RNA VIRUSES AS GENE VECTORS

References


Batten, J.S., Yoshinari, S.,Hemenway C. Potato virus X: a model system for virus replication, movement and gene expression, Mol. Plant Path 4, 125-131 (2003)
Chapman, S., Kavanagh, T., and Baulcombe, D.C. Potato virus X as a vector for gene expression in plants. Plant J. 2, 549-57 (1992)
Dawson, W.O. and Lehto, K.M. Regulation of tobamovirus gene expression. Adv. Virus Res. 38, 307-342 (1990)
Lewandowski, D.J. , and Dawson, W.O. Deletion of internal sequences results in tobacco mosaic virus defective RNAs that accumulate to high levels without interfering with replication of the helper virus. Virology 251, 427-37 (1998)
Mori, M., Fujihara, N., Mise, K., and Furusawa, I. Inducible high-level mRNA amplification system by viral replicase in transgenic plants. Plant J. 27, 79-86 (2001)
Pogue, G.P., Lindbo, S.A., Garger, S.J, Fitzmaurice, W.P. Making an ally from an enemy, Plant virology and the new agriculture, Ann. Rev. Phytopath. 40, 45-74 (2002)
Ratcliff, F., Martin-Hernandez, A.M., Baulcombe, D.C. Tobacco rattle virus as a vector for analysis of gene function by silencing. Plant J. 25, 237-45 (2001)
Shivprasad, S., Pogue, G.P., Lewandowski, D.J., Hidalgo, J., Donson, J., Grill, L.K., and Dawson, W.O. Heterologous sequences greatly affect foreign gene expression in tobacco mosaic virus-based vectors. Virology 255, 312-323 (1999)
Toth, R.L., Pogue, G.P., Chapman, S. Improvement of the movement and host range properties of a plant virus vector through DNA shuffling. Plant J. 30, 593-600 (2002)
Yusibov, V., Shivprasad, S., Turpen, T.H., Dawson, W., Koprowski, H. Plant viral vectors based on tobamoviruses Curr. Topics in Micro. and Immunol. 240, 81-94 (1999).
Outline

I- Why use gene vectors?
1- Make commercially useful product (high level expression)
2- Test effect of a gene on a plant, by either expressing or inactivating
II- How does one make a vector?
1- Insert gene into DNA
2- Transcribe the DNA into infectious RNA and inoculate plant
3- Dawson (TMV), Ahlquist (BMV) first to make infectious transcripts
4- Alternatively express DNA in plant to give infectious RNA
III- General strategies
1- Replace or extend an existing gene
2- Add a new gene
3- Use reporter gene (GFP - 240AA, CAT - 300AA, GUS - 600AA)
4- Express from subgenomic replicon
5- Express from subgenomic promoter
6- Construct a unique restriction site at insertion position
7- Fiddle promoter to get maximum expression
IV - TMV as a vector (Dawson)
1- Insert new subgenomic promoter between M and CP
2- Simplest construct is TMV with two CP genes (construct unstable)
3- Next try a heterologous CP (more stable)
4- 3' most CP expressed 10 to 20 the internal CP
5- Replace internal CP with GFP
6- Construct infects N benthamiana, but not N tabacum
V- Optimizing GFP expression from TMV
1- Longer promoter
2- Internal pseudoknots (better expression of internal genes)
3- GFPC3 (improved GFP [more fluorescent])
4- Optimize codon usage (hasn't been done yet)
VI - PVX vector
1- Cut PVX at unique restriction site
2 - Insert new gene (with PVX promoter)
3 - High level expression and active virus (infects tobacco)
4 - Homologous constructs of other potexviruses express poorly
VII - BMV as vector
1- Express genome segments from plant genes
2- Have one inducible gene (so replication can be turned on at will)
3- Replace CP with gene of interest
VIII - Is high level expression of inserts compatible with virus replication?
1- Modified viruses generally replicate poorly
2- DNA shuffling (selective mutagenesis) is a potential solution.