Arabidopsis thaliana class II poly(A)-binding proteins are required for efficient multiplication of turnip mosaic virus.

The poly(A)-binding protein (PABP) is an important translation initiation factor that binds to the polyadenylated 3' end of mRNA. We have previously shown that PABP2 interacts with the RNA-dependent RNA polymerase (RdRp) and VPg-Pro of turnip mosaic virus (TuMV) within virus-induced vesicles. At least eight PABP isoforms are produced in Arabidopsis thaliana, three of which (PABP2, PABP4 and PABP8) are highly and broadly expressed and probably constitute the bulk of PABP required for cellular functions. Upon TuMV infection, an increase in protein and mRNA expression from PAB2, PAB4 and PAB8 genes was recorded. In vitro binding assays revealed that RdRp and the viral genome-linked protein (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). To assess whether PABP is required for potyvirus replication, A. thaliana single and double pab knockouts were isolated and inoculated with TuMV. All lines showed susceptibility to TuMV. However, when precise monitoring of viral RNA accumulation was performed, it was found to be reduced by 2.2- and 3.5-fold in pab2 pab4 and pab2 pab8 mutants, respectively, when compared with wild-type plants. PABP levels were most significantly reduced in the membrane-associated fraction in both of these mutants. TuMV mRNA levels thus correlated with cellular PABP concentrations in these A. thaliana knockout lines. These data provide further support for a role of PABP in potyvirus replication.

Eukaryotic elongation factor 1A interacts with Turnip mosaic virus RNA-dependent RNA polymerase and VPg-Pro in virus-induced vesicles.

Eukaryotic elongation factor 1-alpha (eEF1A) was identified as an interactor of Turnip mosaic virus (TuMV) RNA-dependent RNA polymerase (RdRp) and VPg-protease (VPg-Pro) using tandem affinity purification and/or in vitro assays. Subcellular fractionation experiments revealed that the level of eEF1A substantially increased in membrane fractions upon TuMV infection. Replication of TuMV occurs in cytoplasmic membrane vesicles, which are induced by 6K-VPg-Pro. Confocal microscopy indicated that eEF1A was included in these vesicles. To confirm that eEF1A was found in replication vesicles, we constructed an infectious recombinant TuMV that contains an additional copy of the 6K protein fused to the green fluorescent protein (GFP). In cells infected with this recombinant TuMV, fluorescence emitted by 6KGFP was associated with cytoplasmic membrane vesicles that contained VPg-Pro, the eukaryotic initiation factor (iso) 4E, the poly(A)-binding protein, the heat shock cognate 70-3 protein, and eEF1A. These results suggest that TuMV-induced membrane vesicles host at least three plant translation factors in addition to the viral replication proteins.

Heat shock 70 protein interaction with Turnip mosaic virus RNA-dependent RNA polymerase within virus-induced membrane vesicles.

Tandem affinity purification was used in Arabidopsis thaliana to identify cellular interactors of Turnip mosaic virus (TuMV) RNA-dependent RNA polymerase (RdRp). The heat shock cognate 70-3 (Hsc70-3) and poly(A)-binding (PABP) host proteins were recovered and shown to interact with the RdRp in vitro. As previously shown for PABP, Hsc70-3 was redistributed to nuclear and membranous fractions in infected plants and both RdRp interactors were co-immunoprecipitated from a membrane-enriched extract using RdRp-specific antibodies. Fluorescently tagged RdRp and Hsc70-3 localized to the cytoplasm and the nucleus when expressed alone or in combination in Nicotiana benthamiana. However, they were redistributed to large perinuclear ER-derived vesicles when co-expressed with the membrane binding 6K-VPg-Pro protein of TuMV. The association of Hsc70-3 with the RdRp could possibly take place in membrane-derived replication complexes. Thus, Hsc70-3 and PABP2 are potentially integral components of the replicase complex and could have important roles to play in the regulation of potyviral RdRp functions.

Hypocholesterolaemic effects of plant sterol analogues are independent of ABCG5 and ABCG8 transporter expressions in hamsters.

