 and ORF3 of FCV, amplified by PCR with the infectious cDNA clone for FCV 2024 pIK12 (32) and primers Ol-FCVT7ORF2 and Ol-FCV43, and cut with KpnI and SspI.

The construct pCH1 served as a starting point for the establishment of all other constructs. QuikChange mutagenesis (Stratagene, Heidelberg, Germany) was used to introduce point mutations and small deletions. In addition, standard cloning procedures combined with PCR approaches were employed. PCR fragments were amplified in 30 cycles (45 s at 95 °C, 60 s at 55 °C, and 60 s at 72 °C) with Vent polymerase (New England Biolabs). The PCR for QuikChange mutagenesis was conducted in 16 cycles (60 s at 95 °C, 45 s at 55 °C, and 12 min at 68 °C) with Pfu polymerase (Promega) using the buffer and conditions proposed by the manufacturer. The primers were purchased from Invitrogen.

The cloned PCR products were all verified by nucleotide sequencing with the BigDye Terminator Cycle Sequencing kit (PE Applied Biosystems, Weiterstadt, Germany). Sequence analysis and alignments were done with Genetics Computer Group software (33). Further details of the cloning procedure and the sequences of the primers are available upon request to the authors.

Expression, Detection, and Quantification of Proteins—Transient expression of plasmids in BHK-21 cells using vaccinia virus MVA-T7, metabolic labeling with [35S]cysteine or [35S]methionine (ICN, Eschwege, Germany), preparation of cell extracts, and recovery of immunoprecipitates with double precipitation were done as described (25). VP2 expression efficiency was quantified after SDS-PAGE separation of VP1 and VP2 precipitated with antisera V1 (32) and V2 (raised against bacterially expressed VP2), respectively. The gels were analyzed with a Fujifilm BAS-1500 phosphorimager, and intensities of the signals were determined with TINA 2.0 software (Raytest, Straubenhardt, Germany). The molar ratio of VP1 and VP2 was calculated based on the number of labeled residues within the proteins and the measured radioactivity. For comparison of expression efficiencies of different constructs, the VP2 expression level of the given wild-type construct was defined as 100%. The amount of VP2 expression of the other constructs was normalized using the values determined for L-VP1 or Npro and Npro-VP1 as internal standard. The corrected value for VP2 was then used for calculation of the expression efficiency given as a percentage of the wild-type value. The data presented here represent the averages of at least three independent experiments.

To exclude effects of different mRNA stability on the outcome of the experiments, four selected constructs showing considerably reduced VP2 expression and the wild-type plasmid were analyzed in duplicate for intracellular steady state mRNA levels after transfection of MVA-T7-infected BHK-21 cells via Northern blot. The detected differences with regard to the data for the wild-type construct were within the expected experimental variation and much smaller than the effects observed on the protein level (not shown).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
VP1 Is Expressed with Much Higher Efficiency Than VP2—The FCV capsid proteins are encoded by two ORFs that overlap by four nucleotides and are contained in the genomic and the sg mRNA (Fig. 1). The minor capsid protein VP2 is incorporated into the particle in much lower amounts than the major capsid protein VP1 (35). To analyze, whether it is also expressed to much lower levels, the molar ratio of VP1 and VP2 in infected cells was determined. The analyses were conducted with extracts prepared 6-h postinfection, a time point at which no considerable cytopathogenic effect can be detected.

Both capsid proteins were detected in the lysates of infected CRFK cells by immunoprecipitation with antisera specific for VP1 or VP2, respectively. To assure quantitative protein recovery, precipitation was performed twice, and the two precipitates were combined. The supernatant of the second precipitation did no longer contain relevant amounts of the proteins (data not shown). The molar ratio of VP1 and VP2 in the cells was ~18:1, as calculated from the radioactivity incorporated in the precipitates (average of more than five experiments).

The expression of the FCV capsid proteins does not depend on the presence of non-structural proteins but is driven by the sg mRNA (36). We therefore used a transient expression system to analyze the mechanisms responsible for VP2 expression. A cDNA construct, pCH1, was established that contains the regions coding for the leader protein (L) and the two capsid proteins (Fig. 1). In the RNA derived from pCH1, L-VP1 translation is initiated by an EMCV IRES, whereas the VP2 coding ORF and its upstream region are in the wild-type configuration. Both capsid proteins can be detected in extracts of transfected CRFK (not shown) or BHK-21 cells (Fig. 3, lane pCH1). The molar ratio of VP1 and VP2 (~18:1 in CRFK cells and 23:1 in BHK cells) was similar to that found in infected cells. Because of the higher expression levels, BHK-21 cells were used to analyze the mechanism of VP2 translation.


