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. 2014 Apr 30;15(5):R67.
doi: 10.1186/gb-2014-15-5-r67.

Proof by synthesis of Tobacco mosaic virus

Bret Cooper

Proof by synthesis of Tobacco mosaic virus

Bret Cooper. Genome Biol. .

Abstract

Background: Synthetic biology is a discipline that includes making life forms artificially from chemicals. Here, a DNA molecule was enzymatically synthesized in vitro from DNA templates made from oligonucleotides representing the text of the first Tobacco mosaic virus (TMV) sequence elucidated in 1982. No infectious DNA molecule of that seminal reference sequence exists, so the goal was to synthesize it and then build viral chimeras.

Results: RNA was transcribed from synthetic DNA and encapsidated with capsid protein in vitro to make synthetic virions. Plants inoculated with the virions did not develop symptoms. When two nucleotide mutations present in the original sequence, but not present in most other TMV sequences in GenBank, were altered to reflect the consensus, the derivative synthetic virions produced classic TMV symptoms. Chimeras were then made by exchanging TMV capsid protein DNA with Tomato mosaic virus (ToMV) and Barley stripe mosaic virus (BSMV) capsid protein DNA. Virus expressing ToMV capsid protein exhibited altered, ToMV-like symptoms in Nicotiana sylvestris. A hybrid ORF6 protein unknown to nature, created by substituting the capsid protein genes in the virus, was found to be a major symptom determinant in Nicotiana benthamiana. Virus expressing BSMV capsid protein did not have an extended host range to barley, but did produce novel symptoms in N. benthamiana.

Conclusions: This first report of the chemical synthesis and artificial assembly of a plant virus corrects a long-standing error in the TMV reference genome sequence and reveals that unnatural hybrid virus proteins can alter symptoms unexpectedly.

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Figures

Figure 1
Figure 1
Schematic of synthetic TMV. (A) Genome organization. (B) Long-oligonucleotides with 20 or 40 base overlaps. (C) Overlapping approximately 480 bp molecules made from long-oligonucleotides in (B). (D) Overlapping approximately 1,140 bp molecules made from (C). (E) Full-length DNA made from molecules from (D).
Figure 2
Figure 2
Sequence polymorphisms between the Goelet sequence and FL-TMV-NA. Alignment of DNA nucleotides from positions 615 to 632 and 825 to 833. The corresponding translated amino acids (a.a) are shown, and the differences are in boldface type. This region is in the replicase.
Figure 3
Figure 3
Encapsidation and infectivity of synthetic 1-34CE-30 and 1-34CE-ToMV CP-30. (A) 1-34CE-30 RNA encapsidated in vitro with FL-TMV-NA CP (stained with uranyl acetate and imaged by transmission electron microscopy; distance between first and last black ticks in white bar = 200 nm). (B) FL-TMV-NA CP disks prior to the addition of 1-34CE-30 RNA (stained with uranyl acetate and imaged by transmission electron microscopy; distance between first and last black ticks in white bar = 100 nm). (C) Mosaic symptoms on N. tabacum cv. Xanthi SX plant inoculated with encapsidated 1-34CE-30 (4 weeks after inoculation). (D) N. tabacum cv. Xanthi NN local lesions from 1-34CE-30 are to the left of the midrib and local lesions from 1-34CE-ToMV CP-30 are to the right. (E) 1-34CE-ToMV CP-30 virion from Xanthi SX (stained with uranyl acetate and imaged by transmission electron microscopy; distance between first and last black ticks in white bar = 200 nm). (F) N. sylvestris infected with 1-34CE-ToMV CP-30.
Figure 4
Figure 4
Effects of ORF6 on symptomology. Symptoms on N. benthamiana 14 days after inoculation with the respective viruses.
Figure 5
Figure 5
Needleman-Wunsch amino acid alignment for ORF6 and CP of the respective viruses. (A) ORF6. The nucleotide position for the CP gene start codon is shown by the arrow. (B) CP. Dots indicate identical amino acids; plus signs indicate similar amino acids.
Figure 6
Figure 6
Synthetic 1-34CE-BSMV CP-30. (A) Mosaic symptoms on N. benthamiana. (B) Virion from N. benthamiana (stained with uranyl acetate and imaged by transmission electron microscopy; distance between first and last black ticks in white bar = 100 nm).
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