Detection of the potyviral genome-linked protein VPg in virions and its phosphorylation by host kinases

Abstract

The multifunctional genome-linked protein (VPg) of Potato virus A (PVA; genus Potyvirus) was found to be phosphorylated as a part of the virus particle by a cellular kinase activity from tobacco. Immunoprecipitation, immunolabeling, and immunoelectron microscopy experiments showed that VPg is exposed at one end of the virion and it is accessible to protein-protein interactions. Substitution Ser185Leu at the C-proximal part of VPg reduces accumulation of PVA in inoculated leaves of the wild potato species Solanum commersonii and delays systemic infection, which is not observed in tobacco plants. Our data show that kinases of S. commersonii differentially recognize the VPg containing Ser or Leu at position 185, whereas both forms of VPg are similarly recognized by tobacco kinases. Taken together, our data imply that the virion-bound VPg may interact with host proteins and that phosphorylation of VPg may play a role in the VPg-mediated functions during the infection cycle of potyviruses.

FIG. 1.

Phosphorylation of PVA VPg by plant protein kinase activity is stimulated by Mn²⁺. (A) Increasing concentrations of manganese were introduced into assays containing bacterially expressed PVA VPg, total protein kinase activity from leaves of N. tabacum, and [γ-³³P]ATP. Phosphoproteins were separated by SDS-PAGE and transferred to membranes, and their positions were identified by staining with Ponceau S. Radioactivity associated with phosphoproteins was compared by PhosphorImager densitometry and plotted against Mn²⁺ concentration. In a control experiment, manganese was removed from phosphorylation reaction in a 1:1 molar complex with EDTA. (B) Effect of Mn²⁺, Mg²⁺, or Ca²⁺ on phosphorylation of PVA VPg. Proteins were assayed for phosphorylation in a reconstituted system containing plant enzymes, [γ-³³P]ATP, and 10 mM Mn²⁺, Mg²⁺, or Ca²⁺. Proteins were subjected to SDS-PAGE and transferred to membranes. Autoradiograms of phosphorylated proteins are shown together with stained membranes.

FIG. 2.

PVA VPg is phosphorylated when packaged into virions. PVA particles were phosphorylated with a kinase activity from tobacco leaf extracts, and VPg-RNA complexes were isolated from PVA particles by the LiCl method and treated with RNase A. The virus-derived VPg (lane 1) and the recombinant VPg (lane 2) expressed in E. coli and used as a control in the phosphorylation experiment were subjected to SDS-PAGE and blotted onto a membrane, and phospohorylation was verified by autoradiography (upper panel). VPg was detected with anti-VPg antibodies to compare the blotted amounts of VPg (lower panel).

FIG. 3.

PVA particles can be immunoprecipitated with anti-VPg antibodies. (A) Immunoprecipitation of PVA particles from infected plant material. The protein extracts derived from PVA-infected and mock-inoculated tobacco plants were mixed with anti-VPg antiserum or anti-CP IgG. The antigen-antibody complexes were collected with protein A beads and washed with NET buffer (see Materials and Methods). The immunoprecipitated proteins were resolved by SDS-PAGE and visualized by Coomassie staining. Lanes: 1, extract from a mock-inoculated control plant immunoprecipitated with anti-VPg antiserum; 2, extract from a PVA-infected plant immunoprecipitated with anti-VPg antiserum; 3, extract from a mock-inoculated control plant immunoprecipitated with anti-CP IgG; 4, extract from a PVA-infected plant immunoprecipitated with anti-CP IgG. The position of CP is indicated by an arrowhead, and its molecular mass (32.5 kDa) is indicated to the left. (B) Immunoprecipitation of purified PVA particles and recombinant CP. Purified PVA particles were immunoprecipitated with anti-VPg antiserum. Bacterially expressed CP was used as a control to rule out possible nonspecific interaction of anti-VPg antibodies or Sepharose A with PVA CP. Anti-CI antiserum was used as an additional control. Immunoprecipitated proteins were resolved by SDS-PAGE, blotted to a nylon membrane, and detected with anti-CP IgG by the ECL enhanced chemiluminescence method. Lanes: 1, PVA virions immunoprecipitated with anti-VPg antiserum; 2, six-His-tagged CP immunoprecipitated with anti-VPg antiserum; 3, PVA virions immunoprecipitated with anti-CI antiserum; 4, six-His-tagged CP immunoprecipitated with anti-CI antiserum. The panel to the right represents a Coomassie-stained gel, showing that equal amounts of CP (lane1) and virions (lane2) were subjected to immunoprecipitation. The molecular mass of CP (32.5 kDa) is indicated to the left.

FIG. 4.

Detection of PVA VPg in virions by IEM. The complexes of anti-VPg antibodies and virion-associated VPg were visualized with gold-conjugated protein A. The size of the gold particles is 15 nm. (A) The complexes of gold-conjugated protein A and anti-VPg antibodies are detected at only one end of the virion (bar, 200 nm). (B) Magnified view of a labeled PVA particle. (C) Preimmune IgGs or gold-conjugated protein A does not bind directly to the virions (particles not treated with anti-VPg antibodies; bar, 500 nm). (D) Virus-like particles, which contain neither viral RNA nor VPg, are not recognized by anti-VPg antibodies (bar, 500 nm).

FIG. 5.

Phosphoamino acid composition of PVA VPg. An autoradiogram shows the results from phosphoamino acid analysis of ³³P-labeled VPg assayed by two-dimensional TLE. The positions of phosphoamino acid markers are indicated by circles. S, phosphoserine; T, phosphothreonine; Y, phosphotyrosine.

FIG. 6.

Similar phosphorylation patterns obtained for the VPgs of PVA strains B11 and B11-M by using kinase activity from tobacco. (A) Schematic map of the recombinant VPg molecules. The amino acid differences between VPgs B11^wt, M^wt, B11^Ser185Leu, and M^Leu185Ser and the positions of the changed amino acids in VPg are indicated. (His)₆, six-His tag. (B) Recombinant VPgs B11^wt, M^wt, B11^Ser185Leu, and M^Leu185Ser were phosphorylated in the presence of 5 mM Mn ²⁺ in vitro with a total protein extract of tobacco as the kinase source. Phosphorylated proteins were purified by SDS-PAGE, blotted onto nylon membrane, and visualized with Ponceau S. VPg was digested on the membrane with trypsin, and the released peptides were lyophilized and separated on TLE/TLC plates. Radioactive ³³P-labeled peptides were autoradiographically visualized.

FIG. 7.

Different phosphorylation patterns obtained for the VPgs of PVA strains B11 and B11-M by using kinase activity from S. commersonii. The recombinant VPg proteins and phosphorylation assay were those described in the legend to Fig. 6, except that the kinase source was the total protein extract derived from S. commersonii. The positions at which the differences in the phosphorylation patterns obtained with potato-derived kinase are most obvious are indicated by circles.