Synthetic Life
A common argument used by theists to support their belief in God, is that life is so complicated that it could have only been made by God. Often this is accompanied with the assertion that there is a "vital force" that separates inanimate objects from living things, and that God is the source of this "vital force". This philosophy, called "vitalism", has now been totally discredited. The following research report in the highly respected peer-reviewed journal Science details the de novo creation of a complete functional virus from synthetic chemicals.
Some people will probably say that a virus is not a living thing, but that all depends on how you define life. Clearly a simple virus is not as complex as a mammal, but it does have much more properties associated with living things than properties associated with inanimate objects. For example viruses reproduce, evolve, contain genetic information, and have a life cycle. These are characteristics in common with a pet dog, not a pet rock.
Even today, the study of chemistry is divided into "organic" and "inorganic" chemistry. This is a throwback to an era when it was believed that only living things could create the carbon bonds that define the difference between organic and inorganic chemistry. This has been discredited in 1828, when Wöhler synthesized urea. The year 2002 marks another milestone in science, with man's first creation of a virus, a living thing, from nonliving material.
I have copied the complete article here along with the author's supplemental material at the end. (If you need to reference this paper, use a format like this: "Cello, J; Paul, A; Wimmer, E, Chemical Synthesis of Poliovirus cDNA: Generation of Infectious Virus in the Absence of Natural Template, Science 2002 297: 1016-1018")
Copyright © 2002 by the American Association for the Advancement of Science
Volume 297(5583) 9 August 2002 pp 1016-1018
Chemical Synthesis of Poliovirus cDNA: Generation of Infectious Virus in the Absence of Natural Template
[Research: Reports]
Cello, Jeronimo; Paul, Aniko V.; Wimmer, Eckard*
Abstract
Full-length poliovirus complementary DNA (cDNA) was synthesized by assembling
oligonucleotides of plus and minus strand polarity. The synthetic poliovirus
cDNA was transcribed by RNA polymerase into viral RNA, which translated and
replicated in a cell-free extract, resulting in the de novo synthesis of
infectious poliovirus. Experiments in tissue culture using neutralizing
antibodies and CD155 receptor-specific antibodies and neurovirulence tests in
CD155 transgenic mice confirmed that the synthetic virus had biochemical and
pathogenic characteristics of poliovirus. Our results show that it is possible
to synthesize an infectious agent by in vitro chemical-biochemical means solely
by following instructions from a written sequence.
Research on viruses is driven not only by an urgent need to understand, prevent,
and cure viral disease. It is also fueled by a strong curiosity about the minute
particles that we can view both as chemicals and as "living" entities.
Poliovirus can be crystallized (1) and its empirical formula can be calculated
(2), yet this "chemical" replicates naturally in humans with high efficiency,
occasionally causing the paralyzing and lethal poliomyelitis.
Poliovirus, an enterovirus of the Picornaviridae, is a small, nonenveloped,
icosahedral virus consisting of five different macromolecules: 60 copies each of
capsid polypeptides VP1, -2, -3, and -4 and one copy of the positive-sense,
single-stranded RNA genome (~7.5 kilobases in length) (Fig. 1A) (3). The
chemical sequence (4, 5), the genetic map of the genome (4), and the
three-dimensional crystal structure of the virion (6) were determined 2 decades
ago. Poliovirus employs one of the simplest genetic systems known for
proliferation (3, 7). The virus enters the cell after attaching to the cellular
receptor CD155 (8, 9). Immediately after the virus particle uncoats inside the
cell, the genomic RNA is translated under the control of the internal ribosomal
entry site (IRES) into a single polypeptide, the polyprotein (10, 11). The
polyprotein is then processed into functional proteins by two viral proteinases
(3, 7). With the aid of viral proteins, most notably the RNA-dependent RNA
polymerase 3Dpol and the genome-linked protein VPg, along with cellular
components, the viral RNA is transcribed into minus-strand copies that serve as
templates for the synthesis of new viral genomes (plus-strand RNA). Newly
synthesized plus-strand RNA can serve as messenger RNA for more protein
synthesis, engage further in RNA replication, or be encapsidated by an
increasing pool of capsid proteins (7, 12). In suitable tissue culture cells
(for example, HeLa cells), the entire replication cycle is complete in only 6 to
8 hours and yields 104 to 105 progeny virions per cell.
