Introduction
Cap‐dependent translation initiates through a complex set of protein–protein and RNA–protein interactions that begin with binding of initiation factor eIF4F to the 5′ terminal 7‐methylguanosine (m
7G) cap structure on the mRNA (reviewed in
Hershey and Merrick, 2000). eIF4F is composed of subunits eIF4E, eIF4GI and eIF4A. Recruitment of the 43S pre‐initiation complex, consisting of the Met‐tRNA
iMet–eIF2–GTP ternary complex and eIF3 bound to the 40S ribosomal subunit, to capped mRNA is mediated primarily through interactions between eIF4GI and eIF3 (
Hentze, 1997;
Gingras et al., 1999). This 48S complex scans to the AUG in the appropriate context to initiate protein synthesis. Translation initiation also can be cap‐ and end‐independent. This mechanism is exemplified by internal ribosomal entry sites (IRES) of picornavirus RNA genomes (
Pestova et al., 2001), although internal initiation on some cellular mRNAs also occurs (
Carter et al., 2000;
Hellen and Sarnow, 2001). IRES‐mediated assembly of initiation complexes occurs through RNA–protein interactions, and protein synthesis begins at initiation codons downstream of the IRES. Thus, ribosome recruitment and translation initiation on IRES‐containing mRNAs is independent of the 5′ terminus and an m
7G cap.
Positive‐strand RNA viruses in the families
Picornaviridae,
Potyviridae,
Luteoviridae,
Comoviridae and
Caliciviridae lack m
7G cap structures. Instead, their RNAs are covalently linked at the 5′ end to a small protein called VPg (
viral
protein
genome linked) (reviewed in
Sadowy et al., 2001). VPg of the picornaviruses does not function in initiation of translation on the viral RNA, as initiation is IRES driven (
Jang et al., 1990). The potyviral VPg is multifunctional, and binds to eIF(iso)4E (
Wittmann et al., 1997), but the consequences of this interaction with respect to translation initiation are not clear, as elements in the 5′ untranslated region (UTR) of the potyviral RNA direct cap‐independent translation (
Carrington and Freed, 1990). In contrast to these examples, there is significant suggestive evidence of a role for VPg in initiation of protein synthesis on calicivirus RNA. Removal of VPg from calicivirus RNA results in loss of infectivity (
Burroughs and Brown, 1978), and dramatically reduces translation of feline calicivirus (FCV) RNA
in vitro (
Herbert et al., 1997). These observations suggested VPg was important in initiation of protein synthesis, perhaps functioning as a cap analogue, as proposed by Herbert and co‐workers (
Herbert et al., 1997). However, a putative VPg‐directed mechanism must deviate from that displayed by m
7G cap‐dependent initiation, because addition of m
7G cap analogue to translation reactions of VPg‐linked FCV RNA had no effect on protein synthesis.
The
Caliciviridae include the prototype human calicivirus Norwalk (NV), FCV, rabbit hemorrhagic disease virus (RHDV), vesicular exanthema of swine virus (VESV) and others (
Clarke and Lambden, 2000). Calici virus RNA genomes are 7–8 kb in length, positive sense, polyadenylated and covalently linked to a 12–15 kDa VPg at the 5′ ends of both genomic and subgenomic RNAs (
Clarke and Lambden, 2000). The genomes of NV, FCV and RHDV have been completely sequenced (
Meyers et al., 1991;
Carter et al., 1992;
Jiang et al., 1993;
Hardy, 1999). The first strong context initiation codon in each of the viral genomic RNAs is near the 5′ terminus, at nucleotide 11, 20 and 10, respectively. The N‐terminal protein encoded in the first open reading frame is expressed
in vitro and in infected cells (
Liu et al., 1996;
Wirblich et al., 1996;
Clarke and Lambden, 2000), suggesting the absence of an IRES that would initiate translation at downstream AUGs. Such features of calicivirus genomes raise interesting questions regarding how translation on calicivirus RNA is initiated in the absence of a cap or an IRES.
