The test‐tube synthesis of a chemical called poliovirus

Science & Society1 July 2006free access The test-tube synthesis of a chemical called poliovirus The simple synthesis of a virus has far-reaching societal implications Eckard Wimmer Eckard Wimmer Department of Molecular Genetics and Microbiology, School of Medicine, Stony Brook University, Stony Brook, NY, USA Search for more papers by this author Eckard Wimmer Eckard Wimmer Department of Molecular Genetics and Microbiology, School of Medicine, Stony Brook University, Stony Brook, NY, USA Search for more papers by this author Author Information Eckard Wimmer1 1Department of Molecular Genetics and Microbiology, School of Medicine, Stony Brook University, Stony Brook, NY, USA EMBO Reports (2006)7:S3-S9https://doi.org/10.1038/sj.embor.7400728 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info In July 2002, newspapers across the globe reported that scientists had created a virus in a test tube. This unexpected news struck a raw nerve among lay people and scientists alike. The work was condemned as dangerous and irresponsible, scorned as a stunt and perceived as a challenge to divine power. It was also hailed as a milestone in biology. What really happened? Guided by the nucleotide sequence, which my colleagues and I determined in 1981 (Kitamura et al, 1981), Jeronimo Cello, Aniko Paul and I described the chemical synthesis of a DNA molecule equivalent to the poliovirus genome (Cello et al, 2002). Using methods that we developed in the 1980s and 1990s (van der Werf et al, 1986; Molla et al, 1991), we then converted the virus-specific DNA by simple in vitro biochemical manipulations into authentic poliovirus particles (Cello et al, 2002). On paper, the synthesis looked simple and, not surprisingly, it was immediately predicted—correctly—that a similar method could be used to synthesize any virus, including smallpox. Poliovirus, which was discovered nearly a century ago by Karl Landsteiner and Erwin Popper (1909), is a human virus that replicates in the gastrointestinal tract after ingestion. In rare instances, the virus invades the central nervous system where it destroys motor neurons that control muscle movement. This results in a terrifying disease, called poliomyelitis, which leads to irreversible paralysis and even death (Mueller et al, 2005a). Although the virus is relatively benign—the ratio of infection to neurological complication ranges from 1:100 to 1:1,000—the speed with which it spreads caused frightening epidemics among unprotected populations during the first half of the twentieth century. The development of two vaccines, the inactivated poliovirus vaccine (IPV) of Jonas Salk in 1954 and the attenuated oral vaccine (OPV) of Albert Sabin in 1957, broke the terrifying grip of poliomyelitis in the mid-1950s. The distinct mode of oral administration and the mechanism of eliciting a protective immune response—both mucosal and humoral—made the OPV the favourite for mass vaccination. Encouraged by the reduction of poliomyelitis in the Americas, the World Health Organization (WHO; Geneva, Switzerland) started a global vaccination campaign in 1988 with the aim of eradicating poliovirus. So far, it has been a success: the global incidence of poliomyelitis decreased from more than 350,000 cases in 150 countries in 1988 to 1,255 cases in 18 countries in 2004 (Kew et al, 2005). In spite of the unexpected resilience of the virus (Heymann et al, 2005), there is great optimism among the officials conducting the global eradication campaign that all polioviruses will eventually be eliminated. Not everybody, however, shares this optimism. Moreover, the question arises as to whether a virus whose 'formula'—genome sequence—is known can ever be eradicated. The empirical formula of poliovirus (Fig 1A; Molla et al, 1991) is C332,652H492,388N98,245O131,196P7,501S2,340. Because poliovirus is a quasi-species (Wimmer et al, 1993), the number of atoms in viral particles represents an average from a large population of different viruses. There might be little practical use in describing poliovirus by its empirical formula, but it persuasively portrays the virus as a chemical. Placing the atoms in order, a particle of high symmetry emerges (Fig 1B; Hogle et al, 1985), with all the properties required for its proliferation and survival in nature. These properties are encoded in the viral genome, a single-stranded RNA molecule of about 7,500 nucleotides (Fig 2; Kitamura et al, 1981). There might be little practical use in describing poliovirus by its empirical formula, but it persuasively portrays the virus as a chemical Figure 1.Poliovirus and its empirical formula. (A) Empirical formula of the organic matter of poliovirus (Molla et al, 1991). (B) Depiction