| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
1 Department of Plant Science, McGill University, Ste-Anne-de-Bellevue, Québec H9X 3V9, Canada
2 Institut national de la recherche scientifique, Institut Armand-Frappier, Laval, Québec H7V 1B7, Canada
Correspondence
Jean-François Laliberté
jean-francois.laliberte{at}iaf.inrs.ca
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
A supplementary table and two supplementary figures are available with the online version of this paper.
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
; Wells et al., 1998). PABP is also important for stability (Behm-Ansmant et al., 2007; Parker & Song, 2004), biogenesis (Amrani et al., 1997; Brown & Sachs, 1998) and nuclear export of cellular mRNAs (Brune et al., 2005).
Multiple PABP isoforms are normally found in animals and plants (Mangus et al., 2003). Eight PABP genes have been identified in Arabidopsis thaliana (Belostotsky, 2003). The corresponding proteins are divided into four classes based on gene expression and similarity. Class II genes (PAB2, PAB4 and PAB8) are highly and broadly expressed and probably encode the bulk of PABP required for cellular functions (Belostotsky, 2003).
In animal cells, PABP is cleaved by proteinases of picornaviruses (Joachims et al., 1999; Kerekatte et al., 1999; Kuyumcu-Martinez et al., 2002, 2004b; Rodriguez Pulido et al., 2007; Zhang et al., 2007), caliciviruses (Kuyumcu-Martinez et al., 2004a) and retroviruses (Alvarez et al., 2006). The resultant cleavage leads to host translational shutdown (Joachims et al., 1999; Kuyumcu-Martinez et al., 2004a, b; Zhang et al., 2007). Rotaviruses also inhibit PABP functions through an alternative pathway. The binding of the viral non-structural protein 3 to eIF4G prevents PABP from interacting with proteins bound to the 5' cap and leads to a decrease in the translation of cellular mRNAs (Montero et al., 2006; Piron et al., 1998).
Paradoxically, PABP also appears to be required for efficient viral RNA synthesis/translation of a number of animal viruses. Accumulating evidence suggests that most RNA viruses adopt a closed-loop conformation for efficient translation and transcription of their genome (for review see Thivierge et al., 2005). This is achieved by recruitment of translation initiation factors at the 5' and 3' ends of the viral RNA and often involves PABP. Poliovirus replication requires genome circularization and proceeds through a protein bridge that involves PABP (Herold & Andino, 2001). Interactions between viral internal ribosomal entry site (IRES), eIF4G, PABP and the poly(A) tail are also important for picornavirus cap-independent translation. Disruption of PABP–eIF4G or PABP–poly(A) tail interactions on IRES-driven reporter mRNAs leads to reduced rates of translation (Bradrick et al., 2007; Khaleghpour et al., 2001; Michel et al., 2000; Svitkin et al., 2001). Similar results were also obtained using IRES derived from the Tobacco etch virus (Gallie, 2001; Gallie et al., 1995).
Turnip mosaic virus (TuMV) is a potyvirus (Fauquet et al., 2005). Its positive single-stranded RNA genome of almost 10 kb contains one long open reading frame and bears a viral genome-linked protein (VPg) covalently linked at its 5' terminus and a poly(A) tail at the 3' terminus (Nicolas & Laliberté, 1992). Several lines of evidence indicate that components of the eIF4F complex are required for potyvirus replication. Potyviruses interact with eIF4E or its eIF(iso)4E isomers via VPg-Pro (Schaad et al., 2000; Wittmann et al., 1997) and disruption of the interaction leads to resistance (Léonard et al., 2000). Requirement for eIF4E/4G or eIF(iso)4E/4G isomers depends on the virus being studied, as A. thaliana knockout plants for eIF(iso)4E or eIF(iso)4G1/2 genes are resistant to TuMV, lettuce mosaic virus and plum pox virus infection, while disruption of eIF4E1 or eIF4G results in resistance to clover yellow vein virus (Decroocq et al., 2006; Duprat et al., 2002; Lellis et al., 2002; Nicaise et al., 2007; Sato et al., 2005). These results were also confirmed in multiple plant species with the identification of eIF4E and eIF4G isoforms as recessive resistance gene products against potyviruses (Gao et al., 2004; Kang et al., 2005; Nicaise et al., 2003; Ruffel et al., 2002, 2005). Together, these results are indicative of an essential link between potyvirus replication and components of the translation initiation complex.
We have recently reported the interaction of A. thaliana PABP2 with the RNA-dependent RNA polymerase (RdRp) and VPg-Pro of TuMV within virus-induced vesicles, where replication probably takes place (Beauchemin & Laliberté, 2007). This suggests that PABP might also be a required factor for potyvirus infection. The present work shows that VPg-Pro and RdRp of TuMV interact not only with PABP2 but also with PABP4 and PABP8 in A. thaliana, which explains why single pab2, pab4 or pab8 knockout plants were all susceptible to TuMV infection. However, further depletion of PABP in double-knockout plants resulted in reduced TuMV RNA accumulation. These data support a role for PABP in potyvirus infection.
|
METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
) were amplified by using PCR with primers PABP4-BamHI and PABP4-NotI, or PABP8-BamHI and PABP8-NotI (see Supplementary Table S1, available in JGV Online). The amplified fragments were digested with BamHI/NotI and cloned into similarly digested pGEX-6P1 (GE Healthcare) and used for purification as described for PABP2-GST (Dufresne et al., 2008). TuMV VPg-Pro and histidine-tagged RdRp were purified as previously described by Dufresne et al. (2008) and Menard et al. (1995).
RdRp and VPg-Pro binding assays with PABP.