NV and other human caliciviruses cause epidemic outbreaks of acute gastroenteritis (
Fankhauser et al., 1998;
Hardy, 1999). These viruses do not grow in cell culture, and thus the molecular mechanisms by which the proteins encoded in their genomes are expressed are not well understood. We formulated the hypothesis that initiation of protein synthesis on NV RNA proceeds by a unique mechanism that is dependent on interactions between VPg and the cellular translation machinery. In this study, we show that VPg directly binds to initiation factor eIF3. We also show that VPg inhibits cap‐dependent and IRES‐driven translation of reporter mRNAs in a dose‐dependent manner. Based on data described herein, we propose VPg may function to recruit translation initiation complexes to calicivirus mRNA through direct protein–protein interactions with eIF3, and potentially other eIFs as well.
Discussion
Mechanisms of eukaryotic ribosome recruitment to mRNA are distinguishable by their requirements for initiation factors and 5′ end dependence (
Jackson, 2000;
Pestova et al., 2001). The mechanism utilized by the majority of cellular and viral mRNAs is 5′ end and m
7G cap dependent, and the 43S pre‐initiation complex is recruited to the mRNA through its interactions with the eIF4GI component of the cap‐binding complex eIF4F. 5′ terminus‐independent internal initiation driven by an IRES, typified by those present in some positive strand RNA virus genomes, has different initiation factor requirements depending on the IRES analyzed (
Pestova et al., 1996,
1998;
Jackson, 2000). We put forth the hypothesis that translation initiation on calicivirus RNA may proceed by a unique mechanism that involves protein–protein interactions between VPg and the cellular translation machinery. In this study, we have shown that recombinant VPg binds to purified eIF3, and to native eIF3 in mammalian cell lysates; the C‐terminal half of VPg is the primary mediator of the interaction; and eIF4GI and other translation initiation factors are present in VPg‐interacting complexes. These data, interpreted in the context of those previously reported for translation of the FCV genomic RNA, suggest that VPg may function as a cap analogue through protein–protein interactions distinct from those thus far described as important in m
7G cap‐dependent or IRES‐mediated translation initiation. Herbert and co‐workers have postulated a role for VPg in direct ribosome recruitment (
Herbert et al., 1997), and our data so far support this model. Future studies will address the current hypothesis that the interactions described herein function as an end‐dependent mechanism of ribosome recruitment to calicivirus RNA, mediated by interactions between a 5′ terminus‐linked viral protein VPg and host cell translation initiation complexes.
The organization of calicivirus genomic and subgenomic RNA, specifically a 5′‐genome‐linked protein and short 5′ UTRs, led to the hypothesis that VPg may function in translation initiation. Generally, short 5′ UTRs confer an inefficiency of translation on the mRNAs in which they are present (
Kozak, 1987,
1991a,
b,
1994). The 5′ UTRs of all the calicivirus genomes sequenced to date are less than 20 nucleotides long. In addition, an equally short 5′ UTR is present on the subgenomic RNA encoding the calicivirus capsid protein (
Clarke and Lambden, 2000). It is possible then, that a direct interaction between VPg and eIF3 bound to 40S subunits positions the ribosome precisely at the initiating AUG, and thus these viral RNAs would not be subject to the diminutive effects of a short 5′ leader sequence on translational efficiency. Such a direct recruitment has been proposed for the HCV IRES, where 40S subunits and eIF3 directly bind the IRES in the absence of other initiation factors (
Pestova et al., 1998). In this study, VPg bound directly to purified eIF3. Furthermore, the ribosomal subunit protein S6 was detected in pull‐down eluates of full‐length VPg, and both the C‐ and N‐terminal VPg domains. It may be that binding to 40S subunits is mediated through the N‐terminal domain of VPg. It is tempting to draw a parallel with the HCV IRES with respect to these interactions functioning to position the ribosome at or near the initiator codon. Still, the mechanisms and initiation factor requirements must be different between the two virus systems, as the HCV IRES–eIF3–40S interaction is RNA–protein, whereas the VPg interactions are protein–protein.
