Triple gene block protein interactions involved in movement of Barley stripe mosaic virus

Abstract

Barley stripe mosaic virus (BSMV) encodes three movement proteins in an overlapping triple gene block (TGB), but little is known about the physical interactions of these proteins. We have characterized a ribonucleoprotein (RNP) complex consisting of the TGB1 protein and plus-sense BSMV RNAs from infected barley plants and have identified TGB1 complexes in planta and in vitro. Homologous TGB1 binding was disrupted by site-specific mutations in each of the first two N-terminal helicase motifs but not by mutations in two C-terminal helicase motifs. The TGB2 and TGB3 proteins were not detected in the RNP, but affinity chromatography and yeast two-hybrid experiments demonstrated that TGB1 binds to TGB3 and that TGB2 and TGB3 form heterologous interactions. These interactions required the TGB2 glycine 40 and the TGB3 isoleucine 108 residues, and BSMV mutants containing these amino acid substitution were unable to move from cell to cell. Infectivity experiments indicated that TGB1 separated on a different genomic RNA from TGB2 and TGB3 could function in limited cell-to-cell movement but that the rates of movement depended on the levels of expression of the proteins and the contexts in which they are expressed. Moreover, elevated expression of the wild-type TGB3 protein interfered with cell-to-cell movement but movement was not affected by the similar expression of a TGB3 mutant that fails to interact with TGB2. These experiments suggest that BSMV movement requires physical interactions of TGB2 and TGB3 and that substantial deviation from the TGB protein ratios expressed by the wild-type virus compromises movement.

FIG. 1.

Analysis of a nucleoprotein complex present in BSMV-infected plants. (A) BSMV RNAβ containing a deletion in the CP ORF and a His-tagged TGB1 protein. (B) Sedimentation of nucleoprotein complexes isolated from BSMV-infected plants expressing a TGB1_wt or TGB_His derivative. (C) Immunoblot assays with TGB1 antiserum to proteins present in fractions 13 to 20 collected from the major peaks of A₂₅₄. (D) Dot blot assay of BSMV RNAs in fractions 13 to 20 visualized with a ³²P-labeled riboprobe complementary to the conserved 238-nt 3′ termini of the BSMV gRNA and sgRNA.

FIG. 2.

Composition and stability of TGB1_wt and TGB1_His complexes recovered after metal affinity chromatography of pooled sucrose density gradient fractions 15 to 18. (A) Immunoblot analyses of metal affinity chromatography fractions with TGB1 antiserum. (B) Dot blot analyses of BSMV RNAs present in metal affinity chromatography fractions with a ³²P-labeled riboprobe complementary to the conserved 238-nt 3′ termini of the BSMV gRNA and sgRNA. (C) Northern blot analyses of RNAs recovered after metal affinity chromatography with ³²P riboprobes complementary to plus- or minus-sense BSMV RNA 3′ termini. (D) Polyacrylamide gel analyses of proteins recovered after metal affinity chromatography by silver staining and Western blotting with TGB1 antibody. (E and F) Western blot assays with TGB1 antisera of BSMV nucleoprotein complexes after sucrose density gradient fractionation in the presence of 100 or 500 mM NaCl (E) or in the presence or absence of 50 mM EGTA (F). Note that in panel C, the designations α, β, and γ to the left of the panel refer to the electrophoresis of BSMV RNA.

FIG. 3.

TGB1 interactions present in Ni(II)/GGH-cross-linked extracts from BSMV-infected plants. Extracts recovered at 7 dpi were treated as follows: lane 1, no cross-linking; lane 2, cross-linking with Ni(II)/GGH; lane 3, treatment with Ni(II) in the absence of GGH. Treated samples were heated in the presence of SDS and 8 M urea, separated on polyacrylamide gels, blotted to nitrocellulose, and visualized with TGB1 antisera. Ab, antibody. The values on the left are molecular sizes in kilodaltons.

FIG. 4.

Glutathione affinity chromatography detection of TGB protein interactions. TGB proteins were expressed in yeast cells, and protein extracts were separated by SDS-PAGE before (panels A and C, crude extracts) and after (panels B and D, column eluates) glutathione affinity chromatography. Proteins were reacted with anti-TGB1 and visualized with a goat anti-mouse secondary antibody (Ab) linked to horseradish peroxidase or with an anti-GST or anti-poly-His primary antibody reacted with a rabbit secondary antibody. (A and B) Proteins were coexpressed in the following combinations: lane 1, GST and TGB1; lane 2, GST-TGB1 and TGB1; lane 3, GST and TGB1; lane 4, GST-TGB3 and TGB1; lane 5, GST and His-TGB2; lane 6, GST-TGB1 and HisTGB2; lane 7, GST-TGB3 and His-TGB2. (C and D) GST-TGB1 was coexpressed in yeast with WT TGB1 or one of the mutant TGB1 proteins containing substitutions within conserved motif 1, 2, 4, or 6 of the helicase domain. Crude extracts (C) were subjected to glutathione affinity chromatography (D) to determine whether the mutant helicases were competent to interact with GST-TGB1. Note that the crude extract panels represent ∼12% of the total protein extracted from yeast and ∼80% of the total protein extracted was applied to the GSH matrix. The eluate panels were loaded with ∼30% of the protein eluting from the GSH matrix. Therefore, we estimate that ∼50% of the protein applied to the GSH was bound to the column and recovered during elution. The boxed regions show extracts that were fractionated on different gels.