Figure 1
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  		FIGURE 1.
Structure of plasmid pCH1. Schematic representation of the basic structure and organization of the FCV genomic and subgenomic RNA and the important features of the pCH1 construct (not to scale). In the upper part, gray bars represent ORF2 and ORF3 coding for the capsid proteins. The white bar symbolizes the nonstructural protein coding region (ORF1). The sequence in the middle shows the region where ORF2 and ORF3 overlap. The stop of ORF2 and the start of ORF3 are underlined and written in bold face. Protein VPg present at the 5'-ends of both the genomic and subgenomic RNAs is symbolized by a black circle at the end of a line representing the NTR. Below, the plasmid pCH1 is shown with white bars representing the ORF2 and ORF3 containing DNA and black lines symbolizing the noncoding flanking regions. Hel, putative helicase domain; Pro, 3C-like protease; Pol, RNA-dependent RNA polymerase; VP1, major capsid protein; VP2, minor capsid protein.

 


Figure 2
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  		FIGURE 2.
Importance of AUG as a start codon for VP2 translation. The upper region shows a schematic drawing of constructs pCH1 (wildtype) and pCnC63 with mutation of the second methionine in ORF3 and fusion of a reporter sequence coding for the protein Npro to the 3'-end of ORF3. Below the bar representing pCnC63, the changed 5'-terminal sequences of the ORF3 start codon mutants are shown (mutated residues in bold face, underlined). Below the scheme, an autoradiograph shows the expression products labeled with cysteine (left) or methionine (right) and precipitated with antisera specific for VP1 (upper panel) and VP2 (lower panel). The sequences of the mutated initiation codons are given above the gel. Below the gel, VP2-Npro expression levels relative to the pCnC63 control are given (mean values of at least three independent experiments, data normalized relative to the expression levels of L-VP1). Cells infected with MVA-T7 served as a negative control (Vacc).

 
AUG Is Not Essential for VP2 Translation—For most mechanisms of translational initiation, the presence of the initiation codon AUG is crucial or at least preferred, but the use of socalled alternative initiation codons has also been reported. To analyze the importance of an initiator AUG for VP2 translation, the codon was exchanged in pCH1 for ATA, CTG, GTG, CTA, or TGC, respectively. Except for the triple mutant (TGC), all variants showed VP2 translation, but at somewhat reduced rates (data not shown). To analyze this effect quantitatively, the pCnC constructs were generated, in which the Npro gene from a pestivirus was fused in-frame to the 3'-end of the VP2 coding sequence to serve as a reporter (Fig. 2). The VP2-Npro fusion protein expressed from these constructs contains 7 or 8 (TGC mutant) cysteines. The total amount of VP2-Npro translated from these constructs was quantified after cysteine labeling. In all cases, translation of the fusion protein was detected (Fig. 2, left side). The single base substitutions showed expression levels of 47–58% compared with the ATG construct (pCnC63) and the two-base substitution 35%. Even the exchange of the complete triplet still led to a translational efficiency of 12% of the wild-type level.

To determine whether the fusion proteins initiate with a methionine or the amino acid encoded by the changed codon, a second methionine present in the VP2 part of the above described fusion protein was exchanged for a cysteine (pCnC constructs, Fig. 2). The resulting VP2-Npro fusion protein contains no internal methionine. After labeling with [35S]methionine, VP2-Npro was detected for all constructs except the triple mutant (Fig. 2, right side). Comparison of quantification results after cysteine and methionine labeling (Fig. 2 left and right part, respectively) showed that indeed most of the proteins derived from the single and double mutants contained an initiator methionine. The failure to detect the protein from the TGC mutant after methionine labeling might be caused by the inefficient VP2 expression from this construct. Taken together, VP2 translation is not dependent on an AUG initiation codon but is driven by a methionine tRNA-dependent initiation mechanism. The latter is also true for most initiations on the recombinant RNAs with altered AUG codons.


Figure 3
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  		FIGURE 3.
Influence of VP1 translation on VP2 expression. A, insertion of four extra nucleotides (written in bold face and underlined) at a BglII site (BglII*) 275 nucleotides upstream of the ORF2 stop codon results in premature termination of ORF2. A gray bar indicates the protein encoded by ORF2. The L-VP1* expressed from the frameshift mutant pCH72 is truncated by 70 amino acids, and the C-terminal 18 amino acids of the protein represent nonFCV residues (light gray area). Below the scheme, autoradiographs show the immunoprecipitated products of the frameshift construct, compared with wild type construct pCH1. B, partial sequences of pCH1 and two different mutants (pCH17, pCH18) with one extra cytosine residue (bold face and underlined), leading to the fusion of the ORF2 and ORF3 frame (overlapping region of ORF2 and ORF3 in bold face, underlined). Below the sequences, autoradiographs show the products expressed from the fusion constructs.