Fig. 1. Genomic structure of PV1(M) and strategy for the synthesis of its full-length cDNA. (A) The positive-stranded RNA of poliovirus is shown with VPg at the 5' end of the NTR. In the cDNA, VPg is replaced by the T7 RNA polymerase promoter. The polyprotein contains one structural (P1) and two nonstructural (P2 and P3) domains. The 3' NTR contains a heteropolymeric region and is polyadenylated (shown as AAAn). (B) PV1(M) cDNA carrying a T7 RNA polymerase promoter at the 5' NTR end was subdivided into three large fragments for the synthesis of full-length sPV1(M) cDNA. The sizes of the fragments (in bp) are depicted above or below each rectangle that represents the respective fragment. The genome sequence encoded by each fragment was described in (13). (C) The three DNA fragments were synthesized as described in the text. The DNA fragments were assembled stepwise via common unique restriction endonuclease cleavage sites to yield full-length sPV(M) cDNA (F1-2-3 pBR322). The sequence of sPV(M) cDNA was confirmed by automated sequence analyses.
Here we describe the de novo chemical-biochemical synthesis of infectious
poliovirus from basic chemical building blocks, independent of viral components
previously formed in vivo and with the use of the known sequence as the only
instruction for engineering the genome. The succession of macromolecular events
in an infected cell was reproduced in a test tube containing a cell-free extract
devoid of nuclei, mitochondria, and other cellular organelles and seeded with
viral RNA. This result confirms that the genome sequence originally deciphered
from virion RNA is correct (4, 5) and demonstrates the feasibility of
chemical-biochemical synthesis of an infectious agent in the absence of a
natural template.
The strategy of synthesizing the genome of poliovirus type 1 (Mahoney) [PV1(M)]
began with the assembly of a full-length cDNA carrying a phage T7 RNA polymerase
promoter at the (left) 5' end (Fig. 1) from three large, overlapping DNA
fragments (F1, -2, and -3). Each DNA fragment was obtained by combining
overlapping segments of 400 to 600 base pairs (bp). The segments were
synthesized by assembling purified oligonucleotides [average length, 69
nucleotides (nt)] of plus and minus polarity with overlapping complementary
sequences at their termini, and the segments were then ligated into a plasmid
vector (13). Five to 15 clones were sequenced to identify either the correct DNA
segments or the segments containing small numbers of errors that could be
eliminated, either by combining the error-free portions of segments by an
internal cleavage site or by standard site-directed mutagenesis (13). To
ascertain the authenticity of the synthesized viral genome [sPV1(M)] and to
distinguish it from the wild-type (wt) sequence of PV1(M) [wt PV1(M)] (4, 5), we
engineered nucleotide substitutions into the sPV1(M) cDNA as genetic markers
(13).
We have shown previously that poliovirus cDNA carrying a phage T7 promoter for
the phage RNA polymerase can be transcribed with T7 RNA polymerase into highly
infectious RNA (14). Accordingly, the sPV1(M) cDNA and wt PV1(M) cDNA were
transcribed (13) and were found to yield transcript RNAs of the same length as
virion RNA (15). De novo synthesis of poliovirus from transcript RNA of wt
PV1(M) cDNA in a cell-free extract of uninfected HeLa cells has been previously
described by Molla et al. (2). Therefore, the incubation of transcript RNA from
sPV1(M) cDNA in cytoplasmic extracts of uninfected HeLa cells should result in
the generation of poliovirus. To examine this possibility, transcript RNA
derived from sPV1(M) cDNA was incubated with a cytoplasmic extract of HeLa S3
cells, and the synthesis of virus-specific proteins and infectious viruses were
monitored. The products of sPV1(M) cDNA-derived RNA translation and proteolytic
processing were the same as those obtained with wt PV1(M) RNA (Fig. 2), an
observation suggesting that the open reading frame (ORF) of the sPV1(M)-specific
RNA is intact. We then tested for the presence of infectious virus particles in
the cell-free incubation mixture by adding aliquots of the incubation mixture to
monolayers of HeLa cells. After 48 hours, plaques appeared [0.5 to 1 × 105
plaque-forming units (PFU) per µg of transcript RNA in 50 µl of reaction] whose
heterogeneous morphology was characteristic of those produced by authentic
poliovirus (Fig. 3). All together, these results indicate that the input
synthetic RNA was translated and replicated in the cell-free extract and that
newly synthesized RNA was encapsidated into newly synthesized coat proteins,
resulting in the de novo synthesis of infectious poliovirus.