of a poliovirus particle generated from X-ray crystallographic data (Hogle et al, 1985). Download figure Download PowerPoint Figure 2.Genome sequence of poliovirus, an RNA virus (Kitamura et al, 1981; Racaniello & Baltimore, 1981a), shown here in the corresponding DNA sequence. Download figure Download PowerPoint At present, it is impossible to synthesize RNA of such length chemically. We therefore rerouted the synthesis through double-stranded DNA, using mail-ordered complementary oligonucleotides, which can be assembled in a linear fashion. After many steps of elongating the DNA strand by adding new oligonucleotides, we obtained a genome-length double-stranded complementary DNA (cDNA) of about 7,500 base pairs that contained all the genetic information of the viral RNA genome (Fig 3A). This synthetic cDNA was transcribed into viral RNA using a specific RNA transcriptase (van der Werf et al, 1986), thereby yielding infectious viral RNA (Fig 3A). We could have simply transfected this RNA into human cells to obtain authentic poliovirus; instead, we opted to violate the normal replicative cycle of the virus by mixing the RNA with a cell-free extract of uninfected human cells that was devoid of nuclei, mitochondria and other cellular organelles. As we showed a decade earlier (Molla et al, 1991), the RNA was both translated and replicated to form viral proteins and new genomes until a critical mass of viral products was reached; at this stage, poliovirus particles spontaneously assembled (Fig 3A). Figure 3.Synthesis of poliovirus in the absence of natural template. (A) Short complementary segments of synthetic DNA (oligonucleotides) are annealed, and enzymatically extended and ligated (connected). A full-length complementary DNA (cDNA) is assembled stepwise to represent the entire genetic information of the poliovirus RNA genome in the form of DNA. The cDNA is then transcribed into infectious viral RNA by a T7 RNA transcriptase. This RNA is used to seed a HeLa cell-free extract that will replicate, just like in intact cells, to form progeny virions (Cello et al, 2002; Molla et al, 1991). (B,C) Evidence for de novo synthesized virus is provided by plaque assays. Poliovirus plaques derived from synthetic virus (sPV1) and wild-type virus, respectively, are formed on monolayers of HeLa cells (Cello et al, 2002). Reproduced from Mueller et al (2005b), with permission. Download figure Download PowerPoint Our experiment has thus overthrown one axiom in biology—namely, that the proliferation of cells or, for that matter, viruses depends on the physical presence of a functional genome to instruct the replication process. It was believed that without parental genomes, no daughter cells or progeny viruses would arise. We have broken this fundamental law of biology by reducing poliovirus to a chemical entity, which can be synthesized on the basis of information stored in the public domain—an experimental proof of principle that is applicable to the synthesis of all viruses (Kitamura et al, 1981; van der Werf et al, 1986; Molla et al, 1991; Cello et al, 2002). Indeed, any DNA molecule of any length could be assembled from oligonucleotides; thus, a virus might be resurrected from pure genomic information. We were probably among the first to realize the potential dangers associated with this technology—the possible misuse of viral synthesis in bioterrorism. The US Defense Advanced Research Project Agency (Arlington, VA, USA) took the same stand and provided funding for our project, an endeavour we considered as a wake-up call. Indeed, the widespread attention generated by our publication raised the overall awareness of the new reality of synthetic viruses and its possible consequences. …the widespread attention generated by our publication raised the overall awareness of the new reality of synthetic viruses… Chemists have historically considered de novo synthesis to be the ultimate proof for any deciphered chemical structure. If the synthetic product, such as an antibiotic, had the same properties as the natural isolate in vitro and in vivo, the structure was considered proven. For the chemist, the synthesis of authentic poliovirus provides proof that the sequence originally deciphered from genomic RNA is correct (Cello et al, 2002; Kitamura et al, 1981; Racaniello & Baltimore, 1981a). Although nobody really doubted the accuracy of the poliovirus genome sequence because it had been determined multiple times, there are cases in which DNA synthesis might be the only way to ascertain that a genome sequence is correct, as in the resurrection of the Spanish influenza virus from archaeological samples (Tumpey et al, 2005; see below). The synthesis of a replicating 'organism' in the absence of a natural template was without