Proof by synthesis of Tobacco mosaic virus
© Cooper; licensee BioMed Central Ltd. 2014
Received: 23 January 2014
Accepted: 30 April 2014
Published: 30 April 2014
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.
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.
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.
Tobacco mosaic virus (TMV) has been a model system in biology for more than 100 years and has led to the discovery of some of the basic underpinnings of life [1–4]. It has contributed to fundamental biology and to the applied sciences, being used to establish genetic foundations of mutation and plant immunity and to create the first transgenic crop plant with an improved agricultural trait [5–7].
The sequence of TMV variant V01408.1, herein dubbed the Goelet sequence, became a reference by which all other plant viruses were compared, and it facilitated the creation of TMV cDNA from which infectious RNA transcripts could be expressed , opening new possibilities for characterizing the encoded genes and for transiently expressing foreign genes in plants . Amidst all this success, however, is the almost forgotten fact that the Goelet sequence does not represent a single molecule of a TMV genome. Rather, it is a composite consensus of more than 400 independent clones of cDNA fragments reverse transcribed from a population of TMV RNA molecules exhibiting nucleotide polymorphisms consistent with the inherent error rate of viral RNA-dependent replication. Complicating matters is the likelihood that the virus that Goelet et al. sequenced was a mixture of closely related strains . Today, the Goelet sequence is the National Center for Biotechnology Information (NCBI) reference sequence for TMV (NC_001367.1). In 2009, its accession was updated to include the ORF6 protein, but to this date the genomic nucleotide sequence has not been amended. Furthermore, sequences for more than a hundred TMV variants or strains have been reported and none has been merged with NC_001367.1. Consequently, BLAST alignment of NC_001367.1 to all other full-length TMV genomic sequences in the partially non-redundant NCBI Nucleotide collection yields only one hit with perfect identity: itself. Meanwhile, other TMV variants have been molecularly cloned and demonstrated to be infectious [11, 14], but there is no known infectious clone for the Goelet sequence, and it remains unknown whether the Goelet sequence represents an infectious viral molecule.
The molecular biology techniques that first enabled the cDNA cloning of TMV 32 years ago have matured to the point where it is now possible to synthesize DNA molecules from basic chemicals in the absence of precursor biological template molecules such as RNA. This advance is part of the domain called synthetic biology, a discipline that can be used to verify the veracity of genome sequences, perform archeology, and assemble genomes de novo[15, 16]. Poliovirus was the first virus to have its genome chemically synthesized based on an alphabetic text blueprint of its nucleotide sequence, a feat later performed for other animal and bacterial viruses [16–25]. The complete chemical synthesis of a plant virus genome, however, has not yet been reported. To achieve this mark, DNA was enzymatically synthesized from overlapping oligonucleotides based on the Goelet alphabetic rendering of TMV and was then transcribed into RNA that was artificially packaged into virions with purified TMV CP in vitro. The man-made virus particles encoding the Goelet sequence, a mutant thereof, and two viral chimeras were tested in plants.
Results and discussion
Goelet sequence DNA synthesis from modular clones
Thirty sets of long-oligonucleotides with complementary, overlapping ends were chemically synthesized for construction of a linear DNA molecule encoding the Goelet TMV sequence (Figure 1). The DNA molecule also was designed to encode the T7 RNA polymerase promoter for RNA transcription in vitro and to have flanking sequences for PCR amplification. Sets of long-oligonucleotides were assembled to create 15, approximately 480 bp DNA modules (Figure 1) that were cloned in plasmids and sequenced. Only six clones for each construct needed to be screened to find at least one with a perfect module sequence, except in one case where it required the screening of 13 clones. The frequency of correct to incorrect modules and the error rate were similar to those previously reported for modules used to synthesize the mouse mitochondrial genome .
DNA was amplified from the modules, and the full-length DNA molecule of the Goelet sequence, called 1-30, was enzymatically synthesized from overlapping intermediates using 3 PCR steps for a total of 60 cycles (Figure 1) and was accomplished in 2 to 3 days by the author (fragment size selection by gel purification was the most time consuming step). This time estimate is consistent with the 5-day estimate reported for the PCR synthesis and enzymatic assembly of the mouse mitochondrial genome, which is roughly double the size .