Fig. 2. Products of in vitro translation and proteolytic processing of poliovirus RNAs in a HeLa cell-free extract. Transcript RNA derived from sPVM cDNA (13) and virion RNA derived from wt PV1(M) were translated and analyzed as described in (13). Lane 1, wt PV1(M) marker (M) displaying a lysate of [35S] methionine-labeled poliovirus-infected HeLa extract; lane 2, virion RNA derived from wt PV1(M); lane 3, RNA derived from sPV1(M) cDNA. Bands correspond with the segments in Fig. 1A. VPO, 2BC, 3AB, and 3CD are precursor polypeptides.
Fig. 3. Plaque phenotypes of polioviruses generated in the HeLa cell-free
extract. De novo synthesis of poliovirus from transcript RNA in cell-free
extract of uninfected HeLa cells was done as described in (13). (A) Plaque
phenotype of virus derived from transcript sPV1(M) RNA. (B) Plaque phenotype of
virus derived from virion wt PV1(M) RNA.
Experiments were then carried out to confirm that the infectious material
isolated from the cell-free extract was indeed sPV1(M), as designated by the
oligonucleotide sequence. Restriction enzyme digestion of the reverse
transcriptase-polymerase chain reaction (RT-PCR) product of the viral RNA
recovered from sPV1(M)-infected HeLa cells revealed the presence of all
engineered markers (fig. S1, lanes 1 and 2).
We also tested the effects of the poliovirus receptor-specific monoclonal
antibody (Mab) D171 and type-specific hyperimmune sera on plaque formation by
sPV1(M) (Table 1). Mab D171 has been shown to completely block infection of all
three serotypes by specifically binding to CD155, the cellular receptor of
poliovirus (8, 9, 16). The treatment of HeLa cells with Mab D171 before the
addition of sPV1(M) completely abolished plaque formation (Table 1). Similarly,
no plaques were observed when sPV1(M) was incubated with poliovirus type
1-specific rabbit hyperimmune serum [anti-PV1(M)]. Neutralization of the
synthetic virus was type-specific because hyperimmune serum to poliovirus type 2
(Lansing) [PV2(L)] did not inhibit plaque formation (Table 1). These results
were in full agreement with those obtained with wt PV1(M) (Table 1). They imply
that the de novo poliovirus particles synthesized in the cell-free extract were
serotype 1, requiring the authentic poliovirus receptor for infection.
Table 1. Biological characterization of sPV1(M). Plaque reduction assay in the presence (+) and absence (-) of antibodies as described in (13). Anti-PV1(M) and anti-PV2(L) are neutralizing polyclonal antibodies specific for types 1 and 2 poliovirus, respectively. Neuropathogenicity of sPV1(M) and wt PV1(M) was assayed in hPVR-tg mice as described in (13). PLD50 is defined as the amount of virus that caused paralysis or death in 50% of the inoculated mice.
The sPV1(M) virus was assayed to determine whether it expresses a
neurovirulent phenotype in mice transgenic for the human poliovirus receptor
[CD155 tg mice strain ICR.PVR.tg I (17)]. When injected with wt poliovirus
strains, these animals develop a neurological disease indistinguishable,
clinically and histologically, from primate poliomyelitis (17-19). Intracerebral
injection of sPV1(M) caused flaccid paralysis or death in CD155 tg mice,
resembling the disease produced by wt PV1(M) (13). However, a larger inoculum of
sPV1(M) than PV1(M) was necessary to paralyze or kill the animals (Table 1). The
increase in the magnitude of attenuation was unexpected, because all nucleotide
substitutions introduced into sPV1(M) resulted in silent mutations in the ORF,
except for the newly created Xma I and Stu I sites in the 5' nontranslated
region (NTR) and 2B region, respectively. These latter changes had been shown
previously to have no influence on viral replication in tissue culture (20, 21).
However, the silent mutations that we introduced into the poliovirus genome may
exert a strong influence on pathogenesis by hitherto unknown mechanisms.
The presence or absence of genetic markers in the inoculated virus and the virus
isolated from the spinal cords of paralyzed mice was confirmed by amplification
of the viral RNA by RT-PCR and restriction enzyme analysis. Our results show
that the viruses isolated from the spinal cords of paralyzed mice resembled the
inoculated virus (fig. S1). Our data also confirm that the synthetic virus was
the causative agent of the flaccid paralysis observed in the sPV1(M)-infected
mice.