The presence of additional components of translation initiation complexes, such as eIF4GI, eIF2α and eIF4E, when binding assays were performed with cell extracts instead of purified eIF3 suggests one alternative mechanism that involves VPg binding to multiple initiation factors (including 40S subunits) to assemble an initiation complex that more closely resembles that recruited by an m
7G cap. Asano and co‐workers reported the presence of a multifactor intermediate complex composed of eIF3, eIF1, eIF5 and eIF2–GTP–Met‐tRNA
iMet in cell extracts that exist free of 40S subunits (
Asano et al., 2000). It is conceivable that the presence of one or more of the additional eIFs detected by immunoblot in the pull‐down assays reflects their associations with eIF3 instead of direct interactions with VPg. Still, detection of components of eIF4F, including eIF4GI and eIF4E, point to the possibility of a complex set of interactions that finally would result in assembly of a functional initiation complex on VPg‐linked RNA.
The effects of VPg on translation of reporter RNA
in vitro were tested to investigate the functional consequences of the eIF3 interactions. VPg inhibited translation of a capped reporter RNA and a reporter RNA containing an EMCV IRES. We considered the possibility that the mechanism of inhibition of translation of these two RNAs may involve disruption of the interaction between eIF4GI and eIF3. eIF4GI was detected in GST–VPg pull‐down eluates, but we were unable to show, or exclude, a direct interaction through a two‐hybrid analysis of eIF4GI and VPg. These two proteins may bind the same sites on eIF3, leading to an inhibition of translation in the presence of VPg. The binding site(s) of eIF4GI on eIF3 is not known, but if the mechanism of translation inhibition by VPg includes preventing the eIF4GI–eIF3 interaction, the eIF3d subunit of eIF3 may play a role. VPg may sequester eIF3, and potentially other initiation factors bound to eIF3, making them unavailable to form functional initiation complexes. Obvious candidates for inhibition targets include those required for cap‐dependent translation, such as components of eIF4F. An alternative and equally plausible hypothesis is provided by the ability of VPg to inhibit translation mediated by the CrPV IGR‐IRES, which can assemble 80S ribosomes from purified 40S and 60S subunits in the absence of any other eIF (
Wilson et al., 2000a). These data suggest that inhibition of translation from this mRNA (and perhaps the others) by VPg is mediated through the 40S subunit. However, the presence of endogenous eIFs in the reticulocyte lysates used for these experiments complicates this straightforward interpretation. Thus the mechanism by which VPg is able to inhibit translation of reporter RNAs that have different factor requirements remains unclear at this point. Studies that add purified initiation factors to VPg‐inhibited translation reactions to rescue protein synthesis are ongoing in order to define the inhibitory mechanism reported here.
VPg of positive‐strand RNA viruses in families other than the
Caliciviridae has been ascribed a number of functions (
Sadowy et al., 2001). The picornavirus VPg is comparatively small (2–4 kDa), functions in protein‐primed RNA synthesis (
Paul et al., 1998) and is not necessary for infectivity of transfected viral RNA. The function of VPg linked to potyvirus genomes appears more complex. Potyviral VPg is larger than the calicivirus VPg (24–26 kDa). In addition to its role in RNA synthesis (
Hong et al., 1995), VPg has been found in the nucleus (
Restrepo et al., 1990), functions in long‐distance cell–cell movement (
Schaad et al., 1997) and binds eIF(iso)4E (
Wittmann et al., 1997;
Leonard et al., 2000). The role of potyvirus VPg binding to eIF(iso)4E with respect to protein synthesis is not known, yet a recent study has confirmed the importance of this interaction by showing that mutant
Arabidopsis thaliana resistant to potyviral infection do not express eIF(iso)4E (
Lellis et al., 2002). Whether potyviral VPg functions in translation initiation remains to be established, as it is clear that sequences in the 5′ UTR of potyviral RNA direct cap‐independent translation enhancement (
Carrington and Freed, 1990). Thus far, there is no evidence that NV RNA contains an IRES to direct cap‐ or 5′ terminus‐independent translation. Assays to address directly if and how the NV VPg may function in ribosome recruitment are challenging. This virus does not grow in cell culture and, consequently, there is no source of NV VPg‐linked RNA to perform recruitment studies. Efforts to establish a system with a cultivatable calicivirus from which native VPg‐linked RNA can be obtained are in progress. Such a system will allow us to address directly translation initiation complex recruitment through functional assays.