 
VP2 Expression Is Dependent on VP1 Translation—Assuming that, similar to the situation in RHDV, VP2 is translated by a termination/reinitiation mechanism, the translation of VP1 should be essential for VP2 expression. To investigate if this is true for FCV, a construct was generated with a frameshift in the ORF2 sequence leading to a stop of VP1 translation about 200-nucleotides upstream of the ORF3 start. Thus, the transfer of ribosomes to the VP2 start site via translation of VP1 was blocked. As expected, a truncated VP1 was expressed from pCH72. Importantly, VP2 was not translated from the pCH72-derived RNA (Fig. 3A).

In addition, we wanted to analyze whether the ribosome has only to get to the start of VP2 via translation or whether it also has to terminate before initiation of VP2 translation. For this purpose ORF2 and ORF3 were fused by insertion of one nucleotide close to the start/stop site (Fig. 3B), moving the termination site far away from the VP2 start site to the end of ORF3, but leaving the ORF3 sequence unchanged. Two constructs with a cytosine residue inserted at different positions were established (Fig. 3B, pCH17, pCH18). Both the VP1 and VP2 antisera precipitated the fusion protein L-VP1-VP2 expressed from these constructs, but VP2 alone could not be found (Fig. 3B). These experiments showed that VP2 expression is dependent on translation and termination of translation of the upstream ORF2.


Figure 4
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  		FIGURE 4.
Importance of the ORF2 stop codon for expression of ORF3. On top, partial sequences of wild type pCH1 and mutants with elimination of the ORF2 stop codon (pCH14-16) and of in-frame termination signals downstream of the original stop (pCH24-26, pCH70) are shown. ORF2 in-frame stop codons are underlined, and the mutated nucleotides are written in bold face. Below, autoradiographs show the products precipitated after transient expression. The VP2 expression efficiency is given below the gel for each construct as the percentage of the value determined for pCH1 (data normalized relative to the levels of L-VP1 expression).

 
A Termination Signal near the ORF3 Start Is Crucial for VP2 Translation—As shown before for the fusion constructs, long distance separation of the ORF2 stop and ORF3 start signals blocks ORF3 translation. To analyze the importance of the original ORF2 termination signal for VP2 expression in more detail, the stop codon of ORF2 was deleted by changing the TGA codon to TGT, TGC, or TGG (Fig. 4, pCH14-16). In contrast to the situation in RHDV, the elimination of the bona fide ORF2 termination signal had only a mild effect on VP2 expression (reduction to ~70–90% of the wild-type level). We hypothesized that this could be caused by the presence of two additional in-frame termination signals located at positions 10–12 and 16–18 downstream of the genuine ORF2 stop codon. In another approach, these downstream termination signals were eliminated in addition to the genuine stop codon. After transfection of the resulting triple mutants, VP2 expression could no longer be detected (Fig. 4, pCH24-26). The loss of VP2 translation is not a consequence of the destruction of an essential downstream signal, because the sole mutation of the two downstream stop codons resulted in VP2 translation. The change of the sequence context is nevertheless of some importance, because the VP2 translation level of this mutant was reduced to 42% compared with the wild-type construct (Fig. 4, pCH70). Taken together, a termination signal near the ORF3 start is crucial for VP2 translation.


Figure 5
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  		FIGURE 5.
Importance of the ORF2 sequence for translation of VP2. Schematic representation of a series of constructs with in-frame deletions in ORF2. The sequence coding for Npro was fused to the ORF2 sequence at different positions so that the newly established reading frames contained the 810, 84, 72, 69, and 66 3'-terminal residues of ORF2, respectively. Below the scheme, autoradiographs show the products precipitated after transient expression of the pCnN constructs with antisera against VP2 and Npro. As Npro is an autoprotease cleaving at its own C terminus, the shortened Npro-VP1 fusion proteins (Npro-VP1* and Npro-VP1{Delta}) and Npro alone can be detected. Below the left gel, calculated VP2 expression efficiencies are given (relative to pCNn1, normalized to the expression levels of Npro-VP1{Delta}/Npro).

