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. 2013 Jul;87(14):7933-9.
doi: 10.1128/JVI.00743-13. Epub 2013 May 8.

The resistance protein Tm-1 inhibits formation of a Tomato mosaic virus replication protein-host membrane protein complex

Kazuhiro Ishibashi  1 , Masayuki Ishikawa
Affiliations

The resistance protein Tm-1 inhibits formation of a Tomato mosaic virus replication protein-host membrane protein complex

Kazuhiro Ishibashi et al. J Virol. 2013 Jul.

Abstract

The Tm-1 gene of tomato confers resistance to Tomato mosaic virus (ToMV). Tm-1 encodes a protein that binds ToMV replication proteins and inhibits the RNA-dependent RNA replication of ToMV. The replication proteins of resistance-breaking mutants of ToMV do not bind Tm-1, indicating that the binding is important for inhibition. In this study, we analyzed how Tm-1 inhibits ToMV RNA replication in a cell-free system using evacuolated tobacco protoplast extracts. In this system, ToMV RNA replication is catalyzed by replication proteins bound to membranes, and the RNA polymerase activity is unaffected by treatment with 0.5 M NaCl-containing buffer and remains associated with membranes. We show that in the presence of Tm-1, negative-strand RNA synthesis is inhibited; the replication proteins associate with membranes with binding that is sensitive to 0.5 M NaCl; the viral genomic RNA used as a translation template is not protected from nuclease digestion; and host membrane proteins TOM1, TOM2A, and ARL8 are not copurified with the membrane-bound 130K replication protein. Deletion of the polymerase read-through domain or of the 3' untranslated region (UTR) of the genome did not prevent the formation of complexes between the 130K protein and the host membrane proteins, the 0.5 M NaCl-resistant binding of the replication proteins to membranes, and the protection of the genomic RNA from nucleases. These results indicate that Tm-1 binds ToMV replication proteins to inhibit key events in replication complex formation on membranes that precede negative-strand RNA synthesis.

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Figures

Fig 1
Fig 1
Tm-1 inhibits negative-strand RNA synthesis. In vitro translation and replication of Tm-1-sensitive (Ile) and resistance-breaking (LT1) ToMV strains were performed in the presence (+) or absence (−) of Tm-1. Accumulation of the negative-strand RNA was examined by RNase protection assay using a 32P-labeled probe.
Fig 2
Fig 2
Effect of Tm-1 addition on the binding of ToMV replication proteins to membranes. (A) Fractionation of ToMV replication proteins by centrifugation. Genomic RNAs of TLIle or LT1 were translated in mdBYL, mixed with mdBYL from Tm-1-expressing (+) or -nonexpressing (−) BY-2 cells, incubated with BYL membranes, and fractionated into soluble (S) and membrane-containing (P) fractions by centrifugation. ToMV replication proteins and the Tm-1 protein were detected by Western blotting. To examine RdRp activity, fractions were incubated with [α-32P]CTP and other ribonucleoside triphosphates. The 32P-labeled RNA products were separated by 8 M urea-2.4% PAGE and visualized by autoradiography. G, genomic RNA; RF, replicative-form RNA. The asterisks represent background signals. (B) Membrane flotation analysis. Membrane-containing pellets prepared as described for panel A were suspended with TR buffer (lanes 1 to 8) or TR buffer containing 0.5 M NaCl (lanes 9 to 16) and subjected to membrane flotation analysis. The membrane (M) fractions and the soluble (S) fractions were collected, and the amounts of the indicated proteins and the activity of ToMV RdRp were examined.
Fig 3
Fig 3
Tm-1 inhibits sequestration of ToMV RNA in an isolated membranous compartment. 32P-labeled TLIle and LT1 RNAs were translated in mdBYL. The translation mixtures were mixed with mdBYL from Tm-1-expressing (+) and -nonexpressing (−) BY-2 cells and incubated with membranes. The samples were then divided into two aliquots. One aliquot was treated with MNase, and RNA was extracted and analyzed by PAGE and autoradiography. The other aliquot was directly analyzed for RNA without MNase treatment. For the sample marked *1, puromycin was added before the translation reaction. For the sample marked *2, Triton X-100 was added before MNase treatment. G, genomic RNA.
Fig 4
Fig 4
Negative-strand RNA synthesis is not required for the sequestration of ToMV RNA in the membranous compartment or NaCl-resistant membrane binding of the 130K protein. (A) Schematic representation of ToMV RNA derivatives used in Fig. 4 and 5. TLIle130F lacks the read-through region of the 180K polymerase, the 30K protein-coding region, and the 5′ half of the coat protein (CP)-coding region. TLIleΔ3′ is a transcript from a PmlI-linearized plasmid carrying full-length TLIle cDNA and lacks the 3′-terminal 158-nucleotide sequence. TLIle130FΔ3′ is a transcript from a PmlI-linearized plasmid encoding TLIle130F. (B) The read-through region for the 180K protein and the 3′ UTR of ToMV RNA are not required for the nuclease resistance of the genome RNA. 32P-labeled TLIle, TLIle130F, and TLIleΔ3′ RNAs were used as translation templates and analyzed as for Fig. 3. (C) The read-through region for the 180K protein and the 3′ UTR of ToMV RNA are not required for the 130K protein to bind membranes in a 0.5 M NaCl-resistant manner. TLIle, TLIle130F, and TLIleΔ3′ RNAs were translated in mdBYL, incubated with mdBYL from Tm-1-expressing (+) or -nonexpressing (−) BY-2 cells, and then incubated with BYL membranes. After incubation, membrane-containing pellets were prepared by centrifugation at 16,000 × g, suspended in 0.5 M NaCl-containing buffer, and centrifuged again at 16,000 × g to obtain the supernatant (S) and pellet (P) fractions. ToMV replication proteins in each fraction were detected by Western blotting and quantified with a LAS-3000 (Fujifilm, Japan). A typical set of results is shown, and the percentages of the 130K protein recovered in the P16 fractions are indicated. The asterisk represents background signals.
Fig 5
Fig 5
Tm-1 inhibits complex formation of ToMV replication proteins with host membrane proteins. ToMV RNA derivatives encoding the FLAG-tagged 130K protein of either Tm-1-sensitive (Ile) or resistance-breaking (LT1) type with (130F) or without (130Δ3′) the 3′ UTR (Fig. 4A) were translated in mdBYL, incubated with mdBYL from Tm-1-expressing cells, and then incubated with BYL membranes. The mixtures were fractionated into soluble (S16) and membrane (P16) fractions. The 130K protein in the P16 fraction was solubilized with LPC and immunoprecipitated (IP) with anti-FLAG antibody. The precipitates were analyzed by Western blotting to detect the indicated proteins.
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