precedent at the time of its publication in 2002, and provoked unusual and widespread responses. Leaving the scientific aspects aside, there were two factors that we believe contributed to the emotional, and sometimes contradictory, reactions. The first was the format in which the manuscript was presented to the public. During the editing process, our paper was stripped bare of our original discussion of the ethical and societal implications. In fact, we lost a battle with the editors of Science and agreed to a final text that resembled a laboratory report. The importance of how scientific data reach the public should not be underestimated; in our case, the brevity led commentators to twist the story in any desired direction—sometimes inflammatory and of little substance—and left us, the authors, with little defence. The second reason was the timing of the publication. The manuscript appeared less than a year after the terrorist attacks on 11 September 2001 and the anthrax bioterrorist attacks in 2001. Consequently, news of the poliovirus synthesis reached a highly irritated public, particularly in the USA, which was ready to link it with bioterrorism. The US public worried that our synthesis provided a blueprint for generating dangerous biological weapons that might endanger national security. …news of the poliovirus synthesis reached a highly irritated public, particularly in the USA, which was ready to link it with bioterrorism The different reactions to the poliovirus synthesis were astounding and perplexing. In general, they fell into one of the following categories: positive reactions, ethical concerns, questions about the scientific value of the experiment, concerns about jeopardizing the global eradication of poliovirus, issues of national security, and issues of freedom and censorship of biological research. Thankfully, the majority of responses were positive. Considering ethical concerns, our research has fuelled a long-standing debate on whether viruses are alive. So far, the relationship of viruses to life has been described in vague terms at best: "viruses are parasites that skirt the boundaries between life and inert matter," said Luis P. Villarreal; "viruses lead a kind of borrowed life," Marc H. V. van Regenmortel and Brian W. Mahy maintained; and "whether or not viruses should be regarded as organisms is a matter of taste," according to André Lwoff (Villarreal, 2004). However, these descriptions are inconclusive, as humans have not reached a consensus on how to define life, and will probably never agree on a single definition. Not surprisingly, there has been a deluge of different definitions of life formulated by individuals of every province in society, including scientists, philosophers and theologians. A quote in 1975 by John Maynard Smith, favoured by many scientists including myself, describes life "as any population of entities which has the properties of multiplication, heredity and variation" (Lahav, 1999). Viruses qualify for this classification. By contrast, viruses have been excluded from classification as living entities because they do not consume 'food' or produce 'waste', because they lack metabolism and, as intracellular parasites, because they depend upon a supply of energy. Robert Hazen, however, has argued that "such a taxonomic question [that is, life versus non-life] is fruitless given the likely sequence of steps of increasing complexity that must have characterized the transition from a geochemical to a biochemical world" (R. Hazen, personal communication; Hazen, 2005). When I am asked whether poliovirus is a non-living or a living entity, my answer is yes. I regard viruses as entities that alternate between non-living and living phases. Outside the host cell, poliovirus is as dead as a ping-pong ball. It is a chemical that has been purified to homogeneity and crystallized (Schaffer & Schwerdt, 1955), with its physical and chemical properties largely determined (Wimmer et al, 1993), and its three-dimensional structure solved. Just like a common chemical, poliovirus has been synthesized in the test tube. Once poliovirus, the chemical, has entered the cell, however, it has a plan for survival. Its proliferation is then subject to evolutionary laws: heredity, genetic variation, selection towards fitness, evolution into different species and so forth—that is, poliovirus obeys the same rules that apply to living entities. One could even argue that poliovirus undergoes sexual reproduction in the infected cell, as it readily recombines with sibling progeny or with other related viruses (P. Jiang, J.A.J. Faase, H. Toyoda, A.E. Gorbalenya and E. Wimmer, unpublished data) to exchange genetic information (Wimmer et al, 1993). Barring religious beliefs, scientific wisdom