Helper component proteinase of the genus Potyvirus is an interaction partner of translation initiation factors eIF(iso)4E and eIF4E and contains a 4E binding motif

Abstract

The multifunctional helper component proteinase (HCpro) of potyviruses (genus Potyvirus; Potyviridae) shows self-interaction and interacts with other potyviral and host plant proteins. Host proteins that are pivotal to potyvirus infection include the eukaryotic translation initiation factor eIF4E and the isoform eIF(iso)4E, which interact with viral genome-linked protein (VPg). Here we show that HCpro of Potato virus A (PVA) interacts with both eIF4E and eIF(iso)4E, with interactions with eIF(iso)4E being stronger, as judged by the data of a yeast two-hybrid system assay. A bimolecular fluorescence complementation assay on leaves of Nicotiana benthamiana showed that HCpro from three potyviruses (PVA, Potato virus Y, and Tobacco etch virus) interacted with the eIF(iso)4E and eIF4E of tobacco (Nicotiana tabacum); interactions with eIF(iso)4E and eIF4E of potato (Solanum tuberosum) were weaker. In PVA-infected cells, interactions between HCpro and tobacco eIF(iso)4E were confined to round structures that colocalized with 6K2-induced vesicles. Point mutations introduced to a 4E binding motif identified in the C-terminal region of HCpro debilitated interactions of HCpro with translation initiation factors and were detrimental to the virulence of PVA in plants. The 4E binding motif conserved in HCpro of potyviruses and HCpro-initiation factor interactions suggest new roles for HCpro and/or translation factors in the potyvirus infection cycle.

Fig. 1.

Alignment of amino acid sequences of the translation initiation factors. (A) Alignment of the sequence variants (a and b) of eIF(iso)4E from tobacco (Tiso4E) and potato (Piso4E). (B) Alignment of the sequence variants (a and b) of eIF4E from tobacco (T4E) and potato (P4E). Identical amino acids are indicated by dots, and deletions are indicated by hyphens.

Fig. 2.

Interactions between HCpro and the translation initiation factors of tobacco and potato using the GAL4 YTHS. Two variants (a and b) of the translation initiation factors eIF(iso)4E and eIF4E (4E) from tobacco (Tiso4Ea and Tiso4Eb; T3Ea and T4Eb) and potato (Piso4Ea and Piso4Eb; P3Ea and P4Eb) were tested for interactions with the HCpro of PVA. HCpro and initiation factors were fused to the AD or BD and were expressed following cotransformation of yeast cells with the respective vector plasmids. (A) Growth of yeast (Saccharomyces cerevisiae AH109) 10 days after plating on stringent selective medium lacking adenine, histidine, leucine, and tryptophan indicates an interaction between the tested proteins. T4Ea and T4Eb autoactivated the reporter genes when fused to the BD vector and were not included in the assay. Western blot analysis with monoclonal antibodies specific to the AD (B) or the BD (C) was used to detect the expressed proteins. Coomassie blue-stained SDS-PAGE gel served as a protein loading control.

Fig. 3.

Detection of protein-protein interactions in leaves of N. benthamiana using BiFC. HCpro proteins of three potyviruses, PVA, PVY, and TEV, were expressed with YN fused to the HCpro N terminus. Translation initiation factors eIF(iso)4E (Tiso4Eb and Piso4Eb) and eIF4E (T4Ea and P4Eb) of tobacco (T) and potato (P) were expressed with YC fused to the N or C terminus. Leaves were infiltrated with pairs of Agrobacterium strains expressing tester proteins tagged with the opposite halves of YFP (the corresponding bacterial cultures were mixed in a 1:1 ratio for infiltration). YFP fluorescence indicating protein-protein interactions was detected in epidermal cells using an epifluorescence microscope 3 days postinfiltration. Images were taken using the same exposure time. For detection of the expressed recombinant proteins using protein-specific antibodies, see Fig. S1 in the supplemental material.

Fig. 4.

Detection of YFP fluorescence resulting from interaction between PVA HCpro and Tiso4Eb, and its association with 6K2-induced viral vesicles in N. benthamiana. (A) YN-HCpro was expressed from an engineered infectious clone of PVA during the course of virus infection, and Tiso4Eb-YC was coexpressed from a binary vector under 35S promoter. The fluorescence resulting from the interaction between HCpro and Tiso4Eb fluorescence was partly confined to round structures or bodies. Scale bars represent 10 μm. (B) Schematic presentations of the constructs used for protein coexpression. The mature viral proteins: P1, the first protein; HC-Pro; P3, third protein; 6K1 and 6K2, 6-kDa proteins; CI, cylindrical inclusion protein; VPg; NIaPro, the main viral proteinase; NIb, replicase; and CP, coat protein. AA indicates the poly(A) tail. The 5′ and 3′ untranslated regions are not depicted. (Ci to iii) Localization of interacting HCpro and Tiso4E with 6K2-induced viral vesicles. Arrows highlight fluorescence signals and colocalization. Confocal images are single optical sections selected from z-series for viewing with LAS AF Lite.

Fig. 5.

The putative 4E binding protein (4E-BP) motif of HCpro. (A) The amino acids 345 and 351 of PVA HCpro correspond to the conserved residues in the 4E-BP consensus motif YXXXXLΦ, where X is a variable amino acid and Φ is a hydrophobic residue, and were substituted for alanine (underlined) in the PVA HCpro [HC4Emut (MUT)]. (B) Interactions of the wild-type (WT) HCpro of PVA and HC4Emut (MUT) with the eIF(iso)4E of tobacco (Nicotiana tabacum) (Tiso4Eb) illustrated by the β-galactosidase enzyme activity in cotransformed yeast (96-well microtiter plate format) following overnight incubation. The results from three technical replicates of four independent yeast colonies are shown. (+) and (-), the positive and negative control provided by Clontech, respectively. (C) Mean relative β-galactosidase activities corresponding to the samples shown in panel B and quantified by Image J (National Institutes of Health). Standard deviation is indicted with error bars.