RdRp or VPg-Pro binding assays were performed as previously described by Dufresne et al. (2008) with GST-PABP2, GST-PABP4 or GST-PABP8 proteins. Detection of retained protein was achieved with a mouse monoclonal (Novagen) or polyclonal (Molecular Probes) antibody.
Isolation of single and double pab mutants.
PAB2 (SALK_026293), PAB4 (SALK_113383) and PAB8 (SALK_022160) T-DNA insertion lines were obtained from the SALK T-DNA collection (Alonso et al., 2003). pab homozygous mutant plants were identified by PCR using a T-DNA left border primer and gene-specific primers (see Supplementary Table S1, available in JGV Online).
pab2 pab4 and pab4 pab8 double mutants were obtained by crossing the pab4 null mutant with pab2 or pab8 mutants; pab2 pab8 mutants were obtained by crossing the pab8 mutant line with a pab2 mutant.
TuMV inoculation of A. thaliana.
Rosette leaves of 3-week-old A. thaliana plants were inoculated as previously described by Dufresne et al. (2008).
RNA isolation and RT-PCR amplification.
Total RNA was isolated from 3-week-old uninfected, mock-inoculated or TuMV-infected plants using the RNeasy plant Mini kit (Qiagen). RNA aliquots (1 µg) were reverse-transcribed to cDNA using the Omniscript RT kit (Qiagen) and used for PCR. The ACTIN2 (ACT2) (At3g18780; GenBank accession number NM_180280
[GenBank]
) mRNA was used as positive control for amplification. Primers are provided in Supplementary Table S1.
Quantitative real-time RT-PCR analysis (qRT-PCR).
qRT-PCR was performed using SYBr Green MasterMix (Stratagene). Relative amounts of all mRNAs were calculated from threshold cycle values. The ACT2 reference gene was used for normalization and differences in PCR efficiency were taken into account according to the formula: ratio=(Etarget)
CPtarget(control–sample)/(Eref)CPref(control–sample) (Pfaffl, 2001). RNA samples for each genotype were extracted from three different pools of four different plants and qRT-PCR was repeated twice. Primers CP-F (5'-TGGCTGATTACGAACTGACG-3') and CP-R (5'-CTGCCTAAATGTGGGTTTGG-3') were used for TuMV detection; the other primers are provided in Supplementary Table S1.
Protein extraction and membrane fractionation.
Protein extraction and membrane fractionation were performed as previously described on uninfected, mock inoculated and TuMV-infected A. thaliana (Dufresne et al., 2008). Proteins were separated on an 11 % polyacrylamide SDS gel (Laemmli, 1970) and analysed by immunoblotting as previously described (Dufresne et al., 2008).
Statistical analyses.
PABP densitometric levels on immunoblots were compared using Student's t-test. Data from ELISA binding assays were analysed using one-way ANOVA and Tukey's test using SAS 9.0 software. Statistical analyses of qRT-PCR were conducted using REST MCS relative expression software (Pfaffl et al., 2002).
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
; Kuyumcu-Martinez et al., 2004b). Analysis of TuMV-infected Brassica perviridis (Beauchemin & Laliberté, 2007) indicated that PABP cellular levels were increased when compared with mock-inoculated plants. In order to test if this was also the case in TuMV-infected A. thaliana, leaves were homogenized and nuclei, chloroplasts and cell wall debris were removed by centrifugation at 3700 g. Soluble proteins were then separated from membrane-associated proteins by centrifugation at 27 000 g and resuspended in the same volume to allow quantitative evaluation. Total, soluble (S30) and membrane-associated (P30) proteins were separated by SDS-PAGE and subjected to immunoblot analysis with a rabbit serum raised against recombinant AtPABP2. This serum detects A. thaliana PABP2 (68.7 kDa), PABP4 (71.7 kDa) and PABP8 (72.8 kDa). Due to a similar molecular mass, PABP4 cannot be distinguished from PABP8 using one-dimensional SDS-PAGE (Figs 1a and 3c). This serum recognized recombinant GST-tagged PABP2, PABP4 and PABP8 equally well (data not shown). Therefore, it is likely that the intensity of the bands reflects the abundance of all three isoforms in A. thaliana. Fig. 1 shows that a higher level of PABP2 was present in TuMV-infected extracts. Densitometric analysis indicated a 1.37-fold (±SEM, 0.03) increase in total PABP levels (Student's t-test, P>0.0001, n=3). In both healthy and infected extracts, UDP-glucose pyrophosphorylase (UGPase), which is a marker for the cytoplasm, was detected only in soluble and total (data not shown) protein extracts, which indicates the absence of cytoplasmic protein contamination in the membrane-enriched fraction. In contrast to the predominantly cytosolic PABP detected in healthy B. perviridis (Beauchemin & Laliberté, 2007; Dufresne et al., 2008), we found that class II PABP isoforms were abundant in both S30 and P30 fractions in healthy and TuMV-infected A. thaliana Col-0.
|
|
, P>0.05, n=3). Both PAB4 and PAB8 mRNAs showed the largest increases in expression at 17 days p.i., with inductions of 7- and 5.1- fold, respectively. Thus, the observed increase in PAB relative mRNA expression following virus infection correlates with the higher levels of PABP observed in TuMV-infected A. thaliana. It is interesting to note that the mRNA levels (obtained by qRT-PCR) of remaining functional class II PAB genes were unchanged in pab single or double knockout plants when compared with that of wild-type A. thaliana plants. Similarly, PAB expression remained unchanged following exposure to mannitol stress (data not shown).