holds that living entities will irreversibly die. I suggest that viruses do not follow this destiny, but rather switch between non-living and living phases. These seemingly incompatible qualities of viruses might be difficult to comprehend, but think of the electron: it took physicists decades to accept that it is both a wave and a particle. In 1828, Friedrich Wöhler, a German chemist, shattered the doctrine of vitalism by synthesizing an organic compound (urea) from an inorganic compound (ammonium cyanate; Wöhler, 1828). Vitalism states that organic compounds possess properties that cannot be explained in physical or chemical terms. Similarly to the synthesis of urea, no 'vital force' was necessary to 'instruct' the poliovirus genome during the chemical synthesis and transcription of the cDNA en route to infectious viral RNA. There is nothing transcendental about the sequence of the poliovirus genome shown in Fig 2. We have concluded that vitalism is not necessary to explain the properties of poliovirus or any virus (Cello et al, 2002). It must be emphasized, however, that we have merely reproduced poliovirus by following the blueprint of the viral genome (Kitamura et al, 1981). We did not 'create' this virus. The complexity of virus structure and replication currently makes it impossible to design an entirely new virus, regardless of whether the hosts are bacteria, plants or mammals. I understand that my view of poliovirus as an inanimate chemical with a life cycle is not uniformly shared. It might be criticized for adopting a "reductionist approach limiting our scientific understanding of living organisms" (Cho et al, 1999). Religious considerations might also override my position on viruses and life. I do not contest the divergent views of others, and I expect that my views—and our scientific data—will be equally respected. Avoiding dialogues or even ignoring ethical issues can lead not only to misunderstandings but also to communal upheaval and violence. In 1978, Charles Weissmann and colleagues revolutionized RNA virology by inventing reverse genetics (Taniguchi et al, 1978). To use the methods developed for DNA molecular biology, the researchers converted the purified genomic RNA of phage Qβ, a virus of bacteria, into full-length cDNA with the enzyme reverse transcriptase. This virus-specific cDNA yielded authentic RNA Qβ phages after transfection into bacteria (Fig 4A). After 3 years, Racaniello & Baltimore (1981b) repeated this experiment using purified poliovirus RNA and human cancer cells as hosts (Fig 4A). They too obtained authentic virus. After our publication of synthetic poliovirus in 2002, the question was asked: why bother to chemically synthesize cDNA (Fig 4B) when this can be done faster and much more cheaply with the help of enzymes? In relation to this, a perplexing view was aired in the journal Science, claiming that the chemical synthesis of poliovirus cDNA was just a publicity stunt (Block, 2002). Figure 4.Two different strategies for generating RNA viruses via complementary DNA (cDNA) intermediates. (A) Synthesis of cDNA catalysed with reverse transcriptase followed by transfection of the cDNA into host cells (Taniguchi et al, 1978; Racaniello & Baltimore, 1981b). (B) Chemical synthesis of cDNA followed by transcription and incubation of the infectious viral RNA in a cell-free extract (Cello et al, 2002; Molla et al, 1991). Download figure Download PowerPoint Critics in this frame of mind ignore the fact that generating poliovirus cDNA per se was not the message of our 2002 paper. A major point was that viruses can be looked at as chemicals and, can be synthesized from information with the entire of the virus can living It should also be that we in 2002 given the in it would be possible to synthesize poliovirus in a As I will the has also argued that the poliovirus synthesis was (Block, 2002). with the sequence of the wild-type genome, the synthetic cDNA had base that were into the nucleotide as genetic These base had no on the replication of the virus in HeLa cells (Fig However, one of the attenuated the of the synthetic virus in to poliovirus infection et al, 2005). This has a new of in our Although that we did not the poliovirus synthesis in fact yielded scientific information about the genetics of viral Indeed, our of synthesizing poliovirus has to viral genome synthesis for for to strategies in vaccine development E. Wimmer, unpublished eradication of an infectious is the way to control the it The of the eradication campaign the to poliomyelitis by eradicating poliovirus. As the population is poliomyelitis than ever the poliovirus synthesis in 2002 no However, a different question the test-tube synthesis to The answer to this is yes. Poliovirus cannot be because the