Institut de Biologie Moléculaire des Plantes, UPR CNRS 2357, Université Louis Pasteur, 12 rue du Général Zimmer, 67084 Strasbourg Cedex, France
Correspondence
Mario Keller
mario.keller{at}ibmp-ulp.u-strasbg.fr
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ABSTRACT |
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Present address: Laboratoire de Dynamique, Evolution et Expression des Génomes de Microorganismes, FRE CNRS 2326, Université Louis Pasteur, 28 rue Goethe, 67084 Strasbourg Cedex, France.
Present address: UPR CNRS 9050, Département Récepteurs et Protéines Membranaires, ESBS, boulevard Sébastient Brant, 67412 Illkirch Cedex, France.
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INTRODUCTION |
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P6 is a multifunctional 62 kDa protein that plays a pivotal role in the CaMV infectious cycle. In particular it is involved in host-range determination (Schoelz & Shepherd, 1988), symptomatology (Daubert & Routh, 1990
; Wintermantel et al., 1993
; Broglio, 1995
), and probably viral morphogenesis (Himmelbach et al., 1996
). Moreover, P6 is also the major component of the membrane-free cytoplasmic inclusion bodies called viroplasms (Xiong et al., 1982
), where the main steps of the replication cycle occur (Mazzolini et al., 1989
).
Transactivation has been shown to depend on the interaction of P6 with polysomes and initiation factor eIF3 (Park et al., 2001), which is an essential component in the translation initiation process. P6 physically interacts with the g subunit of eIF3 (Park et al., 2001
) and with the 60S ribosomal proteins L18 (Leh et al., 2000
) and L24 (Park et al., 2001
). L18 binds to the miniTAV domain (Leh et al., 2000
), which corresponds to the minimal sequence required for translational transactivation (De Tapia et al., 1993
), while L24 and eIF3 subunit g interact with an RNA-binding region located downstream of the miniTAV. The interaction between P6 and eIF3 on the 60S ribosomal subunit is apparently mediated by at least two ribosomal proteins, L18 and L24. The exact mechanism of P6-mediated reinitiation of translation is not yet fully understood, but it has been proposed that P6 could maintain eIF3 bound to the ribosome during the elongation phase by a shuttling process between the 40S and 60S ribosomal subunits (Park et al., 2001
). Translocation of eIF3 back to the 40S ribosomal subunit shortly before the termination of translation would prevent the release of eIF3 from the ribosome and thus permit a ternary complex to reinitiate translation of the next ORF. L24 is one of the proteins involved in bridging interactions between the 40S and 60S ribosomal subunits (Spahn et al., 2001
). Whether the P6L24 interaction is involved in ribosome formation during translationreinitiation, and/or might affect the association of the two ribosomal subunits late in infection to favour reverse transcription of the CaMV genome, is still unclear (Park et al., 2001
).
It has been shown by far-Western assays performed on proteins from a ribosome-enriched fraction of Arabidopsis thaliana that P6 binds to several polypeptides (Leh et al., 2000). This finding suggests that other components of the cellular translation machinery might be implicated in translation of the 35S RNA and its spliced versions. In this study, we have characterized a novel cellular partner of P6, the ribosomal protein L13 of the 60S ribosomal subunit. We also show that L13 and L18 bind to the same sequence of the miniTAV.
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METHODS |
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Cloning of the A. thaliana L13 coding sequence into pET-KaKS and pGEX-2TK vectors.
The sequence encoding the ribosomal protein L13 of A. thaliana (ecotype Columbia) was amplified by PCR from a cDNA library cloned into the HybridZAP vector (the library was provided by B. Lescure, INRA-UMR 215, Castanet-Tolosan, France) with 5'- and 3'-specific oligonucleotides containing BamHI/KpnI and SacI/CelII restriction sites, respectively, at their 5' termini (5'-GCAGGTACCGGATCCATGAAGCACAACAATGTTATC-3' and 5'-GTCGGATCCGCTAAGCTCACTTCTTCTCTTCTTTCTC-3'). After digestion with appropriate endonucleases, the DNA fragment was cloned into the pET-KaKS vector. The resulting pET-L13 recombinant plasmid generates a fusion protein containing, at its N terminus, a decapeptide, which can be phosphorylated in vitro by heart muscle bovine kinase.