7-Methyl guanosine capped-RNA was transcribed in vitro from gel purified PCR product of 1-30 and was inoculated to Nicotiana tabacum cv. Xanthi NN (tobacco), but no hypersensitive local lesions, the classic marks of infection, appeared. By contrast, the same amounts of RNA from DNA made by PCR amplification of the control plasmid pFL-TMV-NA, a known infectious TMV cDNA clone expressed by a T7 promoter [14, 27], did produce local lesions on NN tobacco. FL-TMV-NA RNA, however, only produced 4 to 10 local lesions per leaf, which suggested low inoculation potential. Because TMV RNA is susceptible to nuclease degradation and is less infectious than packaged virus , the transcribed RNA was encapsidated in vitro with CP isolated from purified FL-TMV-NA virions. On the basis of the number of local lesions, encapsidation improved the infectivity of control FL-TMV-NA RNA by three to five times (purified CP preparations by themselves did not produce symptoms). The CP then was used to encapsidate 1-30 RNA, but the preparation still did not produce local lesions on NN tobacco or mosaic symptoms on Xanthi nn tobacco (SX), the classic systemic-symptom host. The experiment was repeated, but again 1-30 virus was not infectious.
Uncommon polymorphisms in the Goelet sequence
The lack of symptoms implied that 1-30 DNA may not be error-free. To examine its integrity, 1-30 DNA was ligated into a plasmid and four clones were sequenced. Three clones perfectly matched the Goelet sequence whereas the fourth contained a single, silent mutation. Encapsidated RNA transcripts prepared from one plasmid with a perfect Goelet sequence produced no symptoms on NN and SX tobacco. Therefore, the integrity of 1-30 DNA was not to blame. Instead, the Goelet sequence itself was suspicious.
To evaluate the prevalence of the MR polymorphism among TMV isolates, the Goelet amino acid sequence from amino acids 166 to 207 was compared to the NCBI non-redundant protein sequence database by BLASTP. Identical matches were made to the MR amino acids in the first seven reported TMV replicase records, three of which referenced the Goelet sequence. The CE amino acids, however, appeared in 58 of the following TMV replicase records. The next match in the BLASTP report to deviate from CE was that for Tomato mosaic virus (ToMV), a closely related tobamovirus, which encoded CR instead. The C at amino acid position 186 is also conserved in other tobamoviruses such as Bell pepper mottle virus, Tobacco mild green mosaic virus, Sunn-hemp mosaic virus, and Odontoglossum ringspot virus. Hence, the lack of prevalence of the MR polymorphism among TMV isolates and related tobamoviruses implies that the Goelet DNA sequence for nucleotides 624 to 629 may be an artifact of reverse transcription, DNA sequencing, or human error.
An infectious, synthetic TMV
Capsid protein complementation
Virus peptides identified from plants by tandem mass spectrometry
Observed peptide mass
Hypothetical peptide mass
NN local lesion 1
NN local lesion 2
TMV and ToMV can be distinguished by the symptoms they produce on N. sylvestris that has the N’ gene, which confers hypersensitive resistance to ToMV but not to wild-type TMV. N’ encodes a protein with a coiled-coil domain, nucleotide binding site, and a leucine-rich repeat like many other plant disease resistance genes . The elicitor of N’ was mapped indirectly to the ToMV CP gene by Saito et al., who replaced the ToMV CP with an homologous TMV CP gene restriction fragment in an infectious ToMV clone and observed the loss of N’ elicitation. Around the same time, Dawson and colleagues [34, 35] showed that some RNA mutations in the TMV CP gene were sufficient for eliciting N’, and their data also indirectly implicated the ToMV CP gene in elicitation. It was later affirmed that variants of TMV CP, and not TMV CP gene RNA, indeed elicited N’[36, 37], but the same has never been shown for ToMV CP, although this is a technicality since the results for mutant TMV CP clearly implicate ToMV CP as well. Nevertheless, it was necessary to examine synthetic chimera 1-34CE-ToMV CP-30 virus on N. sylvestris to confirm the hypothesis that it would exhibit a ToMV-like phenotype. So leaf extract from 1-34CE-ToMV CP-30 virus-infected Xanthi SX was inoculated to N. sylvestris. Local lesions appeared within 3 days (Figure 3F). By comparison, 1-34CE-30 virus produced systemic symptoms, but no local lesions on N. sylvestris. Thus, this experiment directly demonstrates that the N’ elicitor is included within the coding region of the ToMV CP gene.