TuMV RdRp and VPg-Pro interaction with A. thaliana PABP2, PABP4 and PABP8 in vitro
We have previously shown that two TuMV polypeptides, RdRp and VPg-Pro, interact directly with A. thaliana PABP2 (Beauchemin & Laliberté, 2007; Dufresne et al., 2008; Léonard et al., 2004). Eight PAB genes have been identified in A. thaliana (Belostotsky, 2003). The corresponding proteins are divided into four classes based on gene expression and similarity. Whereas class I, III and IV PABP transcripts are produced at low levels and in a tissue-specific manner, class II members (PABP2, PABP4 and PABP8) are highly and broadly expressed and probably constitute the bulk of PABP required for cellular functions. PABP2 and PABP8 are most similar, exhibiting 68 % identity at the amino acid level, whereas PABP4 bears 60 % identity to PABP2 and 59 % identity to PABP8 (see Supplementary Fig. S1, available in JGV Online). This raises the possibility that TuMV VPg-Pro and RdRp could interact with other class II PABPs during infection.
To test for the direct interaction of VPg-Pro and RdRp with PABP2, PABP4 or PABP8, an ELISA-based binding assay was performed. To validate the assay, ELISA plates were coated with metal chelation-purified TuMV 6xhistidine-tagged RdRp or VPg-Pro. The coated wells were then incubated with increasing concentrations of purified GST-tagged PABP2. Complex retention was detected using an anti-GST antibody. A typical protein–protein interaction saturation binding curve was observed for both VPg-Pro (Fig. 2a) and RdRp (Fig. 2c). No signal was detected when the wells were coated with a metal-chelating chromatography-purified E. coli lysate (data not shown) or when GST-PABP2 was replaced with the GST protein. No synergistic effect on binding was observed when VPg-Pro and RdRp were added simultaneously (data not shown). After validation, we tested the capacity of RdRp and VPg-Pro to bind to PABP class II isoforms. ELISA plates were again coated with recombinant TuMV RdRp or VPg-Pro. The coated wells were then saturated with 25 pmol GST-tagged PABP2, PABP4 or PABP8. For the VPg-Pro-coated plate, the strongest interaction signal was obtained with GST-tagged PABP2 (maximum signal), followed by PABP4 (81 % of maximum signal) and PABP8 (65 % of maximum signal; Fig. 2b). For the RdRp-coated plate, the strongest interaction signal was obtained with GST-tagged PABP2 (maximum signal), followed by PABP8 (51 % of maximum signal), whereas no signal was detected with PABP4 (Fig. 2d). Data from both experiments were obtained for three biological replicates from a minimum of two technical replicates. The differences recorded were statistically significant (Tukey's test; P<0.05). Overall, this suggests that TuMV RdRp and VPg-Pro interact preferentially with PABP2, but are also capable of interaction with one or both of the other class II PABPs (i.e. PABP4 and PABP8).
|
was screened by PCR for T-DNA disruption of the PAB2, PAB4 and PAB8 genes. Viable homozygous T4 knockouts were isolated for all class II PAB genes. T-DNA insertions were found in the first exon of the PAB2 gene, the third exon of the PAB4 gene and the ninth exon of the PAB8 gene (Fig. 3a). The corresponding single null mutants were named pab2, pab4 and pab8. Viable double mutants were obtained by crossing single pab mutants and selfing the F1 hybrids. The corresponding double mutants were named pab2 pab4, pab4 pab8 and pab2 pab8.
The homozygous knockout genotype and the presence of T-DNA insertions were confirmed by PCR on plant genomic DNA (data not shown) and by RT-PCR on expressed mRNA. The PAB2, PAB4 and PAB8 mRNAs were not detected in the corresponding mutants (Fig. 3b). mRNA levels (obtained by qRT-PCR) of remaining functional class II PAB genes were unchanged in pab single or double knockout plants when compared with that of wild-type A. thaliana plants (data not shown). Immunoblotting using an anti-AtPABP2 polyclonal antibody was also performed on plant protein extracts (Fig. 3c). PABP2 protein was not found in the pab2 mutants and the PABP4/PABP8 protein band was not detected in pab4 pab8 mutants. Corresponding PAB gene products (mRNA and protein) were not detected in the single and double pab mutants, which confirms the knockout genotype.
All attempts to isolate a triple pab mutant (by crossing a pab4 pab8 double mutant with a pab2 line) failed. To confirm lethality of the pab2 pab4 pab8 genotype, F3 progenies of selfed hemizygous double knockout pab4 pab8 (PAB2+/–) lines were sown. Again, a viable pab2 pab4 pab8 mutant could not be isolated. PCR genotyping of non-germinating seed allowed identification of dead triple knockouts, which indicates that the pab2 pab4 pab8 genotype is lethal.
TuMV accumulation is reduced in pab2 pab4 and pab2 pab8 double A. thaliana knockouts
The interaction of TuMV VPg-Pro and RdRp with PABP suggests that PABP might be a required factor for virus replication and/or for translation of the viral genome. As seen with TuMV and other potyviruses, the disruption of eukaryotic translation initiation factors such as eIF4E/eIFiso4E and eIF4G/eIF(iso)4G in A. thaliana leads to potyvirus resistance (Duprat et al., 2002; Lellis et al., 2002; Nicaise et al., 2007) which is indicative of their absolute requirement for virus infection.
To test this idea, pab2, pab4 and pab8 A. thaliana single PABP T-DNA knockouts were challenged with TuMV in two independent experiments. Ten plants were inoculated for each genotype. Two weeks p.i., all pab2, pab4 and pab8 plants showed signs of infection similar to those observed for infected wild-type Col-0 (data not shown). Infection was further confirmed by RT-PCR (data not shown). Thus, none of the individual PAB gene products alone was absolutely required for TuMV infection. It is possible that the remaining PAB genes can compensate for the loss of the disrupted PABP gene to allow viral replication.