The recombinant pGST-L13 plasmid was obtained by insertion of a BamHI/EcoRI-digested PCR fragment (generated with the primers 5'-GCAGGTACCGGATCCATGAAGCACAACAATGTTATC-3' and 5'-GGTTGGGAATTCCTCACTTCTTCTCTTCTTTCTCAGC-3') into BamHI- and EcoRI-cleaved pGEX-2TK.
Expression and phosphorylation of recombinant proteins.
L13, GSTP6, P6 and its deleted versions were expressed in E. coli BL21/DE3 (Novagen) modified by transformation with the plasmid pUBS520 (Brinkmann et al., 1989) carrying the E. coli gene argU. L18 and GST were expressed in E. coli BL21/DE3(pLys S) from pETKH-L18 (Leh et al., 2000
) and pGEX-2TK vectors, respectively.
E. coli strains were transformed by electroporation with recombinant plasmids. Once the culture reached the exponential phase, expression of heterologous proteins was induced with 1 mM IPTG for 2 h. Bacteria were collected by centrifugation at 4000 g for 10 min, resuspended in HMK buffer (20 mM Tris/HCl pH 7·5, 100 mM NaCl, 12 mM MgCl2), and lysed by sonication (two pulses for 20 s at 50 W). After centrifugation at 12 000 g for 10 min, the supernatant was discarded and the inclusion bodies containing the proteins of interest were resuspended in 500 µl HMK buffer.
Recombinant proteins were labelled in the presence of [-32P]ATP [3000 Ci mmol1; 10 mCi ml1 (370 MBq)] and 20 U bovine heart muscle protein kinase for 1 h at room temperature, according to the manufacturer's instructions (Sigma). Non-incorporated radioactive ATP was eliminated by filtration through a Sephadex G-50 column (Amersham Pharmacia Biotech). The eluate (300 µl) was treated for 30 min with a mixture of RNase A (40 µg) and DNase (100 U) at 37 °C. Degradation of nucleic acids was verified after fractionation of an aliquot (20 µl) on a 1·5 % agarose gel and ethidium bromide staining.
Western blotting analysis.
Proteins from recombinant bacteria were separated by SDS-PAGE and electrophoretically transferred onto a nitrocellulose membrane (Schleicher and Schuell). The membrane was blocked for 30 min in 5 % non-fat dried milk in PBS buffer (140 mM NaCl, 2·7 mM KCl, 8·1 mM Na2HPO4, 1·5 mM KH2PO4, pH 7·3) containing 1 % Tween 20 and then incubated overnight at 4 °C with specific rabbit polyclonal antibodies raised against P6 (1 : 10 000 dilution) or L13 (1 : 5000 dilution), respectively. The membranes were washed with PBS buffer and treated with goat anti-rabbit antibodies conjugated to alkaline phosphatase at the dilution recommended by the manufacturer.
Far-Western assays.
A blotting protein-overlay technique was used to detect interactions between proteins. Proteins were resolved by SDS-PAGE and transferred onto a nitrocellulose membrane. Membranes were washed several times at 4 °C in PBS buffer containing 5 % non-fat milk and incubated for 12 h at 4 °C with gentle shaking in the same buffer containing the 32P-labelled protein in the overlay and with 50 µg of RNase A and 10 U DNase. After three washes in PBS buffer at room temperature, membranes were dried and radioactive complexes were detected by autoradiography (Kodak XR5 film).
In vitro GST pull-down and competition assays.
