Infectious cucumber mosaic virus RNA transcribed in vitro from clones obtained from cDNA amplified using the polymerase chain reaction

Hayes, Robert J.; Buck, K. W.

doi:10.1099/0022-1317-71-11-2503

Cited by 45 publications

(26 citation statements)

References 42 publications

(38 reference statements)

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“…The lower numbers are amino acid residues in the la protein. (c) Relevant restriction endonuclease sites in the 2a coding region (boxed) and 3' flanking region of pCMV2A (Hayes et al, 1990b). The upper numbers are nucleotide residues corresponding to CMV RNA 2 (Rezaian et al, 1984).…”

Section: Introductionmentioning

confidence: 99%

“…1 b), a full-length cDNA clone of CMV RNA 1 (Hayes & Buck, 1990b), and two oligonucleotide primers, one of which contained an added EcoRI site and the other a BamHI site at the 5' end. After purification with a PrimerErase column (Stratagene), the PCR product was cleaved with EcoRI and BamHI and cloned into the corresponding sites of pMAL-cRI to give pMALla-A, pCMV1A was then cleaved with StuI and BamHI and the 3"2 kb fragment was cloned into the corresponding sites of pMALla-A to give pMALla-1.…”

Section: Introductionmentioning

confidence: 99%

“…1 a) (New England Biolabs), a derivative of pMAL-c (Maina et al, 1988). To clone the whole of the la gene into pMAL-cRI, cDNA corresponding to nucleotides 98 to 250 of RNA 1 was amplified by PCR using pCMV1A (Hayes et al, 1990b). The upper numbers are nucleotide residues corresponding to CMV RNA l (Rezaian et al, 1985).…”

Section: Introductionmentioning

confidence: 99%

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Localization of Functional Regions of the Cucumber Mosaic Virus RNA Replicase Using Monoclonal and Polyclonal Antibodies

Hayes

Pereira²,

McQuillin³

et al. 1994

Journal of General Virology

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Monoclonal antibodies were produced using a purified cucumber mosaic virus (CMV) replicase complex, and Escherichia coli-expressed CMV la and 2a proteins, as immunogens. Five out of eight monoclonal antibodies, which bound to the la and 2a proteins in immunoblots, inhibited the RNA-dependent RNA polymerase (RdRp) activity of the purified replicase complex in vitro. Epitope mapping showed that two of the inhibitory antibodies interacted with regions of the la protein containing putative helicase and methyltransferase domains respectively. Two other inhibitory antibodies mapped to a region of the 2a protein containing the GDD motif which is highly conserved in RdRps. Prior interaction of the latter antibodies with a peptide containing the GDD motif prevented the antibody-mediated inhibition of the replicase. Polyclonal antibodies which inhibited the RdRp activity of the replicase complex were also produced using peptides corresponding to conserved helicase and polymerase motifs in the la and 2a proteins. The greatest inhibition was shown by antibodies to a peptide containing the GDD motif. These results demonstrate the functional importance of the identified sequence motifs in CMV RNA replication and indicate that the motifs are located in the replicase complex at positions accessible to antibodies, consistent with roles in interacting with the RNA template, RNA primer and enzyme substrates.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Localization of Functional Regions of the Cucumber Mosaic Virus RNA Replicase Using Monoclonal and Polyclonal Antibodies

Hayes

Pereira²,

McQuillin³

et al. 1994

Journal of General Virology

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“…The CMV genome consists of the tripartite component RNAs (RNAs 1−3), and encodes five proteins (Palukaitis and Garcia-Arenal, 2003). RNA1 and RNA2 encode the 1a and 2a proteins, which are attributed viral replication (Hayes and Buck, 1990). RNA3 encodes the movement protein (3a) and coat protein (CP).…”

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confidence: 99%

Application of a Reassortant Cucumber mosaic virus Vector for Gene Silencing in Tomato and Chili Pepper Plants

Hong¹,

Rhee²,

Kim³

et al. 2012

The Plant Pathology Journal

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We developed a reassortant RNA virus vector derived from Cucumber mosaic virus (CMV), which has advantages of very wide host range and can efficiently induce gene silencing in a few model plants. Certain CMV isolates, however, show limited host ranges presumably because they naturally co-evolved with their own hosts. We used a reassortant comprised of two strains of CMV, Y-CMV and Gn-CMV, to broaden the host range and to develop a virus vector for virus-induced gene silencing (VIGS). Gn-CMV could infect chili pepper and tomato more efficiently than Y-CMV. Gn-CMV RNA1, 3 and Y-CMV RNA2-A1 vector were newly reconstructed, and the transcript mixture of RNA1 and 3 genomes of Gn-CMV and RNA2 genome of Y-CMV RNA2 containing portions of the endogenous phytoene desaturase (PDS) gene (CMV2A1::PDSs) was inoculated onto chili pepper (cv. Chung-yang), tomato (cvs. Bloody butcher, Tigerella, Silvery fir tree, and Czech bush) and Nicotiana benthamiana. All the tested plants infected by the reassortant CMV vector showed typical photo-bleaching phenotypes and reduced expression levels of PDS mRNA. These results suggest that the reassortant CMV vector would be a useful tool for the rapid induction of the RNA silencing of endogenous genes in chili pepper and tomato plants.

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“…In this paper we describe the use of reverse transcriptase-PCR reactions (RT-PCR) and a high fidelity Vent DNA polymerase (Mattila et al, 1991) for construction of fulllength cDNA clones of Ba, Mo, Suv and Tu strains of BBMV RNA components. Previously, Hayes & Buck (1990) have used a low fidelity Taq DNA polymerase for RT-PCR amplification of full-length CMV cDNAs. Transcribed BBMV RNAs have been used to determine the translational capacity of the individual BBMV segments and to demonstrate the repair of 5' termini in transcript-derived progeny BBMV RNAs.…”

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confidence: 99%

Infectious transcripts from PCR-amplified broad bean mottle bromovirus cDNA clones and variable nature of leader regions in RNA 3 segment

Pogany

Huang

Romero

et al. 1994

Journal of General Virology

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Infectious cucumber mosaic virus RNA transcribed in vitro from clones obtained from cDNA amplified using the polymerase chain reaction

Cited by 45 publications

References 42 publications

Localization of Functional Regions of the Cucumber Mosaic Virus RNA Replicase Using Monoclonal and Polyclonal Antibodies

Localization of Functional Regions of the Cucumber Mosaic Virus RNA Replicase Using Monoclonal and Polyclonal Antibodies

Application of a Reassortant Cucumber mosaic virus Vector for Gene Silencing in Tomato and Chili Pepper Plants

Infectious transcripts from PCR-amplified broad bean mottle bromovirus cDNA clones and variable nature of leader regions in RNA 3 segment

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