In another set of experiments, TuMV inoculation was repeated on the double mutants pab2 pab4, pab2 pab8 and pab4 pab8 to see whether further depletion in PABP cellular level would affect TuMV accumulation. In contrast to pab single and pab4 pab8 mutants, which were usually phenotypically indistinguishable from wild-type plants, pab2 pab4 and pab2 pab8 double mutants had growth and development defects (Fig. 4a and Supplementary Fig. S2, available in JGV Online). The triple pab knockout genotype (pab2 pab4 pab8) was not viable and the effect of complete class II PABP depletion could not be tested. Three week old pab2 pab4, pab4 pab8, pab2 pab8 and wild-type Col-0 control A. thaliana lines were inoculated with TuMV in two independent experiments. All lines showed typical inflorescence stunting that resulted from TuMV infection, indicating that none was completely resistant to TuMV. Despite obvious signs of infection, vegetative tissues from the double mutant line pab2 pab8 were generally less contorted and stunted, which suggested that they were less susceptible to infection (Fig. 4a).
|
). All experiments were replicated independently at least twice. Significant reductions in relative TuMV RNA levels were recorded in the pab2 pab8 line, with reductions of 2- and 3.5-fold at 7 and 17 days p.i., respectively (Fig. 4b). Relative TuMV RNA accumulation in pab2 pab4 was 1.8-fold less than that of infected wild-type Col-0 line at 17 days p.i. (Fig. 4b). No significant difference was measured for the pab4 pab8 line. These experiments indicate that partial PABP depletion, in particular that of PABP2, although not sufficient to confer complete resistance, can impede the replication cycle of TuMV in the cell. The decrease in viral RNA accumulation may well be a reflection of a general alteration in protein synthesis or a non-specific consequence of the developmental defects of these plants. However, the phenotype of the different mutants does not always reflect the level of viral RNA accumulation. For example, pab2 pab4 plants were more affected in their development than pab2 pab8 plants (see Supplementary Fig. S2) but they accumulated wild-type levels of viral RNA after 7 days, while pab2 pab8 plants showed a 50 % decrease in viral RNA level for the same period (Fig. 4b). This suggests that the observed reduction in viral RNA accumulation is not an indirect end result of the physiological state of the mutant plants.
PABP levels in soluble and microsomal fractions correlate with reduced TuMV accumulation
PABP2 represents approximately two-thirds of the class II PABPs produced in A. thaliana (Fig. 3c). Since pab2 pab4 and pab2 pab8 lines (but not the pab4 pab8 mutant) showed reduced viral RNA accumulation, this could be related to a reduction in overall PABP concentration in the cell. However, the possibility exists that the reduced virus accumulation observed might result from a pleiotropic effect of PABP depletion.
We have recently reported redistribution of PABP to membrane-associated fractions in TuMV-infected B. perviridis (Beauchemin & Laliberté, 2007). Through agroinfiltration of fluorescent reporter constructs in Nicotiana benthamiana, we also showed that, upon co-expression of the TuMV 6K-VPg-Pro protein, which induces cytoplasmic vesicle formation, PABP2 was found within these organelles (Beauchemin & Laliberté, 2007; Dufresne et al., 2008). Recruitment of PABP within these virus-induced vesicles derived from endoplasmic reticulum membranes is thought to be important as potyviral replication occurs in association with membranes (Beauchemin et al., 2007; Beauchemin & Laliberté, 2007; Schaad et al., 1997). Therefore, depletion of PABP levels in the membrane fraction would impede the role of PABP in viral replication and result in reduced virus accumulation.
To assess PABP class II isoform levels following TuMV infection in each of the knockout lines, subcellular fractionation experiments were conducted. Leaves of infected plants were harvested 12 days p.i., and soluble (S30) and membrane-enriched (P30) fractions were separated by centrifugation. Proteins from each fraction were separated by SDS-PAGE and subjected to immunoblot analysis with a rabbit serum raised against AtPABP2. Compared with wild-type TuMV-infected Col-0 plants, overall PABP levels were reduced in all pab double A. thaliana mutants (Fig. 5). The TuMV pab4 pab8 line showed PABP levels that corresponded to approximately 86–88 % of what was found in infected Col-0 plants (total, 87.7 %; S30 , 86.63 %; P30, 87.7 %). Both pab2 pab4 and pab2 pab8 lines showed a more pronounced decrease in overall PABP levels. In pab2 pab4 plants, the proportion of PABP compared with the wild-type ranged from 18 to 38 % (total, 38.4 %; S30, 31.4 %; P30, 18.1 %). These proportions were reduced even further (ranging from 26 to 7 %) in pab2 pab8 plants (total, 26.2 %; S30 , 22.6 %; P30, 6.7 %). It must be noted that PABP levels were disproportionately reduced in the membrane-associated fraction in both of these mutants. Thus, TuMV accumulation in these lines correlated with cellular PABP concentration in A. thaliana, and more precisely with the concentration of PABP in membranes, which is anticipated to be important for efficient virus infection.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
; Dufresne et al., 2008). These interactions with PABP appear to be important as they localize within virus-induced vesicles, where potyvirus replication reportedly takes place (Beauchemin & Laliberté, 2007; Dufresne et al., 2008). In the present study, we investigated whether PABP was required for potyvirus propagation in A. thaliana. Using a reverse-genetics approach, none of the single PABP knockout plants that were tested were found to be immune to TuMV infection. However, virus multiplication was significantly inhibited in pab2 pab4 and pab2 pab8 double mutants and was well correlated with PABP levels in membranes. These results suggest that a minimal threshold of PABP is required for efficient multiplication of TuMV.