Materials and methods
Yeast plasmids and transformations
Construction of yeast plasmids and transformations. Matchmaker yeast two‐hybrid vectors were purchased from Clontech. The activation domain vector pGADT7 carries the
LEU2 nutritional marker and the DNA‐binding domain vector pGBKT7 carries the
TRP1 nutritional marker for selection in yeast. The oligo(dT)‐primed MA104 cell cDNA library was cloned into pGADT7 and is described elsewhere (
Graff et al., 2002). The sequence encoding VPg was amplified from a full‐length NV cDNA clone (
Hardy et al., 2002) by PCR with two primers, VPg
EcoRI(+) 5′‐ccg
gaattcggaaagaacaaaggcaagacc‐3′ and VPg‐
BamHI(−) 5′‐cgc
ggatccttcaaaattgatcttttcattataat‐3′. Restriction enzyme sites are underlined. Amplification conditions consisted of 30 cycles of 94°C for 1 min, 50°C for 30 s and 72°C for 30 s. The resulting 400 bp fragment was cloned into pGBKT7 to generate pGBK‐VPg. Binding domain plasmids were transformed into AH109 yeast cells by the lithium acetate/PEG method as described by
Gietz and Woods (1998).
Two‐hybrid interactions were scored by the ability of yeast to grow on SC −L−W medium and to activate reporter genes HIS3, ADE2 and MEL1. Activation of reporter gene expression was indicated by growth in the absence of histidine (H) and adenine (A), and by the ability to metabolize the chromogenic substrate X‐α‐gal (ICN). Approximately 6 × 109 pGBK‐VPg yeast cells were transformed with 120 μg of the MA104 cDNA library by the lithium acetate/PEG procedure. Transformations were plated on SC −L−W−H−A medium and cultured for 2–4 days at 30°C. Colonies then were re‐streaked on the same selective medium with the addition of 400 μg X‐α‐gal.
Isolation of plasmid DNA from yeast and identification of cDNA. Plasmid DNA was isolated from yeast as described previously (
Gietz and Woods, 1998). The activation domain plasmids with cDNAs encoding potential interactors were recovered by electroporation into DH10B cells and culture on LB agar containing 50 μg/ml ampicillin. Small‐scale plasmid purifications were performed with Eppendorf Perfect Prep. DNA was sequenced on an ABI 310 Genetic Analyzer with BigDye Terminator
® chemistry.