 
A Sequence of About 70 Nucleotides Upstream of the Start/Stop Site Is Essential for VP2 Translation—In RHDV, translation reinitiation at the start codon of its 3'-terminal ORF is dependent on an RNA sequence preceding the start/stop site (25). We tried to identify a similar element in FCV RNA with a series of in-frame deletion mutants in ORF2. In a first set of constructs, ~500, 200, 100, 70, or 55 nucleotides of the ORF2 3'-region were preserved. VP2 translation was only observed when more than 55 nucleotides of the ORF2 sequence were still present (data not shown). To map the essential region more precisely, a second set of deletion mutants with an Npro reporter gene fused to the 5'-terminal end was generated preserving 84, 72, 69, or 66 nucleotides of the ORF2 3'-region (Fig. 5). Constructs with at least 72 residual ORF2 nucleotides showed a rather small reduction of the expression level (~75% compared with the pCnN1 construct). A construct with only 69 ORF2 nucleotides yielded 30% of the wild-type level, and a further reduction of the ORF2 sequence to only 66 or fewer nucleotides led to total loss of VP2 translation (Fig. 5). In conclusion, the 69 3'-terminal nucleotides of ORF2 represent the minimal essential sequence required for translation of the following ORF3. In the context of RHDV, we referred to this region as the "termination upstream ribosomal binding site" (TURBS).


Figure 6
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  		FIGURE 6.
Influence of 12-nucleotide deletions in the ORF2 3'-terminal region on VP2 translation. Top, a scheme indicates the positions of the 12-nucleotide deletions (gray bars) contained in the given constructs. In addition, the location of the ATGA start/stop site is shown above the top line. The gels show the proteins precipitated after transient expression. Below the gel, the expression efficiencies compared with pCH1 and normalized relative to the levels of L-VP1 expression are given.

 
Two Regions within the TURBS Are Crucial for VP2 Translation—The above-described results defined the 3'-terminal ~70 nucleotides of FCV ORF2 as TURBS. The signal for translational restart could either represent a monopartite element or could be composed of individual oligonucleotide signals separated by spacer regions. To distinguish between these possibilities, a series of constructs with 12-mer deletions at different positions in the TURBS was established (Fig. 6). In contrast to a deletion upstream of the TURBS (nucleotide –90 to –79), all deletions within the critical region reduced VP2 expression levels (Fig. 6). Two 12-mer deletions in the middle of the TURBS (nucleotides –54 to –31) led to VP2 translation levels of about 40–50%, whereas neighboring deletions upstream and downstream of this central region led to a nearly complete loss of ORF3 translation. Thus, the TURBS contains two essential regions located between nucleotides –78 to –55 and nucleotides –30 to –7 upstream of the start/stop site.

To narrow down these two essential sequences, 3-nucleotide deletions were moved through these regions (Fig. 7). Most of the 3-mer deletion mutants showed reduced levels of VP2 expression. The first and the last 3-mer deletion within the region from nucleotide –78 to –55 led to mildly reduced levels of VP2 translation (80 to 90% of wild-type activity). Three deletions showed levels of about 40% (nucleotides –75 to –73 and –72 to –70) and 55% (nucleotides –60 to –58). A further reduction was observed for a deletion of nucleotides –69 to –67 (22% of wild-type level) and an almost complete loss of VP2 expression was found for two 3-mer deletions located between nucleotides –66 and –61. For the second essential region from nucleotide –30 to –7 the first two 3-mer deletions (nucleotides –30 to –25) showed no negative effect on ORF3 translation. The mutants with deletions within the region –18 to –4 reduced the VP2 expression levels to 43–63%, respectively. Again, two 3-mer deletions were identified that almost abrogated VP2 expression (nucleotides –24 to –19). Taken together, the deletion analyses allowed to identify the sequences –66 to –61 and –24 to –19 as crucial for TURBS function.


Figure 7
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  		FIGURE 7.
Changes in VP2 translation efficiency because of deletions in the two essential regions upstream of the start/stop site on VP2 translation. The sequence on top represents the 3'-terminal end of ORF2 with the two essential regions determined by the 12-nucleotide deletions (written in bold face and underlined). Below, the positions of 3-nucleotide deletions in the first (left) and second (right) motif are given. The gels show the proteins precipitated after transient expression with the calculated VP2 expression efficiencies indicated below the autoradiographs (relative to wild-type pCH1, normalized to the expression levels of L-VP1).