Approximately 1 µg GSTP6 fusion protein (Leh et al., 2000) was mixed with 50 µl glutathioneSepharose 4B beads, previously washed and resuspended in PBS buffer, and incubated for 1 h at 4 °C with gentle shaking in PBS containing 1 % Tween 20. After three washes by brief centrifugation and resuspension in the latter solution, beads were mixed with 30 µl bacterial protein containing 32P-labelled protein. The mixture was adjusted to a final volume of 500 µl and incubated for 3 h at 4 °C with gentle shaking. The beads were then washed three times and resuspended in 20 µl dissociation buffer, boiled for 5 min to dissociate the proteinbead complexes, and finally pelleted at 10 000 g for 2 min. The supernatant was immediately subjected to SDS-PAGE and the 32P-labelled proteins were detected by autoradiography.
For competition assays, equal quantities of Sepharose 4B beads binding GSTP6 were incubated overnight at 4 °C with 32P-labelled L13 and increasing amounts (08 µg) of unlabelled competitor and non-competitor proteins, respectively, in PBS/1 % Tween 20 at a final volume of 500 µl. After thorough washing, the beads were resuspended in 20 µl dissociation buffer as described above, and the proteins contained in the supernatant were separated on SDS-PAGE. The radiolabelled protein was detected by autoradiography for 13 days at 20 °C using an intensifying screen, and the competitor was visualized by Coomassie blue staining of the polyacrylamide gel.
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RESULTS |
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The specificity of the L13P6 interaction was also investigated in a GST-pulldown experiment. For this purpose the sequence encoding L13 was amplified by PCR from pET-L13 and inserted into pGEX-2TK. The sequence of the resulting clone, pGST-L13, revealed two differences from the original sequence of L13 cDNA. The initiator codon was changed into an ATT (isoleucine), and the second codon (normally encoding a lysine) was substituted by AAT specific for asparagine; the mutation of these two amino acids is not expected to have an effect on the GST-pulldown assay.
The proteins GSTL13, GSTP6, HisP6 (a histidine-tagged P6) and GST were expressed in E. coli and used in the GST-pulldown assays (Fig. 1c). GSTP6 protein was bound to Sepharose 4B beads, incubated with 32P-labelled L13 protein and then, after several washes, the retained complexes were analysed by SDS-PAGE. After transfer, the membrane was subjected to autoradiography (Fig. 1c
, bottom left) and staining with Coomassie blue (Fig. 1c
, upper panel). L13 specifically interacted with P6, as evidenced by the strong radioactive signal at the level of a polypeptide of approximately 30 kDa compared with the signals obtained when pulldown assays were performed with HisP6, GST and bacterial proteins as negative controls (Fig. 1c
, bottom panel). Pulldown assays carried out with GSTL13 bound to the beads and 32P-labelled P6 as overlay confirmed this result, as labelled P6 was retained on the beads only in the presence of GSTL13 (Fig. 1c
, bottom right), whereas almost no radioactivity was detected in any of the control experiments. The other radioactive signals in the GSTL13 lane presumably correspond to cleaved products of the P6 protein. The latter were already detected when proteins were analysed on polyacrylamide gel after labelling of the inclusion bodies prepared from E. coli expressing P6 (data not shown), in particular the polypeptide of 42 kDa, a P6-cleavage product which is also found in CaMV-infected plants (Xiong et al., 1982
; Maule et al., 1989
).
L13 interacts with the miniTAV domain of P6
To define the region of CaMV P6 involved in binding of L13, deleted versions of the P6-coding CaMV ORF VI were cloned into the pET-KaKS vector (Leh et al., 2000) except for mutant A, where the coding sequence was inserted into pGEX-2TK (Fig. 2
a). Their capacity to interact in vitro with 32P-labelled L13 was tested by far-Western assays (Fig. 2b
, bottom panel). Only N- and C-terminally truncated P6 and P6 mutants containing the miniTAV domain (residues 112242) were able to bind the ribosomal protein L13 strongly, indicating that L13 interacts with the miniTAV of P6. Far-Western assays with the miniTAV itself could not be performed because this polypeptide is very poorly expressed in E. coli. Mutant H, corresponding to the C-terminal half of P6, interacted weakly with L13, whereas mutants G and
TAV encompassing this region did not, raising the question whether this interaction is specific. The possibility that interaction between L13 and P6 was mediated by nucleic acids (the miniTAV is known to have RNA-binding properties; Cerritelli et al., 1998
) can be excluded because these results were obtained when far-Western experiments were performed in a buffer containing a cocktail of RNase and DNase.