Because hybrid ORF6 influences symptoms in N. benthamiana and is partly encoded by the ToMV CP gene to which N’ gene elicitation is mapped, it was necessary to rule out whether hybrid ORF6 contributes to elicitation. Thus, 1-34CE-ToMVnoorf6-30 virus, isolated from N. benthamiana, was inoculated to N. sylvestris. Local lesions appeared 3 days later. Hence, N’ elicitation is attributed to the part of the gene that encodes the ToMV CP, not the part that encodes ORF6. Like the type strain of ToMV, 1-34CE-ToMV CP-30 virus does not have a mutation in its ToMV CP gene RNA. Thus, RNA structural alterations encoded by the ToMV CP gene do not explain elicitation of N’. Hence, ToMV CP is the elicitor of N’.
The number of organisms with sequenced genomes has grown in recent years, yet the veracity of nearly all of these genome sequences remains hypothetical. Synthetic biology provides a means for proving whether a sequence is a true blueprint for life. To date, only a few viral sequences and a lone prokaryotic genomic sequence have been authenticated [17, 40]. This cannot be said, however, for the original TMV sequence . RNA transcribed from an enzymatically synthesized DNA template of the Goelet sequence is not infectious. The cause appears to be related to insertion/deletion polymorphisms not in most other TMV sequences. The polymorphisms lead to the translation of MR amino acids instead of CE amino acids at positions 186 and 187 of the 126 kDa and 183 kDa replicase components. It is possible that MR disrupts the replicase methyltransferase domain critical for interactions with host proteins or with the 3′ end of TMV RNA [41–44]. The origins of the deleterious polymorphisms are not known with certainty, but it is unlikely that they were native to the TMV studied by Goelet et al. Thus, this report identifies a longstanding error in a model, reference sequence, but also proves that a corrected sequence sustains biological replication (Figure 3C).
Synthetic biology also can be used to identify the functions of genes. The TMV CP gene was exchanged with the homologous ToMV CP gene to test complementation. The chimera virus, 1-34CE-ToMV CP-30, moved systemically through tobacco, and this verified that ToMV CP can complement the long-distance transport function of the TMV CP. Furthermore, the chimera, as expected, produced ToMV-like local lesions rather than TMV-like systemic mosaic in N. sylvestris (Figure 3F). The local lesions, controlled by N’, were elicited by ToMV CP translated from the inserted gene. This insertion, however, also created a hybrid ORF6 gene overlapping the reading frames of the TMV MP gene and the exchanged ToMV CP gene (Figure 5A). Unexpectedly, the novel, hybrid ORF6 protein contributed to symptom severity in another plant, N. benthamiana (Figure 4).
