Abstract
The strategy for processing the polyprotein encoded by plant potyviruses has been mimicked by constructing an expression cassette based on the nuclear inclusion (Nla) proteinase from tobacco etch virus (TEV). This cassette (pPR01),includes the TEV Nla coding region flanked on each side by its heptapeptide cleavage sequence and cloning sites for the in frame insertion of two different open reading frames. pPR01 allows the synthesis, under the control of a single transcriptional promoter, of two proteins in equimolar amounts as part of a polyprotein which is cleaved into individual mature products by the TEV protease. In in vitro reactions the cassette functioned as expected when several different protein-coding sequences were used. The potential uses of pPR01 are discussed.
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Allison R, Johnston R, Dougherty W: The nucleotide sequence of the coding region of tobacco etch virus genomic RNA: Evidence for the synthesis of a single polyprotein. Virology 154: 9–20 (1986).
Carrington J, Cary S, Dougherty W: Mutational analysis of tobacco etch virus polyprotein processing: Cis and trans proteolytic activities of polyproteins containing the 49-kilodalton proteinase. J Virol 62: 2313–2320 (1988).
Carrington J, Dougherty W: Processing of the tobacco etch virus 49K protease requires autoproteolysis. Virology 160: 355–362 (1987).
Carrington J, Dougherty W: Small nuclear inclusion protein encoded by a plant potyvirus genome is a protease. J Virol 61: 2540–2548 (1987).
Carrington J, Dougherty W: A viral cleavage site cassette: Identification of amino acid sequences required for tobacco etch virus polyprotein processing. Proc Natl Acad Sci USA 85: 3391–3395 (1988).
Carrington J, Freed D: Cap-independent enhancement of translation by a plant potyvirus 5 nontranslated region. J Virol 64: 1590–1597 (1990).
Carrington J, Freed D, Oh C: Expression of potyviral polyproteins in transgenic plants reveals three proteolytic activities required for complete processing. EMBO J 9: 1347–1353 (1990).
Dougherty W, Carrington J, Cary S, Parks T: Biochemical and mutational analysis of a plant virus polyprotein cleavage site. EMBO J 7: 1281–1287 (1988).
Dougherty W, Cary S, Parks T: Molecular genetic analysis of a plant virus polyprotein cleavage site: a model. Virology 171: 356–364 (1989).
Dougherty W, Parks T: Molecular genetic and biochemical evidence for the involvement of the heptapeptide cleavage sequence in determining the reaction profile at two tobacco etch virus cleavage sites in cell-free assays. Virology 172: 145–155 (1989).
Fujiwara T, Lessard P, Beachy RN: Inactivation of the nopaline synthase gene by double transformation: Reactivation by segregation of the induced DNA. Plant Cell Rep 12: 133–138 (1993).
Garcia J, Riechmann J, Lain S: Proteolytic activity of the plum pox potyvirus Nla-like protein in Escherichia coli. Virology 170: 362–369 (1989).
Hobbs S, Warkentin T, DeLong C: Transgene copy number can be positively or negatively associated with transgene expression. Plant Mol Biol 21: 17–26 (1993).
Laemmli U: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685 (1970).
Lawson C, Kaniewski W, Haley L, Rozman R, Newell C, Sanders P, Tumer N: Engineering resistance to mixed virus infection in a commercial potato cultivar: resistance to potato virus X and potato virus Y in transgenic russet burbank. Bio/technology 8: 127–134 (1990).
Matzke M, Matzke A: Genomic imprinting in plants: parental effects and trans-inactivation phenomena. Annu Rev Plant Physiol Plant Mol Biol 44: 53–76 (1993).
Matzke M, Primig M, Trnovsky J, Matzke A: Reversible methylation and inactivation of marker genes in sequentially transformed tobacco plants. EMBO J 8: 643–649 (1989).
Parks T, Smith H, Dougherty W: Cleavage profiles of tobacco etch virus (TEV)-derived substrates mediated by precursor and processed forms of the TEV Nla proteinase. J Gen Virol 73: 149–155 (1992).
Restrepo-Hartwig M, Carrington J: Regulation of nuclear transport of a plant potyvirus protein by autoproteolysis. J Virol 66: 5662–5666 (1992).
Riechmann J, Lain S, Garcia J: Highlights and prospects of potyvirus molecular biology. J Gen Virol 73: 1–16 (1992).
Rorrer K, Parks T, Scheffler B, Bevan M, Dougherty W: Autocatalytic activity of the tobacco etch virus Nla proteinase in viral and foreign protein sequences. J Gen Virol 73: 775–783 (1992).
Sambrook J, Fritsch E, Maniatis T: Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989).
Vance V, Moore D, Turpen T, Bracker A, Holowell V: The complete nucleotide sequence of pepper mottle virus genomic RNA: comparison of the encoded polyprotein with those of other sequenced potyviruses. Virology 191: 19–30 (1992).
Verchot J, Koonin E, Carrington J: The 35-kDa protein from the N-terminus of the potyviral polyprotein functions as a third virus-encoded proteinase. Virology 185: 527–535 (1991).
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Marcos, J.F., Beachy, R.N. In vitro characterization of a cassette to accumulate multiple proteins through synthesis of a self-processing polypeptide. Plant Mol Biol 24, 495–503 (1994). https://doi.org/10.1007/BF00024117
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DOI: https://doi.org/10.1007/BF00024117