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DISCUSSION |
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A detailed mutational analysis revealed that the L13 and L18 proteins bind to the same 60 aa long sequence within miniTAV. This interaction domain encompasses the consensus sequence of the RNase H nucleic acid-binding site that can bind in vitro to RNADNA heteroduplexes and double-stranded RNA (Cerritelli et al., 1998). However, treatment of both the membrane and the overlay with nucleases did not impair the interaction of either L13 or L18 with P6, suggesting that binding of these ribosomal proteins to P6 is not mediated by nucleic acids. The involvement of a protein sequence in an interaction with several partners is often observed for viral proteins, and has already been described for CaMV P6 (Park et al., 2001
). These authors demonstrated that both L24 and eIF3 interact with RNA-binding domain A located just downstream of miniTAV, and that these factors compete with one another for binding to this region. As expected, L18 also competes with L13 for binding to miniTAV as both proteins occupy exactly the same sequence and presumably cannot bind simultaneously to the same P6 molecule.
Currently, the only information available concerning the topography of L13 in eukaryotic ribosomes comes from studies performed on the rat 60S subunit using immobilized enzymes and cross-linking experiments (Marion & Marion, 1987). L13 from rat presents a high degree of identity with other eukaryotic L13 and, in particular, with its plant counterpart (75 %). The current model for the spatial rearrangement of proteins within the 60S rat liver ribosomal subunit has L13 linked to L18 by the intermediary of ribosomal protein L31 (Marion & Marion, 1987
). Our finding that L13 binds to L18 in vitro is not inconsistent with this model and, in particular, suggests that these ribosomal proteins could also physically interact on the ribosome surface. The exact localization of L13 and its interaction with L18 can be definitively determined only from crystallographic studies of the 60S subunit structure. However, the crystal structure of the archaeal 50S ribosome has already shown that L18 is located on the outer surface of the subunit near the neck region (Ban et al., 2000
). It is tempting to speculate that L13 and L18 might bind to P6 dimers, as it was recently shown that P6 self-interacts in vitro (Haas et al., 2002b
) and in vivo (Li & Leisner, 2002
), and it has been proposed that the dimers represent the biologically active form of P6 (Haas et al., 2002b
; Li & Leisner, 2002
). Nevertheless, it should be noted that dimerization of P6 is not a prerequisite for L13 and L18 binding as the N-terminal domain, which is absolutely required for P6P6 interaction, is dispensable for L13 binding.
Concerning the role of the L13P6 interaction, this can only be a matter of speculation in the absence of any information on the function of L13 during protein synthesis or in other processes. Several possibilities can be proposed if the hypothesis that L13 physically interacts with L18 on the ribosomal surface is tentatively accepted. Recently, Park et al. (2001) suggested that P6 mediates the interaction between L18 and eIF3 on the large ribosomal subunit and proposed that this interaction permits stalling of the initiation factor during the elongation process to favour the reinitiation of translation of downstream ORFs. The possibility that simultaneous binding of P6 to L13 and L18 reinforces this interaction cannot be excluded. On the other hand, studies in mammals have shown that L18 interacts with a double-stranded RNA-activated protein kinase (PKR) and that this interaction inhibits PKR activity. A putative PKR-like protein was identified in plant extracts using antibodies raised against human PKR (Langland et al., 1995
, 1996
) but no protein containing double-stranded RNA binding and kinase domains has been identified so far (although the two domains could be present on separate proteins in plants). The possible existence of a PKR-like activity in plants was reinforced by the discovery of a plant PKR inhibitor and the fact that its inactivation favours viral pathogenesis (Bilgin et al., 2003
). PKR-like kinases are also involved in the expression of mRNAs bearing short ORFs in their leader (Kumar et al., 1999
). Association of such activities with PKR has led to the hypothesis that the P6L18 interaction might activate the putative plant PKR-like enzyme to favour the translation of the CaMV 35S RNA and spliced versions (Leh et al., 2000
) or, alternatively, sequester the PKR by strengthening its attachment to the ribosome. In either case, L13 could contribute to the function of L18 and would hence act as a cofactor required for the CaMV infectious cycle. Whether PKR activity is implicated in plants in the defence against virus infection, as it is in animals, remains an open question. Finally, we do not exclude the possibility that L13 might carry out extra-ribosomal functions as an independent polypeptide, as described for many eukaryotic and bacteria ribosomal proteins (Wool, 1996
).