While ToMV CP was sufficient for complementing systemic spread of chimera virus 1-34CE-ToMV CP-30 in three tobacco hosts, BSMV CP was not sufficient for enabling the systemic spread of chimera virus 1-34CE-BSMV CP-30 in barley, a non-host for TMV. Thus, transencapsidation of TMV by BSMV in barley may only be a consequence of mixed infection and may not explain spread in the non-host . It is possible that the BSMV triple-gene-block, which is analogous in function to the TMV MP, is needed to support the spread of TMV in barley . Nevertheless, 1-34CE-BSMV CP-30 virus did spread systemically through N. benthamiana and produced unique mosaic symptoms instead of TMV-like systemic necrosis (Figure 6A). It is possible that the novel symptoms were a result of replacing the TMV CP with the BSMV CP, which also led to a potential truncated TMV/BSMV hybrid ORF6 protein (Figure 5A). Thus, it should be clear that the inconspicuous ORF6 and its unnatural derivatives created by synthetic recombination can have pathological consequences. Embedded in the TMV genome are several other small ORFs that have not been examined for pathological importance, possibly because of the past difficulty of using traditional molecular biology techniques to decouple these potential ORFs from the critical replicase, MP, and CP genes in which they are embedded. It should be feasible to use synthetic biology to restructure the ORFs in the TMV genome and separate the genetic responsibilities of these potential, undiscovered genes much in the same way the bacteriophage genome was refactored [20, 21].
Synthetic genomes have been previously created for a few animal and bacterial viruses [16–25]. These genomes were usually maintained in DNA plasmids or were circularized for infectivity. By contrast, with the exception of the non-infectious Goelet replica, the synthetic DNA templates of the infectious plant viruses studied here were not maintained in bacterial plasmids. As linear DNA molecule templates, they are benign and incapable of natural biological replication either in bacteria or plants. Because TMV is an RNA virus, synthetic RNA needed to be transcribed in vitro from DNA. To improve infectivity, virions were constituted in vitro by combining the synthetic RNA with purified TMV CP; virion formation was confirmed by electron microscopy (Figure 3A). These particles may be considered to be the first synthetic virions because they were assembled in vitro from purified CP and synthetic RNA transcribed from synthetic DNA in the absence of potential host factors , although there is opportunity yet to supersede this achievement by formulating fully synthetic virus components entirely by macromolecular chemistry. That detail is trivial, however, compared to the more poignant observation that these man-made plant viruses with simple mutations and recombined genomes altered symptoms in both expected and unexpected ways, portending the ramifications of the artificial manufacture of inherently stable infectious entities . Consequently, all plants and experimental materials that came into contact with synthetic virions and progeny were appropriately disposed.
Materials and methods
The basic nucleotide blueprint was the M13 (-20) primer sequence as an anchor, the T7 minimal promoter sequence immediately followed by the DNA conversion of the Goelet sequence (GenBank Refseq NC_001367.1; 6395 nucleotides), and a terminal KpnI cleavage sequence (total length 6,443 nucleotides; Figure 1). KpnI cleavage of a plasmid encoding TMV adds five nucleotides to the end of the virus but does not inhibit TMV RNA infectivity . The KpnI cleavage site sequence was added for PCR amplification in light of the predicted high degree of secondary structure in the TMV 3′ end . The CP DNA sequences for ToMV (GenBank X02144.1) and BSMV (GenBank U35772.1) were also used.
Long-oligonucleotides with complementary overlapping ends were designed from the blueprint to allow construction of DNA by overlapping PCR . A modular strategy was adopted such that larger DNA molecules could be built from smaller units (Figure 1). Sets of 4 or 6 long-oligonucleotides were designed to overlap each other by 40 bases, with the exception of sets 15 and 16, which overlapped each other by 220 bases, and the first and last, which did not overlap with each other, thereby maintaining the linearity of the molecule (Additional file 1). Long-oligonucleotides were designed regardless of their potential secondary structures. Amplification oligonucleotides (Ampoligos), the lengths of which were optimized for 55 to 65°C annealing temperatures for PCR, anchored the ends of each module. Oligonucleotides were procured from Invitrogen (Carlsbad, CA, USA), ordered on a 25 nmol scale, and diluted to 100 μM in water.
Each long-oligonucleotide (0.5 μl) of two consecutive sets of long-oligonucleotides (Figure 1) was annealed in 16 to 18 μl TE buffer by heating to 95°C for 1 minute and cooling to 30°C at 0.1°C/s. Annealed long-oligonucleotides (0.5 μl) were reacted with 4.5 μl synthesis reaction mix (1 μl 5× isothermal reaction buffer , 0.1 unit Phusion DNA polymerase (Thermo Scientific, Waltham, MA, USA), 16 units Taq DNA ligase (New England Biolabs, Ipswich, MA, USA), and water) at 50°C for 1 h. The assembly (0.5 μl) was amplified by PCR for 20 cycles using amplification oligonucleotides and Phusion Hot Start II DNA polymerase (Thermo Fisher Scientific). DNA was separated by agarose-gel electrophoresis. DNA fragments were purified using UltraClean 15 (MO BIO Laboratories, Carlsbad, CA, USA).