 
To investigate whether the sequence connecting the two crucial motifs serves as a spacer that positions the two elements at a defined distance from each other, deletions of 12, 18, 24, 30, and 36 nucleotides were introduced between the two motifs (Fig. 8A). The 36 N deletion results in a fusion of the two motifs without any spacer sequence. Except for the latter, all the deletion mutants showed VP2 expression with levels varying between ~30 and more than 100%. Interestingly, the highest VP2 expression efficiency was observed with the 30 N deletion mutant. Thus, neither the spacer sequence itself nor the distance between the two motifs is essential for VP2 translation. Deletion of the complete intervening sequence is deleterious, but a spacer of 6 nucleotides is sufficient for full activity.

A similar analysis was also conducted for the sequence between the second motif (nucleotides –24 to –19) and the start of VP2. Two constructs with 6-nucleotide deletions were generated, that both reduced ORF3 translation dramatically (Fig. 8B, pCH66, pCH67). To discriminate between a distance or a primary sequence effect mutant pCH90 was established, in which 6 of 14 residues of the sequence between motif 2 and the start/stop site were changed without affecting the distance between the two. This change had no dramatic effect on VP2 expression efficiency (92% of wild-type level) indicating that the primary sequence of the respective part of the RNA is not important.

TURBS Motif 1 Is Complementary to 18 S rRNA—Sequence comparison studies revealed that a sequence of 9 nucleotides containing the most important hexamer of TURBS motif 1 shows perfect complementarity to a sequence in the 18 S rRNA located at positions 1109–1117 (Fig. 9A). The core sequence of the region complementary to the rRNA is conserved among different calicivirus RNAs and located at very similar positions upstream of the 3'-terminal ORFs of the genomes. To investigate whether the rRNA complementarity is important for VP2 expression a series of 4 mutants with single base exchanges in the crucial region was established (Fig. 9B). 3 of 4 mutants (–62 G to T, –63 G to C, and –66 A to C) disrupted the hypothetical rRNA interaction and showed a dramatic reduction of VP2 expression (more than 90% reduction). In contrast, an exchange at the latter position that preserved the possibility for rRNA hybridization via a G/U pairing instead of the wild-type A/U (–66 A to G) had only minor effects. These results support the model that 18 S rRNA interaction is involved in VP2 expression.


Figure 8
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  		FIGURE 8.
A, importance of the spacer sequence located between the essential motifs. Positions of deletions between the two essential motifs are indicated in the sequence block. In the wild-type sequence, the two essential motifs are written in bold face and are underlined. The lower part shows the proteins precipitated after transient expression and the calculated VP2 expression efficiencies (also see other legends). B, contribution of the sequence between motif 2 and the start/stop site to reinitiation. Two constructs with 6-nucleotide deletions between the second motif and the start of the VP2 gene (pCH66, pCH67) and one mutant with multiple exchanges in this region (pCH90) were tested as in A.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Viruses are dependent on the host cellular translation machinery to achieve protein expression. Initiation of translation is the crucial step at which the decision is made whether an RNA is translated or not. Viruses that replicate in the cytoplasm cannot use the cellular mRNA-modifying enzymes for generation of translatable RNAs, so that development of virus-specific mechanisms of translation initiation was necessary. This necessity offered the opportunity to evolve special control mechanisms for viral gene expression, to generate more proteins from a limited amount of genetic material, and to initiate down-regulation of host cellular protein synthesis (reviewed in Refs. 11–13).

Calicivirus RNAs lack a 5'-terminal cap structure but contain a covalently bound protein VPg instead. VPg was shown to interact with translation initiation factor eIF4E or eIF3 in feline calicivirus or Norwalk virus, respectively, and might serve as a cap substitute in translation of the 5'-terminal ORFs in the viral RNAs (27–29). The 3'-terminal ORF, which gives rise to the minor capsid protein (37, 38), was shown in this report and two publications (25, 36) to be translated from the bicistronic sg mRNA. Similar to RHDV (25) the 3'-terminal ORF in FCV is expressed via a translation termination/reinitiation process. This conclusion is based on the results of several experiments. First, translation of the preceding ORF is essential for expression of the VP2 gene. Thus, the ribosomes translating caliciviral RNAs obviously have to reach the internal initiation site via translation of the upstream sequence, which distinguishes this process clearly from shunting as well as IRES-mediated internal initiation mechanisms.


Figure 9
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  		FIGURE 9.
A, conservation of the first TURBS motif among caliciviruses and putative base pairing with 18 S rRNA. The central part of the first essential motif in the FCV TURBS (nucleotides –66 to –61) is conserved among caliciviruses. The sequences upstream of the start/stop site of FCV, RHDV, Norwalk virus (NW), and Sapporo virus (SV) are shown, and the conserved region is highlighted in gray. At the top of this figure, a part of the rabbit 18 S rRNA sequence (nucleotides 1101–1125, highlighted in light gray) is given in 3'-5' orientation. Possible base pairing between the 18 S rRNA and the conserved FCV TURBS motif is indicated. B, influence of the indicated mutations in the putative 18 S rRNA binding sequence on VP2 translation.