Translation initiation factors in plants are encoded by small gene families. In A. thaliana, four genes [eIF4E1, eIF4E2, eIF4E3 and eIF(iso)4E] encode proteins of the eIF4E family and three genes [eIF4G, eIF4(iso)G1 and eIF4(iso)G2] encode proteins of the eIF4G family (http://research.cm.utexas.edu/kbrowning/fiat/). The different isoforms appear to carry out complementary biological functions as demonstrated by the lack of specific phenotypes in plants with disrupted eIF4E/4G isomers (Duprat et al., 2002; Nicaise et al., 2007; Sato et al., 2005). An analogous role for PABP is demonstrated here, as individual deletion of constitutive and highly expressing PABP paralogues (i.e. PAB2, PAB4 and PAB8 genes) has little effect on A. thaliana growth and development.
Despite apparent functional redundancy, the viral requirement for eIF4E/4G or eIF(iso)4E/4G isoforms is not functionally interchangeable (Robaglia & Caranta, 2006). TuMV, for instance, specifically requires eIF(iso)4E or both eIF(iso)4G1 and eIF(iso)4G2 isoforms for successful infection, while ClYVV recruits eIF4E and eIF4G. The only potyvirus known to deviate from this rule is pepper veinal mottle virus, which can use both eIF4E and eIF(iso)4E to achieve pepper infection (Ruffel et al., 2006). This selective requirement by most viruses is likely to be a consequence of co-evolution between different plant–virus pairs (Charron et al., 2008). Experimental evidence gathered so far has demonstrated that the resistance phenotype relates to the binding capacity of VPg for one or the other of the eIF4E isoforms (Khan et al., 2006; Léonard et al., 2000; Yeam et al., 2007). The important structural differences found in the two eIF4F isoforms probably make it difficult to maintain interaction with both isomers.
The present work shows that VPg-Pro and RdRp of TuMV interact not only with PABP2 but also with PABP4 and/or PABP8 isomers in A. thaliana. This would explain why single pab2, pab4 or pab8 knockout plants were susceptible to TuMV infection; the virus could still utilize remaining PABP paralogues that are expressed in the cell. Further depletion of PABP in double knockout plants resulted in reduced TuMV RNA accumulation and confirms that PABP has a role to play in potyvirus infection. The effect appears to be additive, as it correlated with overall cellular PABP levels; PABP2 was relatively more important as it is the most abundant isoform. Our results indicate that A. thaliana triple pab knockouts are not viable. The absence of complete resistance in double knockouts might therefore result from the presence of residual PABP in the cell. We must underline the fact that VPg-Pro interacted with PABP2, PABP4 and PABP8, while RdRp interacted with PABP2 and PABP8 and apparently not with PABP4. The fact that double mutant pab2 pab8 plants were susceptible to TuMV suggests that the RdRp/PABP interaction is not strictly required for virus replication or that RdRp can possibly interact with the tissue-specific and weakly expressed class I, III and IV PABP.
Rather than a reduction in PABP levels through cleavage, as seen with some animal viruses, PABP concentration in TuMV-infected cells was substantially increased. Results obtained here indicate that it can be related to an increase in PAB mRNA steady-state levels. This upregulation of PAB genes is also interesting; we also found that their expression is not influenced by osmotic stress or by PAB gene disruption (data not shown). This suggests that a virus-specific mechanism could be responsible for PABP upregulation, as seen with the upregulation of other genes associated with protein synthesis following TuMV infection (Yang et al., 2007).
The role of PABP in potyvirus infection remains to be clearly defined. An obvious possibility is that PABP, like the canonical translation initiation factors eIF(iso)4E and eIF(iso)4G, is needed for the closed-loop conformation of viral RNA linking VPg and eIF(iso4)G to the viral poly(A) tail in a VPg–eIF(iso)4E–eIF(iso)4G–PABP protein bridge. The latter would be necessary for efficient cap-independent translation of the viral RNA and a potential means of competing with cellular mRNA for the translation machinery. Since these interactions might also sequester key components from cap-dependent target cellular mRNA, they could also simultaneously inhibit cellular translation.
Another hypothesis is that the dual interaction of the RdRp with both PABP and VPg-Pro may serve to switch from viral translational to replication mode. As the genome is translated, RdRp would accumulate and its combined interaction with VPg-Pro (Léonard et al., 2000) and PABP (Dufresne et al., 2008; Wang et al., 2000) bound to viral RNA would evict or modify conformation of the eIF4F complex, dislodge ribosomes engaged on the viral RNA and allow replication to proceed. The latter explanation is appealing, as VPg-Pro can form a tripartite complex with PABP2 and eIF(iso)4E in vitro (Beauchemin & Laliberté, 2007). However, the ability of RdRp to antagonize this tripartite complex remains to be tested.
PABP is an important regulator of mRNA degradation that acts by protecting the 3' poly(A) tail of mRNAs. This raises the possibility that depletion of PABP cellular levels exposes the viral RNA to increased degradation and would explain the reduced TuMV RNA levels observed in PABP-depleted knockout plants.