Saez-Vasquez et al. (2000) have demonstrated that a significant fraction of L13 from B. napus is located in the nucleus. Transient expression in tobacco BY-2 cells of A. thaliana L13 protein fused to EGFP showed that it also localizes in the nucleus (data not shown). It has been suggested that the tobacco L13 homologue might play a regulatory role in transcription, as a region of L13 can replace the activation domain of the transcription factor GAL4 in yeast double-hybrid experiments (Estruch et al., 1994
). Recently we have discovered that P6, which has previously been considered an exclusively cytoplasmic protein, also localizes in the nucleus of CaMV-infected cells and thus may shuttle between the nucleocytoplasmic compartments using the importexport cellular machinery (Haas et al., 2002b
). The fact that several cellular genes are down- or upregulated in transgenic A. thaliana plants expressing CaMV P6 protein (Geri et al., 1999
) might be indicative of a role for both P6 and L13 in the regulation of cellular gene transcription.
Ribosomal proteins L13, L18 and L24 and initiation factor eIF3 probably represent only some of the components of the translation machinery that are usurped by CaMV P6 protein to fullfil its role in translation reinitiation of polycistronic mRNAs. Results of far-Western experiments indicated that P6 interacts with about a dozen proteins from a ribosomal fraction. It will be interesting to see if other P6 partners are ribosomal proteins of the 40S subunit and/or other initiation factors potentially involved in reinitiation of translation. Involvement of numerous cellular partners suggests that ribosomes bear different P6-binding sites and, consequently, are capable of binding simultaneously to several P6 molecules. P6 might also interact with ribosomes in a coordinated manner during the translation process. Answering these questions will provide new insights concerning the reinitiation mechanism mediated by the CaMV P6 protein.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Ban, N., Nissen, P., Hansen, J., Moore, P. B. & Steitz, T. A. (2000). The complete atomic structure of the large ribosomal subunit at 2.4 A resolution. Science 289, 905920.
Bertauche, N., Leung, J. & Giraudat, J. (1994). Conservation of the human breast basic conserved 1 gene in the plant kingdom: characterization of a cDNA clone from Arabidopsis thaliana. Gene 141, 211214.[CrossRef][Medline]
Bilgin, D. D., Liu, Y., Schiff, M. & Dinesh-Kumar, S. P. (2003). P58IPK, a plant ortholog of double-stranded RNA-dependent protein kinase PKR inhibitor, functions in viral pathogenesis. Dev Cell 4, 651661.[CrossRef][Medline]
Bonneville, J. M., Sanfaçon, H., Fütterer, J. & Hohn, T. (1989). Posttranscriptional trans-activation in Cauliflower mosaic virus. Cell 59, 11351143.[Medline]
Brinkmann, U., Mattes, R. E. & Buckel, P. (1989). High-level expression of recombinant genes in Escherichia coli is dependent on the availability of the dnaY gene product. Gene 85, 109114.[CrossRef][Medline]
Broglio, E. P. (1995). Mutational analysis of cauliflower mosaic virus gene VI: changes in host range, symptoms, and discovery of transactivation-positive, noninfectious mutants. Mol Plant Microbe Interact 8, 755760.[Medline]
Cerritelli, S. M., Fedoroff, O. Y., Reid, B. R. & Crouch, R. J. (1998). A common 40 amino acid motif in eukaryotic RNases H1 and caulimovirus ORF VI proteins binds to duplex RNAs. Nucleic Acids Res 26, 18341840.