 
Second, the detection of an N-terminal methionine in VP2 translated from mutated sequences containing nonAUG start codons shows that translation of VP2 is started by a canonical Met-tRNA-dependent initiation process. This observation represents a strong argument for de novo initiation instead of putative continuous translation processes leading to VP2 via frame-shifting and subsequent proteolytic processing.

Third, the dependence of VP2 translation on the presence of a translational termination signal in close vicinity to the initiation site represents a strong argument for the conclusion that a termination/reinitiation mechanism is responsible for VP2 expression. Interestingly, the genuine stop signal can be substituted by nearby downstream termination signals without considerable reduction of restart efficiency. Thus, the original arrangement with the AUG overlapping the termination signal is slightly preferred, but the coupling between termination and positioning of the ribosome for a restart also works over a certain distance.

Key constructs used in the described transient expression analyses were tested with equivalent results in in vitro translation with rabbit reticulocyte lysate (not shown), so that our data are not biased by the test system. Taken together, the three lines of arguments presented above strongly support the conclusion that a termination/reinitiation mechanism is responsible for the translation of VP2 in FCV. About 5% of the post-termination ribosomes reinitiate translation, which is ~4-fold lower than the value determined for RHDV (25). The process itself is independent from transacting viral factors because the viral non-structural proteins were not present in our assays, and the overwhelming parts of the ORF2 and ORF3 could be deleted or replaced without abrogation of reinitiation. The process also seems not to rely on species-specific cellular factors because it worked with basically the same efficiency in feline and hamster cells.

The molar ratio of VP1 and VP2 was ~18:1, when infected cells were analyzed before the onset of significant CPE. Probably influenced by massive particle formation and cell lysis, the ratio changed to up to 12:1 in late stages of the infectious cycle when the majority of the cells were already dead (not shown). After transient expression, a ratio of 18:1 to 23:1 was determined. That fits well with the value observed in cells before the onset of lysis and also with the ratio of 22:1 that we determined for FCV virions (not shown). Taking into account that calicivirus particles are composed of 180 copies of VP1 (39), the latter value indicates that one virus particle contains about 8 copies of VP2, which is at least 4 times more than previously published (35). Taken together, the results show that the efficiency of the reinitiating process is well adapted to the necessities for particle production.

Examples for protein expression by a termination/reinitiation mechanism can be divided into two types depending on the length of the uORF. Short uORFs were found to be involved in the expression of the following large ORFs, and it appears that these short uORFs have mainly regulatory functions (reviewed in Refs. 41 and 42). The second type of RNAs, for which reinitiation after translation termination is discussed, contain long uORFs that code for functional proteins. Synthetic constructs (43), the influenza B virus RNA segment 7 (44), some of the downstream ORFs in the caulimoviruses RNA (45–47), the ORF-2 of the M2 gene of RSV (48), and the calicivirus 3'-terminal ORF represent examples for this type. The two groups apparently display different mechanisms of translation initiation, because analyses of RNAs with short uORFs revealed that the efficiency of reinitiation decreased considerably when the length of the uORF increased (42, 49). The favorable length of the uORF was in the range of less than 30 codons, and reinitiation was favored by long intercistronic regions. Recent studies provided evidence that resumption of scanning after termination is dependent on maintenance of the interaction between (part of) initiation factor eIF4F and the ribosome during translation of the uORF (50).

In contrast, the uORFs of the second group consist of much more than 30 codons, and the respective RNAs often display only short intercistronic regions or even contain overlaps of the upstream and downstream ORFs (25, 43, 48, 51). It is highly unlikely that initiation factors are still bound to the terminating ribosomes in these cases. Thus, resumption of scanning should either not occur after termination of long uORF translation or should be dependent on prolonged contact between the ribosome and the RNA to allow binding of initiation factors.