Collectively, our data extend previous findings on the importance of PABP in the infectious cycle of TuMV (Beauchemin & Laliberté, 2007; Dufresne et al., 2008). As A. thaliana single and double PABP knockouts are now available, it will be interesting to quantify how other viruses multiply in these mutant backgrounds. Future experiments should aim to carefully dissect whether the requirement for PABP involves viral translation and/or targets RdRp activity. The use of in vitro translation systems and RdRp assays may therefore reveal more precisely the mechanistic function of PABP in the potyvirus life cycle.
|
ACKNOWLEDGEMENTS |
|---|
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
Alvarez, E., Castella, A., Menandez-Arias, L. & Carrasco, L. (2006). HIV protease cleaves poly(A)-binding protein. Biochem J 396, 219–226.[CrossRef][Medline]
Amrani, N., Minet, M., Le Gouar, M., Lacroute, F. & Wyers, F. (1997). Yeast Pab1 interacts with Rna15 and participates in the control of the poly(A) tail length in vitro. Mol Cell Biol 17, 3694–3701.[Abstract]
Beauchemin, C. & Laliberté, J.-F. (2007). The poly(A) binding protein is internalized in virus-induced vesicles or redistributed to the nucleolus during Turnip mosaic virus infection. J Virol 81, 10905–10913.
Beauchemin, C., Boutet, N. & Laliberté, J.-F. (2007). Visualization of the interaction between the precursors of VPg, the viral protein linked to the genome of Turnip mosaic virus, and the translation eukaryotic initiation factor iso 4E in planta. J Virol 81, 775–782.
Behm-Ansmant, I., Gatfield, D., Rehwinkel, J., Hilgers, V. & Izaurralde, E. (2007). A conserved role for cytoplasmic poly(A)-binding protein 1 (PABPC1) in nonsense-mediated mRNA decay. EMBO J 26, 1591–1601.[CrossRef][Medline]
Belostotsky, D. A. (2003). Unexpected complexity of poly(A)-binding protein gene families in flowering plants: three conserved lineages that are at least 200 million years old and possible auto- and cross-regulation. Genetics 163, 311–319.
Bradrick, S. S., Dobrikova, E. Y., Kaiser, C., Shveygert, M. & Gromeier, M. (2007). Poly(A)-binding protein is differentially required for translation mediated by viral internal ribosome entry sites. RNA 13, 1582–1593.
Brown, C. E. & Sachs, A. B. (1998). Poly(A) tail length control in Saccharomyces cerevisiae occurs by message-specific deadenylation. Mol Cell Biol 18, 6548–6559.
Brune, C., Munchel, S. E., Fischer, N., Podtelejnikov, A. V. & Weis, K. (2005). Yeast poly(A)-binding protein Pab1 shuttles between the nucleus and the cytoplasm and functions in mRNA export. RNA 11, 517–531.
Charron, C., Nicolai, M., Gallois, J.-L., Robaglia, C., Moury, B., Palloix, A. & Caranta, C. (2008). Natural variation and functional analyses provide evidence for co-evolution between plant eIF4E and potyviral VPg. Plant J 54, 56–68.[CrossRef][Medline]
Decroocq, V., Sicard, O., Alamillo, J., Lansac, M., Eyquard, J., García, J., Candresse, T., Le Gall, O. & Revers, F. (2006). Multiple resistance traits control Plum pox virus infection in Arabidopsis thaliana. Mol Plant Microbe Interact 19, 541–549.[CrossRef][Medline]
Dufresne, P. J., Thivierge, K., Cotton, S., Beauchemin, C., Ide, C., Ubalijoro, E., Laliberté, J.-F. & Fortin, M. G. (2008). Heat shock 70 protein interaction with Turnip mosaic virus RNA-dependent RNA polymerase within virus-induced membrane vesicles. Virology 374, 217–227.[Medline]
Duprat, A., Caranta, C., Revers, F., Menand, B., Browning, K. S. & Robaglia, C. (2002). The Arabidopsis eukaryotic initiation factor (iso)4E is dispensable for plant growth but required for susceptibility to potyviruses. Plant J 32, 927–934.[CrossRef][Medline]
Fauquet, C. M., Mayo, M. A., Maniloff, J., Desselberger, U. & Ball, L. A. (editors) (2005). Virus Taxonomy: VIIIth Report of the International Committee on Taxonomy of Viruses. San Diego: Elsevier Academic Press.
Gallie, D. R. (2001). Cap-independent translation conferred by the 5' leader of Tobacco etch virus is eukaryotic initiation factor 4G dependent. J Virol 75, 12141–12152.
Gallie, D. R., Tanguay, R. L. & Leathers, V. (1995). The tobacco etch viral 5' leader and poly(A) tail are functionally synergistic regulators of translation. Gene 165, 233–238.[CrossRef][Medline]
Gao, Z., Johansen, E., Eyers, S., Thomas, C. L., Noel Ellis, T. H. & Maule, A. J. (2004). The potyvirus recessive resistance gene, sbm1, identifies a novel role for translation initiation factor eIF4E in cell-to-cell trafficking. Plant J 40, 376–385.[CrossRef][Medline]
Herold, J. & Andino, R. (2001). Poliovirus RNA replication requires genome circularization through a protein-protein bridge. Mol Cell 7, 581–591.[CrossRef][Medline]
Joachims, M., Van Breugel, P. C. & Lloyd, R. E. (1999). Cleavage of poly(A)-binding protein by enterovirus proteases concurrent with inhibition of translation in vitro. J Virol 73, 718–727.
Kang, B.-C., Yeam, I., Frantz, J. D., Murphy, J. F. & Jahn, M. M. (2005). The pvr1 locus in capsicum encodes a translation initiation factor eIF4E that interacts with Tobacco etch virus VPg. Plant J 42, 392–405.[CrossRef][Medline]
Kerekatte, V., Keiper, B. D., Badorff, C., Cai, A., Knowlton, K. U. & Rhoads, R. E. (1999). Cleavage of poly(A)-binding protein by coxsackievirus 2A protease in vitro and in vivo: another mechanism for host protein synthesis shutoff? J Virol 73, 709–717.