Expression and purification of GST–VPg and GST–VPg deletion mutants
To construct vectors expressing GST fusion proteins, sequences encoding NV VPg were amplified by PCR from the full‐length NV cDNA clone with two primers, VPg‐
EcoRI(+) and VPg‐
XhoI(−) 5′‐ccg
ctcgagttcaaaattgatcttttcattataat‐3′ under the same conditions as described above. Sequences encoding VPg of SMV were amplified by PCR from a SMV cDNA clone (
Lochridge and Hardy, 2003) with two primers SMV‐VPg (+) 5′‐ccg
gaattcagtgacatcacgcctgaaggc‐3′ and SMV‐VPg (−) 5′‐acgc
gtcgacctcaaaactgagtttctcatt‐3′. Amplification conditions were 25 cycles of 94°C for 15 s, 55°C for 15 s and 72°C for 1 min. The N‐terminus of NV VPg (amino acids 1–69) was amplified with VPg‐
EcoRI(+) and VPg‐
XhoI(−)N term 5′‐ccg
ctcgagaccatcaccacctgcctgtacctc‐3′ to generate GST–VPg
1–69. The C‐terminus of NV VPg (amino acids 70–138) was amplified with VPg‐
EcoRI(+)C term 5′‐ccg
gaattcggcataggagaaactgaaatgg‐3′ and VPg‐
XhoI(−) to generate GST–VPg
70–138. Amplification conditions were 30 cycles of 94°C for 1 min, 55°C for 1 min and 72°C for 2 min, followed by a 5 min extension at 72°C.
Each PCR fragment was cloned into pGEX‐4T‐1 (Amersham Pharmacia) and expressed as a GST fusion protein in BL21(DE3) cells. Bacteria were cultured to an OD600 of 0.6 and recombinant protein expression was induced with 1 mM IPTG for 2 h. Bacteria were collected by centrifugation and suspended in buffer containing 50 mM Tris–HCl pH 8, 2 mM EDTA, 1% Triton X‐100 and 100 μg/ml lysozyme. This suspension was incubated for 15 min at 30°C and then sonicated three times on ice for 10‐s pulses. Soluble and insoluble proteins were separated by centrifugation for 10 min at 12 000 g. The supernatant containing soluble protein was retained for the purification of GST–VPg. GST was expressed as a control and purified under the same conditions described below.
A 50% slurry of glutathione–Sepharose 4B beads (Amersham Pharmacia) was prepared following instructions provided by the manufacturer. One hundred microliters of prepared beads were mixed with 3 ml soluble bacterial cell lysate from either 50, 15 or 3 ml GST–VPg‐induced culture (volume dependent on the experiment), and rocked for 10 min at room temperature. Beads were washed twice with cold phosphate buffered saline (PBS), and collected by centrifugation for 5 min at 500 g. GST–VPg was eluted from the beads by three 10 min room temperature incubations in elution buffer containing 10 mM reduced glutathione/50 mM Tris–HCl pH 8. Beads were removed by centrifugation for 5 min at 500 g. The eluates were combined, and eluted proteins were evaluated for purity by SDS–PAGE. GST–VPg and GST were quantified with the Bio‐Rad protein assay with bovine serum albumin as the assay standard.
GST pull‐down assay
GST–VPg pull‐down assays employed purified eIF3,
in vitro translated eIF3d and native eIF3 in mammalian cell (CaCo‐2) lysates. Purified eIF3 was obtained from HeLa cells by previously described methods (
Falvey and Staehelin, 1970;
Meyer et al., 1982).
35S‐labeled eIF3d was translated
in vitro as described below for luciferase RNA. Translation reactions were treated with 0.4 μg/μl RNAse A for 30 min at 37°C before use in pull‐down assays. CaCo‐2 colon adenocarcinoma cells were grown to confluency in 60 mm dishes in MEM (Invitrogen) containing 15% fetal bovine serum (Atlanta Biologicals). CaCo‐2 cells were chosen for these experiments because they are intestinal in origin and are relevant to our enteric virus model system. The cells were washed twice with PBS, scraped from the plate and collected in 100 μl of lysis buffer containing 50 mM Tris–HCl pH 8, 15 mM NaCl, 140 mM KCl, 2% NP‐40, 6 μg/ml aprotinin, 6 μg/ml pepstatin A and 6 μg/ml leupeptin. The lysate was brought to a final volume of 1 ml with wash buffer (20 mM Tris–HCl pH 7.5, 15 mM NaCl, 140 mM KCl and 0.1% NP‐40), mixed with the prepared glutathione–Sepharose 4B beads bound to GST–VPg or GST alone, or the indicated mutant, and rotated end‐over‐end for 4 h at 4°C. In some experiments, the lysates were pretreated with 75 U/ml S7 micrococcal nuclease (Roche Biochemicals) in the presence of 1 mM CaCl
2 for 15 min at 20°C. Nuclease digestions were terminated by addition of EGTA to a final concentration of 2 mM. The beads were collected by centrifugation for 5 min at 500
g at 4°C and washed three times with wash buffer. GST–VPg and interactors were eluted from the beads by three 10 min incubations in elution buffer as before.
