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. 2000 Sep;74(17):7730-7.
doi: 10.1128/jvi.74.17.7730-7737.2000.

Complex formation between potyvirus VPg and translation eukaryotic initiation factor 4E correlates with virus infectivity

S Léonard  1 , D Plante, S Wittmann, N Daigneault, M G Fortin, J F Laliberté
Affiliations

Complex formation between potyvirus VPg and translation eukaryotic initiation factor 4E correlates with virus infectivity

S Léonard et al. J Virol. 2000 Sep.

Abstract

The interaction between the viral protein linked to the genome (VPg) of turnip mosaic potyvirus (TuMV) and the translation eukaryotic initiation factor eIF(iso)4E of Arabidopsis thaliana has previously been reported. eIF(iso)4E binds the cap structure (m(7)GpppN, where N is any nucleotide) of mRNAs and has an important role in the regulation in the initiation of translation. In the present study, it was shown that not only did VPg bind eIF(iso)4E but it also interacted with the eIF4E isomer of A. thaliana as well as with eIF(iso)4E of Triticum aestivum (wheat). The interaction domain on VPg was mapped to a stretch of 35 amino acids, and substitution of an aspartic acid residue found within this region completely abolished the interaction. The cap analogue m(7)GTP, but not GTP, inhibited VPg-eIF(iso)4E complex formation, suggesting that VPg and cellular mRNAs compete for eIF(iso)4E binding. The biological significance of this interaction was investigated. Brassica perviridis plants were infected with a TuMV infectious cDNA (p35Tunos) and p35TuD77N, a mutant which contained the aspartic acid substitution in the VPg domain that abolished the interaction with eIF(iso)4E. After 20 days, plants bombarded with p35Tunos showed viral symptoms, while plants bombarded with p35TuD77N remained symptomless. These results suggest that VPg-eIF(iso)4E interaction is a critical element for virus production.

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Figures

FIG. 1
FIG. 1
VPg interaction with eIF4E isomers as demonstrated by ELISA-based binding assay. Wells precoated with 1.0 μg of VPgPro were incubated with 2.0 μg of eIF(iso)4E (lane 1) and eIF4E (lane 2) from A. thaliana, eIF(iso)4E from T. aestivum (lane 3), or no added initiation factor (lane 4). In lane 5, wells were coated with Blotto only and incubated with 2.0 μg of eIF(iso)4E from A. thaliana. Complexes were detected using anti-T7 tag antibodies. Values are averages of two replicates from a typical experiment.
FIG. 2
FIG. 2
VPgPro and VPgΔPro interaction with eIF(iso)4E of A. thaliana as demonstrated by ELISA-based binding assay. (A) Purification of VPgPro and VPgΔPro. Expression and purification were as described in Materials and Methods. Samples were loaded on a sodium dodecyl sulfate-polyacrylamide gel and stained with Coomassie blue. Lane 1, VPgPro (5 μg); lane 2, VPgΔPro (20 μg); lane M, molecular mass standards. (B) ELISA-based binding assay. Wells were coated with 1.0 μg of VPgPro (■) or 4.0 μg of VPgΔPro (●) and then incubated with increasing concentrations of eIF(iso)4E from A. thaliana. Complexes were detected using anti-T7 tag antibodies. Values are averages of two replicates from a typical experiment.
FIG. 3
FIG. 3
Amino acid sequence of the eIF(iso)4E-binding domain of VPg and comparison with corresponding region from other potyviruses. Amino acid sequences were aligned using BLAST software with the BLOSUM62 matrix provided on the NCBI World Wide Web server. The numbers for TuMV represent the first and last residue positions of VPg; for the other viruses, the numbers represent the first and last residue positions of the polyprotein. Dashes indicate amino acids identical to that of the TuMV VPg. PPV, plum pox potyvirus (accession number S47508); LMV, lettuce mosaic potyvirus (P89876); TVMV, tobacco vein mottling potyvirus (P09814); PVY, potato mosaic potyvirus (1906388); TEV, tobacco etch potyvirus (P04517); BCMV, bean common mosaic potyvirus (Q65399); PRSV, papaya ringspot potyvirus (Q01901); ZYMV, zucchini yellow mosaic potyvirus (Q89330).
FIG. 4
FIG. 4
Inhibition by m7GTP of VPg-eIF(iso)4E complex formation as determined by ELISA-based binding assay. (A) Wells were coated with 1.0 μg of VPgPro and incubated with 2.0 μg of eIF(iso)4E from A. thaliana with increasing concentration of m7GTP. Values are averages of two replicates from a typical experiment. (B) Lineweaver-Burk reciprocal representation of binding data. Wells were coated with 1 μg of VPgPro and incubated with increasing concentrations of eIF(iso)4E from A. thaliana in the absence (■) or presence, at 0.5 μM (●) or 1.0 μM (▴), of m7GTP. Values are averages of two replicates from a typical experiment. Solid lines present the best fit of the data to equation y = ax − bx2 + c.
FIG. 5
FIG. 5
Binding of VPg and m7GTP to eIF(iso)4E.
FIG. 6
FIG. 6
Immunoblot analysis of B. perviridis plants bombarded with TuMV plasmid cDNA. After bombardment, plants were placed in a growth chamber for 10 days. Proteins were extracted from the new leaf emerging above the one bombarded, separated on a sodium dodecyl sulfate-polyacrylamide gel, transferred on a nitrocellulose membrane, and incubated with a rabbit anti-TuMV capsid serum. Lane 1, plant bombarded with gold particles not coated with DNA; lanes 2 and 3, plants bombarded with p35Tunos; lanes 4 to 9, plants bombarded with p35TuD77N; lane M, molecular mass standards.
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