Khaleghpour, K., Svitkin, Y. V., Craig, A. W., DeMaria, C. T., Deo, R. C., Burley, S. K. & Sonenberg, N. (2001). Translational repression by a novel partner of human poly(A)-binding protein, Paip2. Mol Cell 7, 205–216.[CrossRef][Medline]
Khan, M. A., Miyoshi, H., Ray, S., Natsuaki, T., Suehiro, N. & Goss, D. J. (2006). Interaction of genome-linked protein (VPg) of Turnip mosaic virus with wheat germ translation initiation factors eIFiso4E and eIFiso4F. J Biol Chem 281, 28002–28010.
Kuyumcu-Martinez, N. M., Joachims, M. & Lloyd, R. E. (2002). Efficient cleavage of ribosome-associated poly(A)-binding protein by enterovirus 3C protease. J Virol 76, 2062–2074.
Kuyumcu-Martinez, M., Belliot, G., Sosnovtsev, S. V., Chang, K.-O., Green, K. Y. & Lloyd, R. E. (2004a). Calicivirus 3C-like proteinase inhibits cellular translation by cleavage of poly(A)-binding protein. J Virol 78, 8172–8182.
Kuyumcu-Martinez, N. M., Van Eden, M. E., Younan, P. & Lloyd, R. E. (2004b). Cleavage of poly(A)-binding protein by poliovirus 3C protease inhibits host cell translation: a novel mechanism for host translation shutoff. Mol Cell Biol 24, 1779–1790.
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685.[CrossRef][Medline]
Lellis, A. D., Kasschau, K. D., Whitham, S. A. & Carrington, J. C. (2002). Loss-of-susceptibility mutants of Arabidopsis thaliana reveal an essential role for eIF(iso)4E during potyvirus infection. Curr Biol 12, 1046–1051.[CrossRef][Medline]
Léonard, S., Plante, D., Wittmann, S., Daigneault, N., Fortin, M. G. & Laliberté, J.-F. (2000). Complex formation between potyvirus VPg and translation eukaryotic initiation factor 4E correlates with virus infectivity. J Virol 74, 7730–7737.
Léonard, S., Viel, C., Beauchemin, C., Daigneault, N., Fortin, M. G. & Laliberté, J.-F. (2004). Interaction of VPg-Pro of Turnip mosaic virus with the translation initiation factor 4E and the poly(A)-binding protein in planta. J Gen Virol 85, 1055–1063.
Mangus, D. A., Evans, M. C. & Jacobson, A. (2003). Poly(A)-binding proteins: multifunctional scaffolds for the post-transcriptional control of gene expression. Genome Biol 4, 223[CrossRef][Medline]
Menard, R., Chatel, H., Dupras, R., Plouffe, C. & Laliberté, J. F. (1995). Purification of Turnip mosaic potyvirus viral protein genome-linked proteinase expressed in Escherichia coli and development of a quantitative assay for proteolytic activity. Eur J Biochem 229, 107–112.[Medline]
Michel, Y. M., Poncet, D., Piron, M., Kean, K. M. & Borman, A. M. (2000). Cap-poly(A) synergy in mammalian cell-free extracts. Investigation of the requirements for poly(A)-mediated stimulation of translation initiation. J Biol Chem 275, 32268–32276.
Montero, H., Arias, C. F. & Lopez, S. (2006). Rotavirus nonstructural protein NSP3 is not required for viral protein synthesis. J Virol 80, 9031–9038.
Nicaise, V., German-Retana, S., Sanjuan, R., Dubrana, M.-P., Mazier, M., Maisonneuve, B., Candresse, T., Caranta, C. & LeGall, O. (2003). The eukaryotic translation initiation factor 4E controls lettuce susceptibility to the potyvirus Lettuce mosaic virus. Plant Physiol 132, 1272–1282.
Nicaise, V., Gallois, J.-L., Chafiai, F., Allen, L. M., Schurdi-Levraud, V., Browning, K. S., Candresse, T., Caranta, C., Le Gall, O. & German-Retana, S. (2007). Coordinated and selective recruitment of eIF4E and eIF4G factors for potyvirus infection in Arabidopsis thaliana. FEBS Lett 581, 1041–1046.[CrossRef][Medline]
Nicolas, O. & Laliberté, J. F. (1992). The complete nucleotide sequence of Turnip mosaic potyvirus RNA. J Gen Virol 73, 2785–2793.