Pull‐down eluates were electrophoresed on SDS‐10% or 8% polyacrylamide gels, and proteins were transferred to nitrocellulose for western immunoblotting. The membranes were blocked for 1 h with 10% BLOTTO (10% non‐fat dry milk in PBS), and then incubated overnight at room temperature with goat anti‐rabbit eIF3 (
Meyer et al., 1982) diluted 1:2000 in 0.5% BLOTTO. The reactivity of the anti‐eIF3 antibody has been described previously (
Brown‐Luedi et al., 1982;
Meyer et al., 1982). This antiserum recognizes all of the subunits of eIF3, in addition to eIF4GI (
Etchison et al., 1982). The titer of antibody to the eIF3d subunit is low. Additional antibodies employed in western blots include rabbit anti‐eIF4GI diluted 1:2000, rabbit anti‐eIF4B diluted 1:1000, rabbit anti‐eIF2α diluted 1:1000 and anti‐S6 diluted 1:1000. All commercial antibodies were purchased from Cell Signaling Technologies. The membranes were washed three times in 0.5% BLOTTO, then incubated for 2 h at room temperature with horseradish peroxidase‐conjugated rabbit anti‐goat, or goat anti‐rabbit, IgG diluted 1:3000 (Jackson Immunoresearch Laboratories) in 0.5% BLOTTO. Proteins that bound antibodies were detected by enhanced chemiluminescence (Amersham Pharmacia).
In vitro translations in RRLs
The Luciferase T7 DNA (Promega) served as template for synthesis of capped mRNA (m
7G‐Luc). The luciferase gene was cloned into the pCITE4a+ vector (Novagen) downstream of the EMCV IRES to generate a template for synthesis of IRES‐containing mRNA (IRES‐Luc). The plasmid pEJ4 that contains the IGR‐IRES of CrPV upstream of the firefly luciferase reporter was kindly provided by Dr P.Sarnow (Stanford University Medical School). Templates were linearized with
XmnI (T7 DNA),
PvuII (pCITE‐Luc) or
BamHI (pEJ4) for 2 h at 37°C, phenol/chloroform extracted and precipitated with 450 mM NH
4OAc and ethanol. m
7G‐Luc mRNA was transcribed with the Ambion mMessage mMachine system, and IRES‐Luc mRNA was transcribed with the Ambion T7 Megascript system following the provided protocols. Plasmid pETp66N was the template for synthesis of eIF3d RNA (
Asano et al., 1997b). Linearized DNA was transcribed using the Ambion T7 Megascript system following provided protocols.
RNA was heated to 65°C for 3 min and quenched on ice before use. Translation reactions were performed in Flexi‐RRL (Promega) as recommended by the manufacturer. Two hundred and fifty nanograms of reporter RNA were translated in 25 μl reactions containing 16.5 μl of lysate, 4 μCi [35S]methionine (1000 Ci/mmol; Amersham Pharmacia), 20 μM amino acid solution minus methionine, 70 mM KCl, and GST or GST–VPg as inhibitor. Translation reactions were incubated for 1 h at 30°C. Translation products were resolved by SDS–PAGE and visualized by autoradiography. Luciferase bands were quantified by densitometric analysis on a Bio‐Rad Molecular Imager FX. Translation inhibition experiments were performed a minimum of three times.