We show here for FCV, that a sequence of ~70 nucleotides located upstream of the start/stop site is crucial for translation of the downstream ORF. In contrast, the ORF3 sequence itself or the 3'-NTR were found to be not essential for reinitiation (not shown). Similar results were also obtained for RHDV, where an upstream element of nearly identical length and location was necessary for translation of the 3'-terminal ORF (25). Influence of upstream sequences on a termination/reinitiation process was also found for the respiratory syncytial virus (RSV) M2 RNA with the two overlapping ORFs M2–1 and M2–2 (24). In RSV, the essential upstream sequence element is somewhat larger and the decrease in translation efficiency of the downstream ORF in consequence of proceeding deletions in M2–1 occurs over a longer distance, so that it is not clear at the moment whether the initiation of translation of M2–2 is mechanistically equivalent to the process described here for caliciviruses. This conclusion is also supported by the observation that in RSV, different upstream AUGs can be used as start sites (48), whereas no use of an alternative start site was observed for RHDV.

Deletion mapping of the sequence preceding the start/stop site in the FCV RNA allowed us to define two short oligonucleotides that are crucial for ORF3 translation. 3-mer deletions within these oligomeric sequences reduced VP2 translation by a factor of 10 or more, whereas the loss of three residues in the flanking regions had considerably lower or almost no effect. 12-mer deletions showed that sequences upstream of motif 1 and downstream of motif 2 are important even though 3-mer deletions had no dramatic effect here. Thus, the FCV TURBS consists of two rather short essential sequences that are separated by a spacer of 36 nucleotides. This spacer is virtually of no importance for ORF3 translation initiation, because deletions of up to 30 nucleotides within this region reduce the VP2 translation rate by only a factor of 3 or even result in enhanced ORF3 expression. The fact that deletions of up to 18 nucleotides in the spacer region did result in reduced VP2 translation whereas longer deletions had no negative effect was somewhat surprising. The most likely explanation for this finding is the induction of secondary structure constraints by the shorter deletions that e.g. reduce the accessibility of the two oligomeric motifs. RNA structure analyses are necessary to elucidate this issue in detail.

The sequence separating the second motif and the ORF3 start codon is also not relevant as a nucleotide sequence itself but is important to position the two elements in the correct distance. This conclusion is based on the fact that five different 3-mer deletions and a whole set of mutations in this region reduced the translational level by only up to 2-fold, whereas two 6-mer deletions were deleterious.

Comparison of the two crucial motifs identified in FCV RNA with other caliciviral sequences revealed that the first element is highly conserved (Fig. 9A). A core sequence of 5 residues is identical in prototype members of all 4 calicivirus genera. The respective sequences are found in the RNA at similar distances upstream of the 3'-terminal ORFs (start of pentamer located 33–61 nucleotides upstream of AUG). In contrast, the second motif located 20–15 nucleotides upstream of the ORF3 start site in FCV is much less conserved and only tentatively localized in many of the sequences (not shown).

According to theoretical considerations, the sequence preceding the start/stop site in calicivirus RNAs should tether the terminating ribosome or its 40 S subunit to the RNA to allow binding of the necessary initiation factors, recognition of the initiation codon, and reinitiation of translation. Contact between the mRNA and the 40 S ribosomal subunit cannot be mediated only by the initiation factors eIF3 and eIF4F, but also by hybridization of the mRNA to homologous sequences in the 18 S rRNA. Such a contact has been shown or at least proposed to play a role in a variety of translation initiation processes encompassing both IRES-dependent and shunting mechanisms (reviewed in Refs. 52–54). Two types of 18 S rRNA interactions can be distinguished. The tripartite leader of late adenoviral RNAs facilitates ribosome shunting and contains sequences able to hybridize to the 3'-terminal hairpin region of 18 S rRNA (55). Three dipartite elements were identified that could hybridize with ~50-paired bases in total. Similar interactions, but with somewhat fewer paired residues, could occur for another adenoviral RNA and human heat shock protein hsp70-1 mRNA. It has not been experimentally proven yet that these sequences indeed hybridize to 18 S rRNA, and even if rRNA interaction is relevant in these cases, the adenovirus example shows that additional factors can also be important in such processes (18).

A prototype for the second group of (putative) mRNA/18 S rRNA base pairing interactions is the mRNA-encoding mouse homeodomain protein Gtx. It contains a 9-nucleotide so-called translation enhancer element (TEE) that can hybridize to an internal linear sequence of 18 S rRNA and was shown to exert IRES function and to aid in ribosome shunting (56, 57). The importance of the Gtx TEE/18 S rRNA interaction was recently directly proven (58). Mutation analysis showed that a TEE with a length of 7 base-pairing nucleotides leads to highest translation efficiency.

