Plant-virus-based vectors for gene transfer will be of limited use because of the high error frequency during viral RNA synthesis

Vloten-Doting, Lous Van; Bol, John F.; Cornelissen, Ben J. C.

doi:10.1007/bf02418253

Cited by 52 publications

(19 citation statements)

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“…3 that levels of extractable NPTII protein were considerably lower than levels of coat protein, the most highly expressed TMV protein (8 (21) that the level of subgenomic RNA from brome mosaic virus RNA 3 was progressively greater the closer promoter insertion was to the 3' terminus. Plant virus RNA polymerases have been proposed to have a low fidelity from the absence of proofreading and, as such, would result in foreign sequences in the viral genome only being maintained under constant selection pressure (6,41). However, the systemic spread of the foreign sequences in TBD4 and TBN62 RNA without selection suggests that sequence fidelity was possible despite replication of the foreign gene by RNA-dependent RNA polymerase.…”

mentioning

confidence: 99%

Systemic expression of a bacterial gene by a tobacco mosaic virus-based vector.

Donson

Kearney

Hilf

et al. 1991

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

189

110

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Tobacco mosaic virus (TMV) produces large quantities of RNA and protein on infection of plant cells. This and other features, attributable to its autonomous repfication, make TMV an attractive candidate for expression of foreign sequences in plants. However, previous attempts to construct expression vectors based on plant RNA viruses, such as TMV, have been unsuccesfl in obtaining systemic and stable movement of foreign genes to uninoculated leaves in whole plants. A hybrid viral RNA (TB2) was constructed, containing sequences from two tobamoviruses (TMV-U1 and odontoglossum ringspot virus). Two bacterial sequences inserted independently into TB2 moved systemically in Nicotiana benthamna, although they differed in their stability on serial passage. Systemic expression of the bacterial protein neomycin phosphotransferase was demonstrated. Hybrid RNAs containing both TMV-U1 and the inserted bacterial gene sequences were encapsidated by the odontoglossum ringspot virus coat protein, facilitating their transmission and amplification on pgin to subsequent plants. The vector TB2 provides a rapid means of expressing genes and gene variants in plants.The expression of foreign genes in plants has proven advantageous to the study of molecular biology (1,2). Stable gene transfer to whole plants can be achieved by a combination of DNA transformation and tissue culture techniques or by virus transfection. With viral-based vectors the ease of infection avoids the time-consuming procedures, "position" effects, and "somaclonal" variation seen when foreign genes are integrated into the plant genome (1). The merits of using a systemically expressing plant RNA virus-based vector have been extensively reviewed (3-7). Several features of tobacco mosaic virus (TMV) (8,9) suggest that it might be usefully adapted for such a purpose: (i) tobamoviruses have a wide host range; (it) they can move cell-to-cell mediated by a virus-encoded peptide; (iii) they exhibit rapid systemic spread in plants; (iv) TMV infections are maintained for the lifetime ofthe plant; (v) TMV RNA is replicated to high levels as autonomous sequences; (vi) this replication results in rapid and productive cytoplasmic gene expression; (vii) temperature-sensitive mutations of RNA synthesis are available to modulate expression of foreign genes; (viii) TMV also lacks the packaging constraints found with nonhelical viruses, including existing DNA plant virus vectors; and (ix) the TMV genome can now be manipulated as a DNA copy and then transcribed in vitro to produce infectious RNA molecules.The TMV genome consists of one 6395-nucleotide (nt) molecule of messenger-sense, single-stranded RNA, encoding at least four proteins (10; for review see ref. 9; Fig. 1). It has similarities of sequence and replication strategy with a series of plant and animal RNA viruses (11). The 126-and 183-kDa replicase proteins are translated directly from the genomic RNA, whereas the 30-kDa cell-to-cell movement protein and 17.5-kDa coat protein are translated from two 3'-coterminal su...

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mentioning

confidence: 99%

Systemic expression of a bacterial gene by a tobacco mosaic virus-based vector.

