Mutational Analysis of Cauliflower Mosaic Virus Gene VI: Changes in Host Range, Symptoms, and Discovery of Transactivation-Positive, Noninfectious Mutants

Broglio, E. P.

doi:10.1094/mpmi-8-0755

Cited by 16 publications

(8 citation statements)

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“…1). Previous transient expression assays in plant protoplasts had shown that the 6-bp insertion within the Pvu II site of YS24 destroys the ability of the gene VI product to transactivate expression of the CAT reporter gene (17). This mutation occurs within the minimal region of gene V7, as delimited by De Tapia et al (28), that will support translational transactivation in plant protoplasts.…”

Section: Methodsmentioning

confidence: 99%

“…The plasmid JS169 was constructed by inserting the gene VI coding region contained in a BamHI-Xho I DNA fragment derived from pKB28B-16,u (16) into the BamHI and Sal I sites of pYES2 (Invitrogen). The plasmid YS24 was constructed by inserting the gene VI coding region contained on an EcoRV-Xho I DNA fragment of p2036T2 (17) into the Sma I and Sal I sites of pUC18 and then subsequently moving a Kpn I-Sph I DNA fragment into pYES2. To construct JS163, a Sal I DNA fragment containing the CAT gene of pCM-1 was inserted into pYES2.…”

Section: Methodsmentioning

confidence: 99%

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Expression of a plant viral polycistronic mRNA in yeast, Saccharomyces cerevisiae, mediated by a plant virus translational transactivator.

Sha¹,

Broglio²,

Cannon³

et al. 1995

Proc. Natl. Acad. Sci. U.S.A.

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We demonstrate that the cauliflower mosaic virus (CaMV) gene VI product can transactivate the expression of a reporter gene in bakers' yeast, Saccharomyces cerevisiae. The gene Vlcoding sequence was placed under the control of the galactose-inducible promoter GALI, which is present in the yeast shuttle vector pYES2, to create plasmid JS169. We also created a chloramphenicol acetyltransferase (CAT) reporter plasmid, JS161, by inserting the CAT reporter gene in-frame into CaMV gene II and subsequently cloning the entire CaMV genome into the yeast vector pRS314. When JS161 was transformed into yeast and subsequently assayed for CAT activity, only a very low level of CAT activity was detected in cellular extracts. To investigate whether the CaMV gene VI product would mediate an increase in CAT activity, we cotransformed yeast with JS169 and JS161. Upon induction with galactose, we found that CAT activity in yeast transformed with JS161 and JS169 was about 19 times higher than the level in the transformants that contained only JS161. CAT activity was dependent on the presence of the gene VI protein, because essentially no CAT activity was detected in yeast cells grown in the presence of glucose, which represses expression from the GAL) promoter. RNase protection assays showed that the gene VI product had no effect on transcription from the 35S RNA promoter, demonstrating that regulation was occurring at the translation level. This yeast system will prove useful for understanding how the gene VI product of CaMV mediates the translation of genes present on a eukaryotic polycistronic mRNA.Cauliflower mosaic virus (CaMV) is a plant pararetrovirus that has a unique strategy for the expression of its six genes. Two RNAs that have sizes of 19S and 35S are transcribed from the circular double-stranded DNA template (1). The monocistronic 19S RNA is responsible for the expression of the CaMV gene VI product, a protein that has been described as a translational transactivator (2). The 35S RNA is a terminally redundant RNA that has dual roles in replication and expression. It serves as a replication intermediate during reverse transcription of the viral genome (1). In addition, it is a polycistronic mRNA that contains a 600-nucleotide leader sequence followed by six closely spaced genes, including gene VI (1). The 35S RNA leader sequence itself is extremely complex. It contains seven to eight small open reading frames, depending on the strain, that vary in size from 9 to 102 nucleotides; it may be folded into an extensive stem-loop structure (3); and it inhibits translation of genes I-V in transiently transfected protoplasts (4, 5).The unusual feature about the CaMV expression strategy is that the CaMV gene VI protein is necessary for efficient translation of genes I-Von the 35S RNA (6-9). Genes I-V, and possibly VI, are believed to be expressed by translation reinitiation; as ribosomes complete the translation of one gene, they reinitiate protein synthesis at the next start codon until all genes are translated (10-12). ...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Expression of a plant viral polycistronic mRNA in yeast, Saccharomyces cerevisiae, mediated by a plant virus translational transactivator.

Sha¹,

Broglio²,

Cannon³

et al. 1995

Proc. Natl. Acad. Sci. U.S.A.

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“…In particular it is involved in host-range determination (Schoelz & Shepherd, 1988), symptomatology (Daubert & Routh, 1990;Wintermantel et al, 1993;Broglio, 1995), and probably viral morphogenesis (Himmelbach et al, 1996). Moreover, P6 is also the major component of the membrane-free cytoplasmic inclusion bodies called viroplasms (Xiong et al, 1982), where the main steps of the replication cycle occur (Mazzolini et al, 1989).…”

Section: Introductionmentioning

confidence: 99%

P6 protein of Cauliflower mosaic virus, a translation reinitiator, interacts with ribosomal protein L13 from Arabidopsis thaliana

Bureau

Leh

Haas

et al. 2004

Journal of General Virology

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The P6 protein of Cauliflower mosaic virus (CaMV) transactivates translation of the CaMV 35S polycistronic pregenomic RNA and its spliced versions, and thus allows synthesis of a complete set of viral proteins. Previous studies have shown that P6 interacts with plant L18 and L24 ribosomal proteins and initiation factor eIF3, and it has been proposed that these interactions are involved in the reinitiation of translation of polycistronic viral RNAs. This study characterizes a novel cellular partner of P6, the ribosomal protein L13 from Arabidopsis thaliana. Far-Western assays performed with several P6 deletion mutants have shown that L13 interacts with the miniTAV of P6, which represents the minimal domain for transactivation, suggesting that the P6-L13 interaction might also be involved in this process. L13 and L18 were found to bind to the same region within the miniTAV. Competition assays between L18 and L13 for binding to miniTAV suggest that interactions between P6 and these ribosomal proteins involve separate P6 molecules, and/or occur at different stages of translation or in the context of another function also mediated by P6.

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“…TAV is a complex protein that appears to be involved in many aspects of the virus life cycle (De Tapia et al, 1993 ). It has been shown to be part of abundant vacuolated inclusion bodies as well as determinant of host specificity and a factor influencing symptom development in infected plants (Broglio, 1995;De Tapia et al, 1993). As mention above, another of its role is to control translation from the polycistronic CaMV 35S RNA.…”

Section: -"Translational Trans-activation" Translation Ofmentioning

confidence: 99%

Plant virus gene expression strategies

Bustamante

Hull

1998

Electron. J. Biotechnol.

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Mutational Analysis of Cauliflower Mosaic Virus Gene VI: Changes in Host Range, Symptoms, and Discovery of Transactivation-Positive, Noninfectious Mutants

Cited by 16 publications

References 0 publications

Expression of a plant viral polycistronic mRNA in yeast, Saccharomyces cerevisiae, mediated by a plant virus translational transactivator.

Expression of a plant viral polycistronic mRNA in yeast, Saccharomyces cerevisiae, mediated by a plant virus translational transactivator.

P6 protein of Cauliflower mosaic virus, a translation reinitiator, interacts with ribosomal protein L13 from Arabidopsis thaliana

Plant virus gene expression strategies

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