Daubert, S. & Routh, G. (1990). Point mutations in cauliflower mosaic virus gene VI confer host-specific symptom changes. Mol Plant Microbe Interact 3, 341345.[Medline]
Delseny, M. & Hull, R. (1983). Isolation and characterization of faithful and altered clones of the genomes of cauliflower mosaic virus isolates Cabb B-JI, CM4-184, and Bari I. Plasmid 9, 3141.[CrossRef][Medline]
De Tapia, M., Himmelbach, A. & Hohn, T. (1993). Molecular dissection of the cauliflower mosaic virus translation transactivator. EMBO J 12, 33053314.[Abstract]
Dieci, G., Bottarelli, L., Ballabeni, A. & Ottonello, S. (2000). tRNA-assisted overproduction of eukaryotic ribosomal proteins. Protein Expr Purif 18, 346354.[CrossRef][Medline]
Estruch, J. J., Crossland, L. & Goff, S. A. (1994). Plant activating sequences: positively charged peptides are functional as transcriptional activation domains. Nucleic Acids Res 22, 39833989.[Abstract]
Fütterer, J. & Hohn, T. (1996). Translation in plants rules and exceptions. Plant Mol Biol 32, 159189.[Medline]
Geri, C., Cecchini, E., Giannakou, M. E., Covey, S. N. & Milner, J. J. (1999). Altered patterns of gene expression in Arabidopsis elicited by cauliflower mosaic virus (CaMV) infection and by a CaMV gene VI transgene. Mol Plant Microbe Interact 12, 377384.[Medline]
Haas, M., Bureau, M., Geldreich, A., Yot, P. & Keller, M. (2002a). Cauliflower mosaic virus: still in the news. Mol Plant Pathol 3, 419429.[CrossRef]
Haas, M., Geldreich, A., Keller, M. & Yot, P. (2002b). Studies on the molecular mechanisms involved in the formation of viroplasms specific on infection by Cauliflower mosaic virus. In XIIth International Congress on Virology, abstract pp. 89-V-576. 27th July1st August. Paris.
Himmelbach, A., Chapdelaine, Y. & Hohn, T. (1996). Interaction between cauliflower mosaic virus inclusion body protein and capsid protein: implications for viral assembly. Virology 217, 147157.[CrossRef][Medline]
Kane, J. F. (1995). Effects of rare codon clusters on high-level expression of heterologous proteins in Escherichia coli. Curr Opin Biotechnol 6, 494500.[CrossRef][Medline]
Kiss-Laszlo, Z., Blanc, S. & Hohn, T. (1995). Splicing of cauliflower mosaic virus 35S RNA is essential for viral infectivity. EMBO J 14, 35523562.[Abstract]
Kumar, K. U., Srivastava, S. P. & Kaufman, R. J. (1999). Double-stranded RNA-activated protein kinase (PKR) is negatively regulated by 60S ribosomal subunit protein L18. Mol Cell Biol 19, 11161125.
Langland, J. O., Jin, S., Jacobs, B. L. & Roth, D. A. (1995). Identification of a plant-encoded analog of PKR, the mammalian double-stranded RNA-dependent protein kinase. Plant Physiol 108, 12591267.
Langland, J. O., Langland, L. A., Browning, K. S. & Roth, D. A. (1996). Phosphorylation of plant eukaryotic initiation factor-2 by the plant-encoded double-stranded RNA-dependent protein kinase, pPKR, and inhibition of protein synthesis in vitro. J Biol Chem 271, 45394544.