FIG. 5.

Deletions and site-specific mutations introduced into the TGB2 and TGB3 proteins to assess yeast two-hybrid interactions and infectivity of the mutant proteins. Arrows above the drawings denote deletion sites. Solid lines extending above or below the rectangles illustrate the locations of site-specific amino acid substitutions, and white boxes represent the membrane-spanning domains within the proteins.

FIG. 6.

Cell-to-cell movement of BSMV derivatives containing the TGB2_G40R,P41R and TGB3_P105R,I108R mutations. (A) C. amaranticolor leaves inoculated with infectious transcripts of WT RNAα and WT RNAγ or (B) N. benthamiana leaves inoculated with WT RNAα and RNAγ-γb-GFP plus either RNAβ_WT or RNAβ transcripts containing the TGB2_G40R,P41R and TGB3_P105R,I108R mutations that disrupt TGB2 and TGB3 heterologous interactions. Photographs were taken at 12 dpi. Note that the small fluorescing spots in the γb-GFP panels represent fluorescence from single cells infected with inocula containing the TGB2 and TGB3 mutant proteins.

FIG. 7.

Effects of overexpression of TGB3 on BSMV movement. (A) Depiction of RNAβ replicons designed for overexpression of TGB3_WT (β_T3) or the TGB3_P105R;I108R double mutant (β_T3mut). Overexpression of the TGB3 derivatives was accomplished by placing the ORF under the control of the TGB1 promoter. The locations of primers used for RT-PCR are designated by black arrows above and below the boxes. (B) C. amaranticolor leaves were inoculated with 20 μg each of infectious RNAα, -β, and -γ transcripts or mock inoculated with buffer lacking RNA. Only leaves inoculated with RNAα, -β, and -γ developed lesions. RT-PCR amplifications with BSMV-specific primers are shown to the right of the leaves. (C) RNAα, -β, and -γ infectious transcripts plus different amounts of the TGB3_WT overexpression replicon were inoculated into leaves. The three leaves on the left show progressive reductions in the numbers of lesions caused by coinoculations of RNAα, -β, and -γ with 3 μg, 6 μg, or 9 μg of the TGB3_WT overexpression replicon, respectively. The control leaf on the right was inoculated with only the TGB3_WT overexpression replicon and hence did not develop lesions. RT-PCR amplifications with specific primers are illustrated below the leaves. (D) Inoculations and PCR were carried out as for panel C, except that the inoculum contained the TGB3_P105R;I108R overexpression replicon. Note that the 0.6-kb PCR product represents amplification from both the full-length RNAβ and RNAβ overexpression replicons. The 1.2-kb PCR product is generated from only the overexpression constructs.

FIG. 8.

Effects of cis and trans expression of the TGB2 and -3 proteins on infection. (A) RNAβ constructs designed for cis and trans expression of TGB2 and -3. Open circles represent the 5′ cap structures, and the internal poly(A) tract is designated at the 3′ end. Note that the 3′ 238-nt tRNA-like structure is not shown in these drawings. β_WT = WT RNAβ. The sgRNAβ1 and sgRNAβ2 mRNAs are illustrated as solid lines below β_WT; βT1_SSMUT = RNAβ containing substitutions for residues at positions 11 and 12 in TGB2 that result in the production of a nonfunctional protein and a mutation in the TGB3 ORF that introduces a premature stop codon to yield a protein truncated at residue 72; β_T1Δ2,3 = RNAβ with a 525-nt deletion of the TGB2/3 ORFs extending from the stop codon of TGB1 to the poly(A) tract; β_T2,3L = RNAβ with a deletion in the TGB1 ORF that maintains expression of the sgRNAβ2 under the control of the sgRNAβ2 promoter to facilitate low-level expression of the TGB2 and -3 proteins; β_T2,3H = RNAβ with a deletion of the TGB1 ORF that permits high-level cis expression of TGB2 and -3 under the control of the sgRNAβ1 promoter; β_T2 = RNAβ containing deletions of the TGB1 and the TGB3 ORFs to facilitate high-level expression of TGB2 under the control of the sgRNAβ1 promoter; β_T3 = RNAβ containing deletions of the TGB1 and TGB2 ORFs to facilitate high-level expression of TGB3 under the control of the sgRNAβ1 promoter. (B) Local lesion development in C. amaranticolor leaves inoculated with RNAα and -γ plus β_WT or different combinations of the cis and trans TGB protein expression derivatives. Leaves were photographed at 4 and 12 dpi. (C) Fluorescence in N. benthamiana leaves at 12 dpi after inoculation with RNAα and RNAγ-GFP plus the RNAβ derivatives designated below the leaves.