Immunological detection of the 8K protein of potato virus X (PVX) in cell walls of PVX-infected tobacco and transgenic potato

Hefferon, Kathleen; Doyle, Steven; AbouHaidar, Mounir G.

doi:10.1007/s007050050089

Cited by 22 publications

(33 citation statements)

References 25 publications

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“…Using synthetic peptides derived from the p7 sequence, we have identified three distinct putative domains within the protein. EMSA showed that the central region, from residue 17 to 35 (represented by peptide p7 [17][18][19][20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35] ), is responsible for the RNA binding properties of CarMV p7. This binding peptide populates a nascent ␣-helix in water solution that is further stabilized in the presence of either secondary structure inducers, such as trifluoroethanol and monomeric SDS, or RNA (which also changes its conformation upon binding to the peptide).…”

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confidence: 99%

“…There are viruses that encode multiple MPs, which, in addition, do not fall in the 30K superfamily. The triple gene block of potexviruses encodes three proteins (open reading frames 2-4) that mediate cell-tocell transport, the first one with RNA-binding and NTPase/ helicase activities and the other two with putative membranespanning domains and, in the case of the third, demonstrated cell wall localization (22,23). Carmoviruses are among the smallest known plant viruses; their genome is a singlestranded RNA of ϳ4 kilobases encoding at least five proteins (24,25).…”

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confidence: 99%

“…The concentration of all of the other peptides was obtained by quantitative amino acid analysis. The CD spectra of the p7 [17][18][19][20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35] -RNA complex were recorded at 5°C with 20 M of p7 [17][18][19][20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35] , and increasing concentrations of CarMV ssRNA transcribed in vitro from clone pCarM.D5 (Ref. 29; this RNA covers exactly the first 181 residues of the CarMV genome) were added (from 1 to 20 ng), until no change in the difference spectrum was observed.…”

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confidence: 99%

“…Spectra were recorded in the far UV region in 5 mM MOPS/NaOH, pH 7.0, buffer. In peptide tritation experiments, increasing concentrations of p7 [17][18][19][20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35] were added (from 0 to 200 M) to a 1 mM RNA solution and left on ice for 30 min before the CD spectrum was recorded in the near UV region (250 -350 nm).…”

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confidence: 99%

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Structural Properties of Carnation Mottle Virus p7 Movement Protein and Its RNA-binding Domain

Vilar

Esteve

Pallás

et al. 2001

Journal of Biological Chemistry

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Plant viral movement proteins (MPs) participate actively in the intra-and intercellular movement of RNA plant viruses to such an extent that MP dysfunction impairs viral infection. However, the molecular mechanism(s) of their interaction with cognate nucleic acids are not well understood, partly due to the lack of structural information. In this work, a protein dissection approach was used to gain information on the structural and RNA-binding properties of this class of proteins, as exemplified by the 61-amino acid residue p7 MP from carnation mottle virus (CarMV). Circular dichroism spectroscopy showed that CarMV p7 is an ␣/␤ RNA-binding soluble protein. Using synthetic peptides derived from the p7 sequence, we have identified three distinct putative domains within the protein. EMSA showed that the central region, from residue 17 to 35 (represented by peptide p7 17-35 ), is responsible for the RNA binding properties of CarMV p7. This binding peptide populates a nascent ␣-helix in water solution that is further stabilized in the presence of either secondary structure inducers, such as trifluoroethanol and monomeric SDS, or RNA (which also changes its conformation upon binding to the peptide). Thus, the RNA recognition appears to occur via an "adaptive binding" mechanism. Interestingly, the amino acid sequence and structural properties of the RNA-binding domain of p7 seem to be conserved among carmoviruses and some other RNAbinding proteins and peptides. The low conserved N terminus of p7 (peptide p7 1-16 ) is unstructured in solution. In contrast, the highly conserved C terminus motif (peptide p7 40 -61 ) adopts a ␤-sheet conformation in aqueous solution. Alanine scanning mutagenesis of the RNAbinding motif showed how selected positive charged amino acids are more relevant than others in the RNA binding process and how hydrophobic amino acid side chains would participate in the stabilization of the protein-RNA complex.Infection of plants by viruses requires viral genome cell-tocell movement through plasmodesmata (the plant intercellular symplastic connections), which is mediated by virus-encoded so-called movement proteins (MPs) 1 (1, 2). MPs participate actively in the intra-and intercellular movement of plant viruses, and mutant virus analyses, reverse genetics, and plant transformation have demonstrated that MP dysfunction impairs viral infection (3, 4). MP properties are best described in the tobacco mosaic tobamovirus (TMV) model system. The 30-kDa TMV MP binds single-stranded (viral) RNA in vitro with no sequence specificity and high cooperativity (5), co-localizes with the cytoskeleton and cell wall (6 -8), is required for the association of viral RNA with endoplasmic reticulum (9), increases the size exclusion limit of plasmodesmata (10), and mediates an active transport of the viral genome to the adjacent cell (4). Sequence deletion and mutagenesis studies have located some of these functions in separate motifs/domains of the protein (11-13). These properties define the tobamo-like model of viral cell t...