The hypolipidaemic effects of plant sterols are well established. However, mechanisms by which plant sterols lower plasma cholesterol levels, particularly at the molecular level, have not been clearly elucidated. The objective of the present study was to determine whether different plant sterol analogues reduce plasma cholesterol levels by up regulating the sterol transporters ABCG5 and ABCG8 in the liver and/or small intestine. Male Golden Syrian hamsters were divided into eight groups. Groups 1 and 2 were fed a maize starch-casein-sucrose-based diet that did not contain cholesterol (control; Con) or the Con diet with the addition of 0.25 % cholesterol (Ch-Con). Groups 3-8 were fed the Ch-Con diet supplemented with 1 % plant sterols, 1 % plant stanols, 1 % of a plant sterol and stanol mixture (50:50), 1.76 % plant sterol-fish oil esters, or 0.71 or 1.43 % stanol-ascorbic acid esters, respectively. After 5 weeks, the Ch-Con diet up regulated the ABCG5 mRNA expression and tended (P = 0.083) to increase ABCG8 mRNA expression in the liver, but did not affect both genes' expression in the small intestine compared with the Con diet. Hamsters fed 0.7 % stanol esters showed lower plasma cholesterol levels (P < 0.001) and also lower liver ABCG5 mRNA expression (P < 0.05) compared with the Ch-Con diet. Plant stanols, stanol esters, and sterol esters did not affect the ABCG5 or ABCG8 mRNA expressions in the liver and intestine although they reduced plasma cholesterol levels. These results suggest that plant sterols and their derivatives reduce plasma cholesterol levels independently from the mRNA expression of ABCG5 and ABCG8 transporters.

The VPgPro protein of Turnip mosaic virus: in vitro inhibition of translation from a ribonuclease activity.

A role for viral encoded genome-linked (VPg) proteins in translation has often been suggested because of their covalent attachment to the 5' end of the viral RNA, reminiscent of the cap structure normally present on most eukaryotic mRNAs. We tested the effect of Turnip mosaic virus (TuMV) VPgPro on translation of reporter RNAs in in vitro translation systems. The presence of VPgPro in either wheat germ extract or rabbit reticulocyte lysate systems lead to inhibition of translation. The inhibition did not appear to be mediated by the interaction of VPg with the eIF(iso)4E translation initiation factor since a VPg mutant that does not interact with eIF(iso)4E still inhibited translation. Monitoring the fate of RNAs revealed that they were degraded as a result of addition of TuMV VPgPro or of Norwalk virus (NV) VPg protein. The RNA degradation was not the result of translation being arrested and was heat labile and partially EDTA sensitive. The capacity of TuMV VPgPro and of (NV) VPg to degrade RNA suggests that these proteins have a ribonucleolytic activity which may contribute to the host RNA translation shutoff associated with many virus infections.

Plant virus RNAs. Coordinated recruitment of conserved host functions by (+) ssRNA viruses during early infection events.

Positive-sense single-stranded RNA viruses have developed strategies to exploit cellular resources at the expense of host mRNAs. The genomes of these viruses display a variety of structures at their 5' and 3' ends that differentiate them from cellular mRNAs. Despite this structural diversity, viral RNAs are still circularized by juxtaposition of their 5' and 3' ends, similar to the process used by cellular mRNAs. Also reminiscent of the mechanisms used by host mRNAs, translation of viral RNAs involves the recruitment of translation initiation factors. However, the roles played by these factors likely differ from those played by cellular mRNAs. In keeping with the general parsimony typical of RNA viruses, these host factors also participate in viral RNA replication. However, the dual use of host factors requires that viral RNA template utilization be regulated to avoid conflict between replication and translation. The molecular composition of the large ribonucleoprotein complexes that form the viral RNA replication and translation machineries likely evolves over the course of infection to allow for switching template use from translation to replication.

Interaction of VPg-Pro of turnip mosaic virus with the translation initiation factor 4E and the poly(A)-binding protein in planta.

The viral protein linked to the genome (VPg) of Turnip mosaic virus (TuMV) interacts in vitro with the translation eukaryotic initiation factor (eIF) 4E. In the present study, we investigated the consequence of TuMV infection on eIF4E expression. Two isomers are present in plants, namely eIF4E and eIF(iso)4E. Expression of the latter was detected in both TuMV-infected and mock-inoculated Brassica perviridis plants, but expression of eIF4E was found only in infected plants. Membranes from TuMV-infected or mock-inoculated tissues were separated by sucrose gradient centrifugation and fractions were collected. Immunoblot analyses showed that 6K(2)-VPg-Pro/VPg-Pro polyproteins were associated with endoplasmic reticulum membranes and were the viral forms likely to interact with eIF(iso)4E and eIF4E. In planta interaction between 6K(2)-VPg-Pro/VPg-Pro and eIF(iso)4E/eIF4E was confirmed by co-purification by metal chelation chromatography. The poly(A)-binding protein (PABP) was also found to co-purify with VPg-Pro. Direct interaction between VPg-Pro and PABP was shown by an ELISA-based binding assay. These experiments suggest that a multi-protein complex may form around VPg-Pro of TuMV.