The chemical synthesis of the viral genome, combined with de novo cell-free
synthesis, has yielded a synthetic virus with biochemical and pathogenic
characteristics of poliovirus. In 1828, when Wöhler synthesized urea, the theory
of vitalism was shattered (22). If the ability to replicate is an attribute of
life, then poliovirus is a chemical
[C332,652H492,388N98,245O131,196-P7501S2340,
see (2)] with a life cycle.
As a result of the World Health Organization's vaccination campaign to eradicate
poliovirus (23), the global population is better protected against poliomyelitis
than ever before. Any threat from bioterrorism will arise only if mass
vaccination stops (23) and herd immunity against poliomyelitis is lost. There is
no doubt that technical advances will permit the rapid synthesis of the
poliovirus genome, given access to sophisticated resources. The potential for
virus synthesis is an important additional factor for consideration in designing
the closing strategies of the poliovirus eradication campaign.
References and Notes
1. F. L. Schaffer, C. E. Schwerdt, Proc. Natl. Acad. Sci. U.S.A. 41, 1020
(1955). [Context Link]
2. A. Molla, A. Paul, E. Wimmer, Science 254, 1647 (1991). Bibliographic Links
[Context Link]
3. T. Pfister, C. Mirzayan, E. Wimmer, in The Encyclopedia of Virology, R. G.
Webster, A. Granoff, Eds. (Academic Press Ltd., London, ed. 2, 1999), pp.
1330-1348. [Context Link]
4. N. Kitamura et al., Nature 291, 547 (1981). Bibliographic Links [Context
Link]
5. V. R. Racaniello, D. Baltimore, Proc. Natl. Acad. Sci. U.S.A. 78, 4887
(1981). Bibliographic Links [Context Link]
6. J. M. Hogle, M. Chow, D. J. Filman, Science 229, 1358 (1985). Bibliographic
Links [Context Link]
7. E. Wimmer, C. U. T. Hellen, X. Cao, Annu. Rev. Genet. 27, 353 (1993).
Bibliographic Links [Context Link]
8. C. L. Mendelsohn, E. Wimmer, V. R. Racaniello, Cell 56, 855 (1989). [Context
Link]
9. S. Koike et al., EMBO J. 9, 3217 (1990). [Context Link]
10. S. K. Jang et al., J. Virol. 62, 2636 (1988). Bibliographic Links [Context
Link]
11. J. Pelletier, N. Sonenberg, Nature 334, 320 (1988). Bibliographic Links
[Context Link]
12. W. K. Xiang, A.V. Paul, E. Wimmer, Semin. Virol. 8, 256 (1987). [Context
Link]
13. Materials and methods are available as supporting material on Science
Online. [Context Link]
14. S. van der Werf, J. Bradley, E. Wimmer, F. W. Studier, J. J. Dunn, Proc.
Natl. Acad. Sci. U.S.A. 82, 2330 (1986). Bibliographic Links [Context Link]
15. J. Cello, A. V. Paul, E. Wimmer, unpublished data. [Context Link]
16. P. Nobis et al., J. Gen. Virol. 66, 2563 (1985). Bibliographic Links
[Context Link]
17. S. Koike et al., Proc. Natl. Acad. Sci. U.S.A. 88, 951 (1991). Bibliographic
Links [Context Link]
18. H. Horie et al., J. Virol. 68, 681 (1994). Bibliographic Links [Context
Link]
19. M. Gromeler, H.-H. Lu, E. Wimmer, Microb. Pathog. 18, 253 (1995). [Context
Link]
20. C. Mirzayan, E. Wimmer, Virology 189, 547 (1992). Bibliographic Links
[Context Link]
21. W. Xiang, K. S. Harris, L. Alexander, E. Wimmer, J. Virol. 69, 3658 (1995).
Bibliographic Links [Context Link]
22. F. Wöhler, Ann. Phys. Chem. 88, 253 (1828). [Context Link]
23. A. Nomoto, I. Arita, Nature Immunol. 3, 205 (2002). [Context Link]
24. We thank A. Wimmer and J. Benach for valuable comments on the manuscript. We
are indebted to B. L. Semler for a sample of cell-free HeLa cell extract.
Supported by Contracts N65236-99-C-5835 and N65236-00-M-3707 from the Defense
Advanced Research Project Agency.