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confidence: 99%

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confidence: 99%

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confidence: 99%

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confidence: 99%

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Structural Properties of Carnation Mottle Virus p7 Movement Protein and Its RNA-binding Domain

Vilar

Esteve

Pallás

et al. 2001

Journal of Biological Chemistry

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“…TGBp2 and TGBp3 both contain stretches of hydrophobic amino acids and are associated with cell wall and membrane fractions both in vivo and in vitro (Donald et al, 1993;Hefferon et al, 1997;Morozov et al, 1990Morozov et al, , 1991NiesbachKlosgen et al, 1990;Solovyev et al, 2000). Using a microinjection and transient expression strategies, TGBp2 was shown to assist the movement of the ribonucleoprotein complex of potexviruses (Lough et al, 1998) and to be capable of increasing the SEL of the plasmodesmata (Tamai & Meshi, 2001).…”

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confidence: 99%

Arg-16 and Arg-21 in the N-terminal region of the triple-gene-block protein 1 of Bamboo mosaic virus are essential for virus movement

Lin

Chang

Liao

et al. 2004

Journal of General Virology

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The protein encoded by the first gene of the triple gene block (TGBp1) of potexviruses is required for movement of the viruses. It has been reported that single ArgRAla substitutions at position 11, 16 or 21 of TGBp1 of Bamboo mosaic virus (BaMV) eliminate its RNA-binding activity, while substitutions at position 16 or 21 only affect its NTPase activity (Liou et al., Virology 277, 336-344, 2000). However, it remains unclear whether these ArgRAla substitutions also affect the movement of BaMV in plants. To address this question, six mutants of BaMV, each containing either a single-or a double-alanine substitution at Arg-11, Arg-16 and Arg-21 of TGBp1, were constructed and used to infect Chenopodium quinoa and Nicotiana benthamiana. We found that all of the BaMV mutants were able to replicate in protoplasts of N. benthamiana. However, only the mutant with an Arg-11RAla substitution in TGBp1 remained capable of movement from cell to cell in plants. Mutants with Arg-16, Arg-21 or both Arg-16 and Arg-21 of TGBp1 replaced with alanine were defective in virus movement. This defect was suppressed when a wild-type TGBp1 allele was co-introduced into the cells using a novel satellite replicon. The ability to trans-complement the movement defect by the wild-type TGBp1 strongly suggests that the ArgRAla substitution at position 16 or 21 of TGBp1, which diminishes the RNA-binding and NTPase activities of TGBp1, also eliminates the capability of BaMV to move from cell to cell in host plants.

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Movement of Hordeivirus Hybrids with Exchanges in the Triple Gene Block

et al. 1999

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The barley stripe mosaic virus (BSMV) triple gene block (TGB) coding for movement proteins (MPs) was replaced with the respective TGB genes from two other hordeiviruses, poa semilatent virus (PSLV) or lychnis ringspot virus (LRSV). The BSMV/LRSV recombinant did not exhibit infectivity on the plants tested, whereas the infection rate and host range of the BSMV/PSLV hybrid were similar to those of BSMV. In particular, the BSMV/PSLV hybrid infected Nicotiana benthamiana, a nonhost plant for PSLV, indicating a contribution of non-MP elements of BSMV genome to host specificity of virus transport. Assuming that the PSLV TGB was functional in the BSMV genome context, a further series of recombinants was constructed, in which smaller portions of the BSMV TGB were replaced by the corresponding PSLV sequences. Examination of the infectivity of the hybrid viruses suggested that the TGB-coded proteins could interact in a host-dependent manner to mediate cell-to-cell movement. Analysis of recombinants with hybrid sequences of the first gene in the TGB (beta b gene) indicated that (i) sequence-independent binding of beta b to viral RNAs could occur during formation of beta b-RNA complexes in vivo, and that (ii) the beta b MP is involved in virus long-distance movement, for which homologous N- and C-terminal beta b domains are required.

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Immunological detection of the 8K protein of potato virus X (PVX) in cell walls of PVX-infected tobacco and transgenic potato

Cited by 22 publications

References 25 publications

Structural Properties of Carnation Mottle Virus p7 Movement Protein and Its RNA-binding Domain

Structural Properties of Carnation Mottle Virus p7 Movement Protein and Its RNA-binding Domain

Arg-16 and Arg-21 in the N-terminal region of the triple-gene-block protein 1 of Bamboo mosaic virus are essential for virus movement

Movement of Hordeivirus Hybrids with Exchanges in the Triple Gene Block

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