For both TURBS motifs, complementarity between the crucial sequences and the 18 S rRNA can be detected. The motif located close to the start/stop site is complementary to several short sequences in the 18 S rRNA, but the lack of conservation of this sequence among caliciviruses makes it difficult to judge the significance of these putative interactions. This is markedly different for the first motif that in FCV contains 9 nucleotides showing perfect complementarity to a sequence in the 18 S rRNA located at positions 1109–1117. The respective FCV sequence includes the highly conserved pentamer (Fig. 9A). A look at the other calicivirus sequences revealed for all analyzed motif 1 sequences a minimum of 7 possible base pairings with the 18 S rRNA motif. Moreover, the putative binding site in the 18 S rRNA is located very close to the known binding site of the Gtx TEE (1124–1132).

The results of mutagenesis studies strongly support the hypothesis that 18 S rRNA interaction of this motif plays a role in the calicivirus translational stop/restart mechanism because all tested mutations that would impair hybridization almost abrogate VP2 expression, whereas a change preserving complementarity by a different mutation at one of the tested positions had only a minor effect.

Support of translation initiation by base pairing between mRNA and 18 S rRNA could probably be best explained by the establishment or stabilization of a contact between the (small subunit of the) ribosome and the mRNA also in the absence of (some) of the canonical eukaryotic translation initiation factors that usually mediate this essential contact during translation initiation. This type of direct interaction between the mRNA and the small ribosomal subunit is reminiscent of the bacterial Shine-Dalgarno sequence, a short oligonucleotide sequence that hybridizes to a sequence in the 3'-region of the 16 S rRNA and positions the prokaryotic ribosome at the translational start site (34, 40). To the best of our knowledge, the calicivirus TURBS-induced VP2 expression is the first example of a termination/reinitiation process in eukaryotes for which evidence for a direct interaction between mRNA and 40 S ribosomal subunits via RNA/RNA hybridization is presented. The rather small effect of VP2 AUG start codon mutations on the initiation rate correlates nicely with the hypothesis that start site selection is here less dependent on the base pairing to the initiator tRNA anticodon because another sequence aids in ribosome positioning and start site selection. This hypothesis is also in accordance with the fact that the position of the start site versus the stop site is quite flexible as can be seen in the present study and the variable arrangement of these signals in the RNAs of different caliciviruses (25).

One special point about the calicivirus TURBS is the fact that the binding sequence is located in the coding region of the mRNA and not in the 5'-NTR. The protein encoded by the respective ORF represents the major capsid protein of the virus, which forms the protein shell mediating the transport of the viral genome from one cell to the other. This special situation with two nonrelated functions combined in one sequence imposes special constraints on the respective sequence. It will be a major task for future work to find out whether hybridization to 18 S rRNA is indeed important for reinitiation of translation, to dissect the functions of the two motifs, and to elucidate the biological function of this special way that caliciviruses use to express their minor capsid proteins.

Viral RNAs interact with the same translational machinery that is active on cellular mRNAs. Thus, translation initiation mechanisms initially identified as unique for a special class of viruses often turned out to be not restricted to some odd viral RNAs but to belong also to the repertoire of the host cell. Reinitiation of translation guided by RNA/RNA hybridization might turn out to represent one of the special ways the cell uses to cope with the different demands made on protein expression.


   FOOTNOTES
 
* This study was supported by Grants Me1367/1 and Me1367/3 from the Deutsche Forschungsgemeinschaft. 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

1 To whom correspondence should be addressed: Institut für Immunologie, Friedrich-Loeffler-Institut, Paul-Ehrlich-Str. 28, D-72076 Tübingen, Germany. Tel.: 49-7071-9670; Fax: 49-7071-967303; E-mail: gregor.meyers{at}fli.bund.de.

2 The abbreviations used are: IRES, internal ribosomal entry site; CRFK cells, Crandell Reese feline kidney cells; eIF, eukaryotic translation initiation factor; EMCV, encephalomyocarditis virus; FCV, feline calicivirus; L protein, FCV leader protein; N, nucleotide; ORF, open reading frame; RHDV, rabbit hemorrhagic disease virus; RSV, respiratory syncytial virus; sg RNA, subgenomic RNA; T7, bacteriophage T7; TEE, translation enhancer element; TURBS, termination upstream ribosomal binding site; VP1, calicivirus major capsid protein; VP2, calicivirus minor capsid protein; VPg, viral protein genome-linked. Back


   ACKNOWLEDGMENTS
 
We thank Maren Ziegler, Petra Wulle, and Janett Wieseler for excellent technical assistance. We are grateful to J. O. Thumfart and H. Schirrmeier for the preparation of the VP2 antiserum.



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 EXPERIMENTAL PROCEDURES
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 DISCUSSION
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