Parker, R. & Song, H. (2004). The enzymes and control of eukaryotic mRNA turnover. Nat Struct Mol Biol 11, 121–127.[CrossRef][Medline]
Pfaffl, M. W. (2001). A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29, e45
Pfaffl, M. W., Horgan, G. W. & Dempfle, L. (2002). Relative expression software tool (REST) for group-wise comparison and statistical analysis of relative expression results in real-time PCR. Nucleic Acids Res 30, e36
Piron, M., Vende, P., Cohen, J. & Poncet, D. (1998). Rotavirus RNA-binding protein NSP3 interacts with eIF4GI and evicts the poly(A) binding protein from eIF4F. EMBO J 17, 5811–5821.[CrossRef][Medline]
Robaglia, C. & Caranta, C. (2006). Translation initiation factors: a weak link in plant RNA virus infection. Trends Plant Sci 11, 40–45.[CrossRef][Medline]
Rodriguez Pulido, M., Serrano, P., Saiz, M. & Martinez-Salas, E. (2007). Foot-and-mouth disease virus infection induces proteolytic cleavage of PTB, eIF3a,b, and PABP RNA-binding proteins. Virology 364, 466–474.[CrossRef][Medline]
Ruffel, S., Dussault, M.-H., Palloix, A., Moury, B., Bendahmane, A., Robaglia, C. & Caranta, C. (2002). A natural recessive resistance gene against Potato virus Y in pepper corresponds to the eukaryotic initiation factor 4E (eIF4E). Plant J 32, 1067–1075.[CrossRef][Medline]
Ruffel, S., Gallois, J., Lesage, M. & Caranta, C. (2005). The recessive potyvirus resistance gene pot-1 is the tomato orthologue of the pepper pvr2-eIF4E gene. Mol Genet Genomics 274, 346–353.[CrossRef][Medline]
Ruffel, S., Gallois, J.-L., Moury, B., Robaglia, C., Palloix, A. & Caranta, C. (2006). Simultaneous mutations in translation initiation factors eIF4E and eIF(iso)4E are required to prevent Pepper veinal mottle virus infection of pepper. J Gen Virol 87, 2089–2098.
Sachs, A. (2000). Physical and functional interactions between the mRNA cap structure and the poly(A) tail. In Translational Control of Gene Expression, pp. 447–465. Edited by N. Sonenberg, J. W. B. Hershey & M. B. Mathews. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Sato, M., Nakahara, K., Yoshii, M., Ishikawa, M. & Uyeda, I. (2005). Selective involvement of members of the eukaryotic initiation factor 4E family in the infection of Arabidopsis thaliana by potyviruses. FEBS Lett 579, 1167–1171.[CrossRef][Medline]
Schaad, M. C., Jensen, P. E. & Carrington, J. C. (1997). Formation of plant RNA virus replication complexes on membranes: role of an endoplasmic reticulum-targeted viral protein. EMBO J 16, 4049–4059.[CrossRef][Medline]
Schaad, M. C., Anderberg, R. J. & Carrington, J. C. (2000). Strain-specific interaction of the Tobacco etch virus NIa protein with the translation initiation factor eIF4E in the yeast two-hybrid system. Virology 273, 300–306.[CrossRef][Medline]
Svitkin, Y. V., Imataka, H., Khaleghpour, K., Kahvejian, A., Liebig, H. D. & Sonenberg, N. (2001). Poly(A)-binding protein interaction with elF4G stimulates picornavirus IRES-dependent translation. RNA 7, 1743–1752.[Abstract]
Thivierge, K., Nicaise, V., Dufresne, P. J., Cotton, S., Laliberté, J. F., Le Gall, O. & Fortin, M. G. (2005). Plant virus RNAs. Coordinated recruitment of conserved host functions by (+) ssRNA viruses during early infection events. Plant Physiol 138, 1822–1827.
Wang, X., Ullah, Z. & Grumet, R. (2000). Interaction between Zucchini yellow mosaic potyvirus RNA-dependent RNA polymerase and host poly(A)-binding protein. Virology 275, 433–443.[CrossRef][Medline]
Wells, S. E., Hillner, P. E., Vale, R. D. & Sachs, A. B. (1998). Circularization of mRNA by eukaryotic translation initiation factors. Mol Cell 2, 135–140.[CrossRef][Medline]
Wittmann, S., Chatel, H., Fortin, M. G. & Laliberte, J. F. (1997). Interaction of the viral protein genome linked of Turnip mosaic potyvirus with the translational eukaryotic initiation factor (iso) 4E of Arabidopsis thaliana using the yeast two-hybrid system. Virology 234, 84–92.[CrossRef][Medline]
Yamada, K., Lim, J., Dale, J. M., Chen, H., Shinn, P., Palm, C. J., Southwick, A. M., Wu, H. C., Kim, C. & other authors (2003). Empirical analysis of transcriptional activity in the Arabidopsis genome. Science 302, 842–846.
Yang, C., Guo, R., Jie, F., Nettleton, D., Peng, J., Carr, T., Yeakley, J. M., Fan, J.-B. & Whitham, S. A. (2007). Spatial analysis of Arabidopsis thaliana gene expression in response to Turnip mosaic virus infection. Mol Plant Microbe Interact 20, 358–370.[CrossRef][Medline]
Yeam, I., Cavatorta, J. R., Ripoll, D. R., Kang, B.-C. & Jahn, M. M. (2007). Functional dissection of naturally occurring amino acid substitutions in eIF4E that confers recessive potyvirus resistance in plants. Plant Cell 19, 2913–2928.
Zhang, B., Morace, G., Gauss-Muller, V. & Kusov, Y. (2007). Poly(A) binding protein, C-terminally truncated by the hepatitis A virus proteinase 3C, inhibits viral translation. Nucleic Acids Res 35, 5975–5984.
Received 17 March 2008;
accepted 13 May 2008.
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
| J MED MICROBIOL | ALL SGM JOURNALS | |
) or GST (
). Means±SEM are given for GST control data where the bars are larger than the symbols. (b, d) Interaction of TuMV VPg-Pro or RdRp polypeptide with different A. thaliana PABP proteins in ELISA-based binding assays. The wells of a microtitre plate were coated with 25 pmol purified VPg-Pro (b) or 6xhistidine-tagged RdRp (d) and saturated with 25 pmol purified GST-tagged PABP2, PABP4, PABP8 or GST. Retention of the complexes was detected with anti-GST antibodies. Columns with different symbols (*, 
, 
and 
) are significantly different according to Tukey's ANOVA test (P<0.05).