sequence of its genome is known and it to be resurrected at any time in This is for all including smallpox. Indeed, the global eradication campaign for in its has much more difficult than from the resilience of wild-type major have as a of properties of the It has the to its as a by in thereby into highly poliovirus after with other (Kew et al, 2005; P. Jiang, J.A.J. Faase, A.E. Gorbalenya and E. Wimmer, unpublished This in different of the of In the OPV can highly poliovirus for et al, 2004). The known number of infected is and the number of poliovirus cannot be determined at the infected individuals a vaccination has been These have led a of to the development of for the control of poliomyelitis Research The for of OPV vaccination 3 after the incidence of The in this are poliomyelitis will as new are have not been infected with wild-type viruses or were not a that has never in human any of poliomyelitis will be whether it is caused by samples of virus stored in by polioviruses or by poliovirus that is chemically synthesized with The scientific and of poliovirus with the new reality of de novo synthesis of to whether the campaign has been a Our are on poliomyelitis rather than its It has been that vaccination poliomyelitis, on developed vaccines, might have to et al, 2005). The scientific and of poliovirus with the new reality of de novo synthesis of to whether the campaign has been a of the publication of the synthesis of poliovirus in Science in July 2002, from scientists and for of national security, the work was and the manuscript should never have been it was and is that the chemical synthesis of poliovirus not a to the the question as to whether the poliovirus synthesis was a blueprint for was and is that the chemical synthesis of poliovirus not a to the population on infectious these on research to the of a through or new The of information is considered to scientific research. it might be concluded that of biological information such and, thus, is the to bioterrorism. this view as It was the of a on and by the US of and the for and in in to a discussion of these editors of scientific of papers in the biological of the US and led to for journal editors and that publication of scientific but also of highly information et al, However, all methods used for the synthesis of poliovirus were the experiment was we described new to synthesize DNA methods to cDNA into infectious viral RNA. The of the poliovirus to a proof of principle and to a wake-up as at the should biological research in be to In a on Research and to the of assembled by the US Research a system for scientists to research that could national Research 2004). The concluded that challenge is for the scientific to a system that fundamental research to research with great potential for the freedom of journal editors to fundamental research the developed in the an of the of at and research These will research and, to the DNA of the of Health the of an experiment in fundamental research in biology, in all cannot be are too to be to for all no matter how the experiment is with viruses are no and as great a in as they do in the but are no of even the scientists when do not the However, results are not but have also led to that have an entire or new Indeed, results might have revolutionized the of the of for These considerations explain why it is difficult to research that a to from the majority of from which we including security. scientists must the for the in research. However, of biological research must be The are such as are to only in the after the poliovirus synthesis was a paper appeared describing the de novo in just of the DNA genome of et al, The DNA was transfected into bacteria, which the from the and This of DNA was a that could be without to any virus, including on the bioterrorist from the US of and the could be used to synthesize poliovirus or virus this fact was lost on the public in the great after the poliovirus synthesis in 2002, which had been largely fuelled by author of the paper, had been publication in virology was the resurrection of the Spanish influenza virus by chemical synthesis (Tumpey et al, 2005). This virus, with the genetic caused the influenza of which an people the of new influenza including the of the highly influenza it was to the Spanish influenza virus and to the molecular by which it its A genome sequence of the Spanish influenza virus was deciphered from decades after the This sequence the de novo synthesis of the virus, in the of its (Tumpey et al, 2005). The publication of the resurrected which caused much public was in multiple of all eventually its synthesis and publication. It was argued that the of the scientific which yielded new into influenza the of I this in the public was much and per