Donson

Kearney

Hilf

et al. 1991

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

189

110

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“…First, each particular virus-mediated expression system is limited to certain plant species due to the host range of plant viruses. Second, plant virus-mediated expression is associated with the potential high error frequency and rapid recombination (van Vloten-Doting et al 1985 ;Drake and Holland 1999 ) . The relatively high mutation rate of the viral RNA-dependent RNA polymerase due to the lack of proofreading ability may result in the production of mixed recombinant proteins containing undesirable proteins.…”

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confidence: 99%

Plant Virus-Mediated Expression in Molecular Farming

Wang

2011

Molecular Farming in Plants: Recent Advances and Future Prospects

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Plant viruses have contrasting abilities. On one hand, they can induce gene silencing, termed as virus-induced gene silencing. On the other hand, they have evolved mechanisms that suppress gene silencing and allow the accumulation of very high levels of viral proteins in infected plants. The latter is the driving force for the manipulation of plant viruses for molecular farming in plants. In comparison to the transgenic approach which is often associated with low levels of expression and the requirement of a time-consuming and labour-intensive genetic transformation process, the plant virus-mediated expression approach has several advantages such as easy manipulation, high yield and fast manufacturing. This approach uses plant virus-based expression vectors as a vehicle to produce therapeutic proteins such as antibodies, enzymes, vaccines, and other recombinant proteins of interest in plants. Over the last two decades, a number of plant viruses have been developed and optimized for expression of a variety of pharmaceutical proteins. Some of these recombinant proteins are currently under pre-clinical or clinical trials. In this chapter, I will summarize recent progress, current challenges and future prospects of plant virus-mediated expression in molecular farming.

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“…Although there is strong selection pressure to maintain the replicon genes responsible for replication and spreading, it has been pointed out that this is not so for a foreign coding sequence and, as a consequence, its function might be lost through mutation (12). This might be particularly likely to happen because of an observed high error frequency of known RNA synthesizing enzymes.…”

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“…It is a common observation, as Van VlotenDoting et al (12) point out, that preparations of the common strain of tobacco mosaic virus contain a small, but detectable, proportion of variants even when the preparation consists of the progeny of what is presumed to be a single particle. ]'his should not be too surprising when one considers that a single tobacco leaf may yield 2 • 1014 virus particles and a twenty leaf plant 4 • 1015 particles.…”

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confidence: 99%

“…Van Vloten-Doting et al (12) have calculated a high mutation rate for alfalfa mosaic virus by analyzing results of an experiment in which virus was transferred from tobacco to bean and then back to tobacco again. A difficulty with this type of experiment is illustrated by the pitfall of an experienced virologist in concluding that the host could transform one strain of tobacco mosaic virus into another when it was tranferred between these same two hosts ( l); a conclusion that was based on results that could not be repeated by others once they had freed both the bean and tobacco strains of variants that would cause systemic infection in the alternate host (14).…”

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Plant-virus-based vectors for gene transfer may be of considerable use despite a presumed high error frequency during RNA synthesis

Siegel

1985

Plant Mol Biol

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The RNA genomes of some plant viruses have properties which make them suitable for development into vectors for gene transfer. It has been claimed that the low fidelity of RNA synthesizing enzymes may limit the usefulness of such vectors. Arguments are presented here to indicate that the problem may not be serious. Examples are presented of plant RNA replicons which carry relatively stable coding sequences whose products are superfluous to either the replicon or the host. In addition, there are numerous instances in which slowly replicating virus strains are maintained in 'pure' culture in a host and are not overgrown by more rapidly replicating strains. It is difficult to estimate the error frequency of RNA replicating enzymes. Methods which rely on host-shift may be subject to error.

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Plant-virus-based vectors for gene transfer will be of limited use because of the high error frequency during viral RNA synthesis

Cited by 52 publications

References 28 publications

Systemic expression of a bacterial gene by a tobacco mosaic virus-based vector.

Systemic expression of a bacterial gene by a tobacco mosaic virus-based vector.

Plant Virus-Mediated Expression in Molecular Farming

Plant-virus-based vectors for gene transfer may be of considerable use despite a presumed high error frequency during RNA synthesis

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