DNA molecules approximately 480 bp long made from 2 sets of overlapping long-oligonucleotides and amplified in 20 PCR cycles were inserted into pCR-Blunt II-TOPO (Invitrogen) and propagated and selected in Escherichia coli. Cloned plasmids were sequenced by Genewiz (Germantown, MD, USA) using the Sanger method. For some 1-30 full-length molecules, DNA was amplified with oligonucleotides with XhoI and KpnI restriction endonuclease sites, digested, and ligated into pUC18 digested with SalI and KpnI, and the plasmids were transformed into Stbl2 E. coli (Invitrogen).
7-Methyl guanosine capped-RNA was produced from 250 to 1,000 ng DNA using the mMESSAGE mMACHINE T7 transcription kit (Ambion/Life Technologies, Carlsbad, CA, USA). Reactions were supplemented with 0.5 μl GTP. A PCR product (using Ampoligos 1 F and 30 R) of plasmid pFL-TMV-NA, a derivative of pU3/12 with unique NdeI and AflII restriction sites near the start and stop codon of the MP gene [14, 27], was evaluated as a control.
Viral RNA encapsidation
Virions of FL-TMV-NA and 1-34CE-ToMV CP-30 were purified from infected Xanthi SX, and virions of 1-34CE-BSMV CP-30 were purified from infected N. benthamiana. FL-TMV-NA CP was purified from virions using the glacial acetic acid degradation method . RNA (2 μg) was encapsidated in a reaction with 100 μg of purified FL-TMV-NA CP .
RNA was extracted from 1 cm2 leaf disks from plants showing viral symptoms using the RNeasy Plant Mini Kit (Qiagen, Germantown, MD, USA). RNA (10 μl) was annealed to Ampoligo 30 R and reverse transcribed using SuperScript III (Life Technologies, Carlsbad, CA, USA). cDNAs of viral genomes were amplified using Ampoligos and were sequenced by Genewiz using the Sanger method.
Protein was extracted from 1 cm2 leaf disks from plants showing viral symptoms by grinding in liquid nitrogen and precipitating in acetone/10% trichloracetic acid/0.07% beta-mercaptoethanol. Extracts were centrifuged at 21,000 × g for 15 minutes and washed two times in acetone. The residue was dried by vacuum centrifugation and resuspended in 100 μl 100 mM Tris base pH 8.5 and 8 M urea. Concentrations of soluble protein were determined by bicinchoninic acid assay (Pierce/Thermo Fisher Scientific, Carlsbad, CA, USA). Soluble protein (300 μg) was reduced, derivatized, digested in trypsin and analyzed by reverse phase-tandem mass spectrometry as previously detailed . Tandem mass spectra were analyzed with Mascot 2.4.0 against virus protein records in the NCBI NR protein sequence database. Peptides with P-values less than 0.05 were accepted. Spectra matching viral peptides were confirmed not to have equivalent or better matches to plant protein records in NR. Tandem mass spectrometry data (RAW files) are archived at .
Barley stripe mosaic virus
National Center for Biotechnology Information
open reading frame
polymerase chain reaction
Xanthi nn tobacco
Tobacco mosaic virus
Tomato mosaic virus.
The research was funded entirely by the USDA-ARS. I thank Kimberly Campbell in my lab for growing plants, Wesley Garrett in the Animal Biosciences and Biotechnology Laboratory for mass spectrometry, and Charles Murphy in the Soybean Genomics and Improvement Laboratory for transmission electron microscopy. This work is dedicated to my TMV forefathers, CM Deom, RN Beachy and JA Dodds.
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