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

fWe report the solution structures of the VPg proteins from feline calicivirus (FCV) and murine norovirus (MNV), which have been determined by nuclear magnetic resonance spectroscopy. In both cases, the core of the protein adopts a compact helical structure flanked by flexible N and C termini. Remarkably, while the core of FCV VPg contains a well-defined three-helix bundle, the MNV VPg core has just the first two of these secondary structure elements. In both cases, the VPg cores are stabilized by networks of hydrophobic and salt bridge interactions. The Tyr residue in VPg that is nucleotidylated by the viral NS7 polymerase (Y24 in FCV, Y26 in MNV) occurs in a conserved position within the first helix of the core. Intriguingly, given its structure, VPg would appear to be unable to bind to the viral polymerase so as to place this Tyr in the active site without a major conformation change to VPg or the polymerase. However, mutations that destabilized the VPg core either had no effect on or reduced both the ability of the protein to be nucleotidylated and virus infectivity and did not reveal a clear structure-activity relationship. The precise role of the calicivirus VPg core in virus replication remains to be determined, but knowledge of its structure will facilitate future investigations.

supporting

confidence: 65%

“…In FCV VPg, the helix ␣2 sequence 40 VEDFLMLRHRA AL 52 is replaced in the MNV protein by 42 IDDYLADREREEELL 56 ( Fig. 3A), which contains many more charged residues.…”

Section: Discussionmentioning

confidence: 99%

Structures of the Compact Helical Core Domains of Feline Calicivirus and Murine Norovirus VPg Proteins

Leen

Kwok

Birtley

et al. 2013

“…A yeast two-hybrid screen has shown that eIF(iso)4E of Arabidopsis thaliana interacts with the VPg of Turnip mosaic virus (TuMV) (57). This interaction (31) has also been reported to take place in infected cells (32). Similar interactions are taking place between the VPg of other potyviruses and either eIF(iso)4E or eIF4E (20,46).…”

mentioning

confidence: 61%

Visualization of the Interaction between the Precursors of VPg, the Viral Protein Linked to the Genome ofTurnip Mosaic Virus, and the Translation Eukaryotic Initiation Factor iso 4E In Planta

Beauchemin

Boutet

Laliberté

2007

Self Cite

150

116

The RNA genome of Turnip mosaic virus is covalently linked at its 5 end to a viral protein known as VPg. This protein binds to the translation eukaryotic initiation factor iso 4E [eIF(iso)4E]. This interaction has been shown to be important for virus infection, although its exact biological function(s) has not been elucidated. In this study, we investigated the subcellular site of the VPg-eIF(iso)4E interaction using bimolecular fluorescence complementation (BiFC). As a first step, eIF(iso)4E, 6K-VPg-Pro, and VPg-Pro were expressed as full-length green fluorescent protein (GFP) fusions in Nicotiana benthamiana, and their subcellular localizations were visualized by confocal microscopy. eIF(iso)4E was predominantly associated with the endoplasmic reticulum (ER), and VPg-Pro was observed in the nucleus and possibly the nucleolus, while 6K-VPg-Pro-GFP induced the formation of cytoplasmic vesicles budding from the ER. In BiFC experiments, reconstituted green fluorescence was observed throughout the nucleus, with a preferential accumulation in subnuclear structures when the GFP split fragments were fused to VPg-Pro and eIF(iso)4E. On the other hand, the interaction of 6K-VPg-Pro with eIF(iso)4E was observed in cytoplasmic vesicles embedded in the ER. These data suggest that the association of VPg with the translation factor might be needed for two different functions, depending of the VPg precursor involved in the interaction. VPg-Pro interaction with eIF(iso)4E may be involved in perturbing normal cellular functions, while 6K-VPg-Pro interaction with the translation factor may be needed for viral RNA translation and/or replication.One of the initial steps in protein synthesis is the recruitment of mRNAs by the translation eukaryotic initiation factor 4F (eIF4F) complex (17). In plants, this complex is composed of two proteins, eIF4E and eIF4G (4). eIF4E is the cap-binding protein, while eIF4G is a large polypeptide containing binding sites for eIF4E, eIF4A, eIF3, and the poly(A) binding protein. eIF4F simultaneously interacts with the cap structure of mRNA (through eIF4E) and the ribosome-associated eIF3 (through eIF4G). Thus, eIF4F executes the pivotal function of bridging mRNAs with ribosomes. Another cap-binding complex, eIF(iso)4F, has been identified in plants (5) and is composed of a smaller eIF(iso)4E subunit and a larger eIF(iso)4G subunit relative to the eIF4F subunits. Those two complexes perform essentially the same task in translation but have different affinities for certain classes of mRNA substrates and may be involved in different cellular events (14).Potyviruses have a positive-strand RNA genome of approximately 10 kb (53). The genome codes for a single long polyprotein that is processed by viral proteinases into 10 mature proteins. The viral RNA is polyadenylated at its 3Ј end and has a covalently linked virus-encoded protein (VPg) instead of a cap structure at the 5Ј end. Being derived from a polyprotein, VPg exists in various forms (i.e., VPg, VPg-Pro, 6K-VPg-Pro) that fulfill specific functions....

“…Higher RNA and VPg levels of replicating viruses may require more P0 and eIF4E/eIF(iso)4E, making their translation more sensitive to silencing of these proteins. P0 transcription is induced early in PVA infection (66), and TuMV infection upregulates eIF4E (67), suggesting that the demand for these proteins is increased during natural potyvirus infection. This was supported by results showing that the silencing of both of these host factors reduced PVA infection similarly (Fig.…”

Section: Fig 10mentioning

confidence: 99%

Ribosomal Protein P0 Promotes Potato Virus A Infection and Functions in Viral Translation Together with VPg and eIF(iso)4E

Hafrén

Eskelin

Mäkinen

2013

We report here that the acidic ribosomal protein P0 is a component of the membrane-associated Potato virus A (PVA) ribonucleoprotein complex. As a constituent of the ribosomal stalk, P0 functions in translation. Although the ribosomal stalk proteins P0, P1, P2, and P3 are all important for PVA infection, P0 appears to have a distinct role from those of the other stalk proteins in infection. Our results indicate that P0 also regulates viral RNA functions as an extraribosomal protein. We reported previously that PVA RNA can be targeted by VPg to a specific gene expression pathway that protects the viral RNA from degradation and facilitates its translation. Here, we show that P0 is essential for this activity of VPg, similar to eIF4E/eIF(iso)4E. We also demonstrate that VPg, P0, and eIF(iso)4E synergistically enhance viral translation. Interestingly, the positive effects of VPg and P0 on viral translation were negatively correlated with the cell-to-cell spread of infection, suggesting that these processes may compete for viral RNA.