Leh, V., Yot, P. & Keller, M. (2000). The cauliflower mosaic virus translational transactivator interacts with the 60S ribosomal subunit protein L18 of Arabidopsis thaliana. Virology 266, 17.[CrossRef][Medline]
Li, Y. & Leisner, S. M. (2002). Multiple domains within the Cauliflower mosaic virus gene VI product interact with the full-length protein. Mol Plant Microbe Interact 15, 10501057.[Medline]
Marion, M. J. & Marion, C. (1987). Localization of ribosomal proteins on the surface of mammalian 60S ribosomal subunits by means of immobilized enzymes. Correlation with chemical cross-linking data. Biochem Biophys Res Commun 149, 10771083.[Medline]
Maule, A. J., Harker, C. L. & Wilson, I. G. (1989). The pattern of accumulation of cauliflower mosaic virus specific products in infected turnips. Virology 169, 436446.[CrossRef][Medline]
Mazzolini, L., Dabos, P., Constantin, S. & Yot, P. (1989). Further evidence that viroplasms are the site of cauliflower mosaic virus genome replication by reverse transcription during viral infection. J Gen Virol 70, 34393449.
Olvera, J. & Wool, I. G. (1994). The primary structure of rat ribosomal protein L13. Biochem Biophys Res Commun 201, 102107.[CrossRef][Medline]
Park, H.-S., Himmelbach, A., Browning, K. S., Hohn, T. & Ryabova, L. A. (2001). A plant viral reinitiation factor interacts with the host translational machinery. Cell 106, 723733.[Medline]
Pooggin, M., Fütterer, J., Skryabin, K. & Hohn, T. (1999). A short open reading frame terminating in front of a stable hairpin is the conserved feature in pregenomic RNA leaders of plant pararetroviruses. J Gen Virol 80, 22172228.
Pooggin, M., Hohn, T. & Fütterer, J. (2000). Role of a short open reading frame in ribosome shunt on the cauliflower mosaic virus RNA leader. J Biol Chem 275, 1728817296.
Ryabova, L. A. & Hohn, T. (2000). Ribosome shunting in the cauliflower mosaic virus 35S RNA leader is a special case of reinitiation of translation functioning in plant and animal systems. Genes Dev 14, 817829.
Ryabova, L. A., Pooggin, M. M. & Hohn, T. (2002). Viral strategies of translation initiation: ribosomal shunt and reinitiation. Prog Nucleic Acid Res Mol Biol 72, 139.[Medline]
Saez-Vasquez, J., Gallois, P. & Delseny, M. (2000). Accumulation and nuclear targeting of BnC24, a Brassica napus ribosomal protein corresponding to a mRNA accumulating in response to cold treatment. Plant Sci 156, 3546.[CrossRef][Medline]
Schoelz, J. E. & Shepherd, R. J. (1988). Host range control of cauliflower mosaic virus. Virology 162, 3037.[Medline]
Scholthof, H. B., Gowda, S., Wu, F. C. & Shepherd, R. J. (1992). The full-length transcript of a caulimovirus is a polycistronic mRNA whose genes are trans activated by the product of gene VI. J Virol 66, 31313139.[Abstract]
Spahn, C. M., Beckmann, R., Eswar, N., Penczek, P. A., Sali, A., Blobel, G. & Frank, J. (2001). Structure of the 80S ribosome from Saccharomyces cerevisiaetRNA-ribosome and subunit-subunit interactions. Cell 107, 373386.[Medline]
Wintermantel, W. M., Anderson, E. J. & Schoeltz, J. E. (1993). Identification of domains within gene VI of cauliflower mosaic virus that influence systemic infection of Nicotiana bigelovii in light-dependent manner. Virology 196, 789798.[CrossRef][Medline]
Wool, I. G. (1996). Extraribosomal functions of ribosomal proteins. Trends Biochem Sci 21, 164165.[CrossRef][Medline]
Xiong, C., Balàzs, E., Lebeurier, G., Hindenlang, C., Stoeckel, M. & Porte, A. (1982). Comparative cytology of two isolates of cauliflower mosaic virus. J Gen Virol 61, 7581.
Received 29 April 2004;
accepted 14 July 2004.